Deepak Chowrasia, Nisha Sharma-Analytical Chemistry. a Qualitative & Quantitative Approach (2015)

Analytical Chemistry A Qualitative & Quantitative Approach (General Techniques)

Deepak Chowrasia

Dr. Nisha Sharma

Assistant Professor Institute of Pharmacy CSJM University, Kanpur, (U.P.), India

Head of Department Institute of Pharmacy CSJM University, Kanpur, (U.P.), India

Analytical Chemistry-A Qualitative & Quantitative Approach, (General Techniques)

Legal Disclaimer This book is written, edited, and printed (soft & hard copy) in India and sold or free circulated (subject to legal written permission from author) with an intention to amplify knowledge of reader irrespective of global territory. Any authorities (Government and/or non-government and/or education institutions and/or Universities and/or other institutions-profitable institutions-profitable or non-profitable non-profitable societies including websites-educational or noneducational) in any respect or under any circumstances is strictly not allowed to store any part of this book in their/other retrieval system, sold, hire-out, lend, resold, circulate, print, reprint, and/or reproduce any part of this book including sections, subsections, figures, tables, and/or any other related written materials by any means (photocopying, (photocopying, recording, mechanical, electronic or otherwise) without the legal written consent from author. Every effort has been made by author/s to avoid error/s (printing, grammatical, and any other kind) or omission/s in this edition. However, any error/mistake or discrepancies if noted may be brought to author and necessary action will be taken in the next edition. It is honestly notified that neither the author/s or the printing press or seller or distributor will be responsible for any damage or loss to anyone of any kind in any manner. Any queries and suggestion regarding this edition is heartiest welcome and can be mailed to [email protected]. This edition is Copyright Copyright ©-2015 edition. All rights reserved with author.

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Preface for First Edition It is difficult to pen down feeling on publication of my book “ Analytical Chemistry-A Qualitative & Quantitative Approach, (General Techniques). The idea of writing

book of this magnitude was encompasses from my own experience & vision while critically analyzing students of science ( B.Sc/M.Sc/Pharmacy/B.Tech.) getting difficulties in searching book predominately in the field of “Analytical Chemistry” although the market is surplus with vast majority of books dealing directly or indirectly under same heading, but their high technical standard, non-soothing language, irrelevant to topic, non-specific course description, lengthy chapters, and insufficient illustration ultimately limits their student friendly applicability. This book thus obscure out all the difficulties a student find in their academic career. While writing this book, an intensive care has been taken to present each chapter in a “lay-man-lucid-languag “lay-man-lucid-languagee (LML) yet not to be get devoid from technical track. A useful categorization of each chapter into section & sub-section renders any jumbling of topics, at the same time maintaining their technical including conceptual background and adhere reader to the main stream at no loss of interest in the subject. The book is enriched with numerous illustrative diagrams, diagrams, concise tabulation of data, minute details, exercise, and multiple choice questions which are important not only from the point of view of student internal & external examination,

but

also

beneficial

for

their

competitive

examination

(GATE/GPAT/CSIR-NET). In the last, I express my worthy thanks & a great success to all academicians, researchers, researchers, and students for their bright future. -Deepak Chowrasia


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Table of Content Chapter Nos.

Title

Author/s

Page Nos.

01

Non Aqueous Titration

Deepak Chowrasia

1

02

Complexometric Complexometric Titration

Deepak Chowrasia

27

03

Karl Fischer Titration

Dr. Nisha Sharma, Deepak Chowrasia

51

04

Diazotization Titration

Deepak Chowrasia, Ajay Kumar

61

05

Kjeldhal Titration


71

06

Paper Chromatography Chromatograph y

Deepak Chowrasia, Chowrasia ,

81

07

Thin Layer Chromatography Chromatography

Deepak Chowrasia

99

08

Column Chromatography Chromatography

Deepak Chowrasia

115

09

HPLC

Deepak Chowrasia

131

10

Gas Chromatography Chromatography

Deepak Chowrasia

155

11

Ion Exchange Chromatography Chromatography

Deepak Chowrasia

195

12

Size Exclusion Chromatography Chromatography

Deepak Chowrasia

209

13

Conductometry


219

14

Coulometry

Deepak Chowrasia

237

15

Potentiometry

Deepak Chowrasia

247

Chapter - 01 NON AQUEOUS TITRATION - Deepak Chowrasia

Chowrasia, Deepak

NON AQUEOUS TITRATION


3

NON AQUEOUS TITRATION (Chapter Overview) 01. INTRODUCTION ......................................................................................................................5 02. THEORY......................................................................................................................................5 03. ADVANTAGES OF NON AQUEOUS TITRATIONS .....................................................6 04. LIMITATIONS OF NON-AQUEOUS TITRATIONS .....................................................6 05. NON AQUEOUS SOLVENTS-PROPERTIES AND PROBLEMS. .............................. 6 06. NON-AQUEOUS SOLVENTS; CLASSIFICATION ....................................................... 7 06.A. 06.B. 06.C. 06.D.

Aprotic or neutral solvents ........................................................................................... 7 Protophilic solvent/proton loving solvent .................................................................7 Protogenic solvent ..........................................................................................................8 Amphiprotic solvent .......................................................................................................8

07. EFFECT OF HEAT ON NON-AQUEOUS TITRATION ............................................... 9 08. END POINT DETECTION IN NON-AQUEOUS TITRATION. ................................... 9 08.A. Visual method .................................................................................................................. 9 08.B Instrumental method ...................................................................................................10 09. APPARATUS ............................................................................................................................10 10. GENERAL PROCEDURE OF NON-AQUEOUS TITRATIONS ...............................11 11. PRECAUTIONS DURING TITRATION .......................................................................... 11 12. METHODS IN NON-AQUEOUS TITRATION...............................................................12 12.A. Acidimetric analysis in non-aqueous titrations .....................................................12 12.A.1. Titrant for acidimetric non-aqueous analysis .............................................. 12 12.A.1.a. Preparation of acetous 0.1 M perchloric acid solution .............12 12.A.1.a.1. Standardization of acetous 0.1M perchloric acid . 13 12.A.1.b. Preparation of 0.1 M perchloric acid solution in dioxane ........ 13 12.A.1.b.1. Standardization of 0.1M perchloric acid solution in dioxane ......................................................................................13

12.A.2. Types of Acidimetric analysis in non aqueous titrations. .........................13 12.A.2.1. Titration of amines and amines salts of organic acid ................13 12.A.2.2. Titration of halogen acid salt of bases .........................................13 12.A.2.3. Assay of few basic chemicals by acidimetric non aqueous titration ...........................................................................................................14

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12.A.2.3.1. Assay of Ephedrine HCl ............................................14 12.A.2.3.2. Assay of adrenaline: ..................................................14 12.A.2.3.3. Assay of Sodium Saccharine. .................................... 14 12.B. Alkalimetric analysis in non aqueous titration ...................................................... 15 12.B.1. Titrant used in alkalimetric analysis in non aqueous titrations .............15 12.B.1.a. Preparation of 0.1 M tetrabutylammonium hydroxide in Toluene-Methanol. ........................................................................................... 15 12.B.1.a.1 Standardization of 0.1 M tetrabutylammonium hydroxide ......................................................................................16 12.B.1.b. Preparation of 0.1M Potassium methoxide in toluenemethanol ...........................................................................................................16 12.B.1.b.1. Standardization of 0.1 M Potassium methoxide in toluene-methanol ............................................................................. 16 12.B.1.c. Preparation of 0.1M lithium methoxide in toluene-methanol ... 16 12.B.1.c.1. Standardization of 0.1 M lithium methoxide in toluene-methanol ............................................................................. 16

12.B.2. Assay of few acidic chemicals by alkalimetric non aqueous titration ....17 12.B.2.a. Assay of Ethosuximide ....................................................................17 12.B.2.b. Assay of benzoic acid .....................................................................17 12.B.2.c. Assay of chlorthalidone ..................................................................17 13. APPLICATIONS OF NON-AQUEOUS TITRATION ...................................................17 14. EXERCISE ................................................................................................................................ 19 15. MULTIPLE CHOICE QUESTIONS ..................................................................................20

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NON AQUEOUS TITRATIONS 01. INTRODUCTION Non aqueous titrations provide a versatile platform for titrating complex organic molecules including therapeutic important pharmaceuticals ( please refer table 05) as well as chemical compounds that possess problem of low solubility and high ionization in aqueous solvents. Also the weakly acidic or weakly basic substances, as most of the therapeutic chemical moieties, that are unable to give sharpe end point in aqueous solvent are far better titrated in non aqueous titration with good equivalence point as their strength is enhanced by dissolving them in suitable non aqueous solvents like carbon tetrachloride, benzene or liquid ammonia etc. 02. THEORY Numerous efforts had been made by different workers in order to justify & establish a unified universal theory regarding acid and bases (see table 01). The Lowery-Bronsted concept (1923) of acid and base can be parallely and equally well imposed to acid base titrations, both in aqueous and non aqueous solvents. Accordingly, acids are the substance that has tendency to donate a proton while base is a substance having tendency to accept proton. Shortly, acids are proton donor while bases are proton acceptor. S.No.

Concept

Acid Property

Base Property

01

Oldest concept

02

Arrhenius concept

03

Lewis theory

Hydrogen or oxygen containing substance, Sour in taste, turn litmus red Proton donor in aqueous solution Electron pair acceptor

Bitter in taste, turn litmus blue

04

Usanovich concept

Accept anion or donate cation Donate anion or accept cation

05

Lux-flood concept

Oxide ion acceptor

Proton acceptor in aqueous solution Electron pair donor

Oxide ion donor

Table 01: Concept of Acid & Base

Let us consider dissociation of acid PB in a solution which gives proton P and conjugate base B. PB acid

P+

+ BProton conjugated base

Likewise a base B can unite with proton P to yield conjugated acid PB

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B

+

P+

6

PB Base

Further expanding the above definition, acid is either an electrically neutral molecule like HCl + or HNO3 or a positive charge cation like C6H5NH3 on other hand base is an electrically neutral molecule like C 6H5NH2 or negative charged anions like Cl , NO3 . 03.

ADVANTAGES OF NON AQUEOUS TITRATIONS A. Simple, handy, accurate, precise, and rapid technique compare to classical method of analysis.

B. Titration of chemicals which shows poor end point in aqueous titrations. C. Poor water soluble substance can be easily and accurately titrated D. Improves reactivity of low or poor reactive substances (weak acids and bases). E. Instant availability of result. F. Requires no special or costlier apparatus. G. Visual detection of end point mostly (except very concentrated or highly coloured solution where potentiometric end point determination has to be done). 04.

LIMITATIONS OF NON-AQUEOUS TITRATIONS A. Utilization of costlier and toxic organic solvents.

B. No recovery of solvent at the end of titration. C. Moisture content should be less than 0.05% during overall process of titration. D. Difficulty in determining visual detection of end point in highly colored solution. E. Required skills for performing titrations. 05. NON AQUEOUS SOLVENTS-PROPERTIES AND PROBLEMS Organic solvents like glacial acetic acid, dioxane, acetonitrile are only the few names among the wide variety of solvents that are used efficiently and effectively in non aqueous titrations. An ideal non aqueous solvent ( did we have?) must possess properties of strict purity, analytical grade, economicity, non reactive, non toxic, dry (water free/anhydrous), good solubility, recyclibility, eco-friendly (tough to achieved due to their organic characteristic) and free from property of interfering with detection of end point. Non aqueous solvents have a unique property of equalizing the strength of weak acidic or weak basic substances to that of strong acidic and strong basic substances and thus this property of solvent equalization is termed as leveling or solvent effect of non aqueous solvents. It is advisable to protect solvent (non aqueous) from exposure to atmospheric air and titration must be performed in closed vessel to render the interference caused by moisture. Since all non aqueous solvents have

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higher value of thermal coefficient of expansion (change in physical property w.r.t. to temperature) compare to water-an aqueous solvent, hence it is recommended to store them at constant temperature and titration must be performed at same temperature to minimized error. 06. NON-AQUEOUS SOLVENTS; CLASSIFICATION Non aqueous solvents are capable of donating and accepting proton under condition of titration, depending upon their nature of donating or accepting protons they are classified as

06.A. Aprotic solvents 06.B. Protophilic solvent 06.C. Protogenic solvents 06.D. Amphiprotic solvents 06.A. Aprotic or neutral solvents These solvent posses a universal characteristic of beings non reactive, non ionizing, low dielectric constant (see table 02), and are neutral in nature. They are unable to generate proton under titrimetric condition and do not take part in overall chemical reaction because of their, this property, they are extensively used for diluting reaction mixtures. For example toluene and carbon tetrachloride. Non aqueous solvents

Dielectric constant

Acetic acid

6.15

Benzene

2.27

Chloroform

4.81

Dioxane

2.21

Acetonitrile

37.5

Sulphuric acid

100

Water*

81 Table 02: Dielectric constant of some solvents

06.B. Protophilic solvent/proton loving solvent These solvents (acetone, ammonia, ether, dioxane, amines-hydrazine, ethylenediamine) have high tendency to accept or gain protons during titration. They are basic in nature and readily react with acid to yield solvated protons. Protophilic solvents like pyridine, ethylenediamine, n-butylamine give high blank titration value due to absorption of atmospheric carbon dioxide hence it is necessary to kept them in tightly closed Pyrex glass containers.

Chowrasia, Deepak

NON AQUEOUS TITRATIONS


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On using protophilic solvent with weak acids, the acidic strength of weak acid increased and get comparable to that of strong acid thus protophilic solvents act as leveling solvent for weak acid. On other hand, titrating weak bases in the presence of acidic solvents (protogenic solvent) the basicity of weak bases enhanced & get equivalent to that of strong bases. This phenomenon of increasing the acidic and basic property of weak acid or weak base with the use of suitable non aqueous solvents is called leveling or solvent effect. 06.C. Protogenic solvent They are proton donating solvent and hence act as acidic in nature. They are, when used with weak bases, increase their basicity by donating protons and exerts a leveling effect on them. Like sulfuric acid, liquid HF, & perchloric acid etc. The acidic strength of non aqueous protogenic solvent follows following order

06.D. Amphiprotic solvent These solvents posses a dual characteristic of protophilic as well as protogenic solvent either they are have tendency to accept and/or donate protons and are dissociate to a slight extent in normally employed titration conditions. Amphiprotic solvents include water, alcohol, and acetic acid. Solvent

Glacial ethanoic acid (anhydrous acetic acid) (CH3COOH)

Acetonitrile (CH3CN)

Dioxan (1,4-dioxan)

Chowrasia, Deepak

Structure

HO O

N

Chemical Properties

Remark

MW-60.05 D-1.049g/mol ◦ MP-16-17 C ◦ BP-118-119 C RI-1.371 Pka-4.76

Commonly used non-aqueous solvent, moisture content (0.1-1.0%) essentially, acetic anhydride (q.s.) is added to convert any water if present to acetic acid, frequently used with ACN, & nitromethane

MW-41.05 D-786mg/ml ◦ MP-(-46 C) ◦ BP- 81 C RI-1.344 Pka-25

Also termed as Cyanomethane or methyl cynide, commonly used with ethanoic acid (frequently) and other solvent like phenol and chloroform, produce good end point for metal ethanoate titrated with perchloric acid.

MW-88.11 D-1.033g/ml

A better substitute for glacial acetic acid while dealing with mixture of



Solvent

Structure

Chemical Properties ◦

O

9

Remark

MP-11.8 C ◦ BP-101.1 C

substances, accepted official solvent for non-aqueous titration, but devoid of leveling effect.

MW-73.10 D-0.948g/ml ◦ MP-(-65 C) ◦ BP-152 C RI-1.4305

Usually abbreviated as DMF, is a protophilic solvent sometime creates difficulties in obtaining end point.

O

Dimethylformamide (CH3)2NC(O)H N

O

Table 03: Non-aqueous solvents & their properties 07. EFFECT OF HEAT ON NON-AQUEOUS TITRATION Non aqueous solvents are organic solvents that have high value for coefficient of thermal expansion compare to an aqueous solvent like water that means even a small change in temperature leads to a greater degree of error during titration. This problem can be efficiently overcome by standardization and carrying out titration at same temperature. If any how this could not be possible then titrant volume can be corrected by using formula given below

Where, VT =true volume of titrant VM=measure volume of tyrant T1=standardized temperature of titrant T2= carried out temperature of titration 08.

END POINT DETECTION IN NON-AQUEOUS TITRATION

08.A. Visual method It is the most commonest, simplest, cheaper, and highly versatile method used to determined end point of non aqueous titrations. The method involved addition of few drops (2-3) of suitable indicator (see table 04) into titration mixture and the end point of titration is determined visually by detecting change in color of titration mixture (one to another).

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08.B Instrumental method Visual method, although, easiest and convenient in end point determination, but shade off its applicability where the titration mixture is either turbid or highly colored in nature. Here the instrumental method, conveniently, potentiometry ( please refer chapter on potentiometry) plays crucial role in determining equivalence point. Potentiometric based end point determination in non aqueous titration involves utilization of two electrodes (reference & indicator electrode) dipped into reaction vessel & connected to a sensitive potentiometer by suitable wired assembly. Addition of titrant into titration mixture present in reaction vessels results in fluctuation in reading in galvanometer or other potential detecting device, data so obtained is then plotted graphically in order to detect end point. S.Nos

Indicator

Acidic

Neutral

Basic

01

Crystal violet(0.5%in GAA)

Yellowish green

Blue green

Violet

02

1-nephtholbenzene (0.2% in GAA)

Dark green

Orange

Blue or blue green

03

Nile blue A (1% in GAA)

Blue green

-

Blue

04

Oracet blue B (0.5% in GAA)

Pink

Purple

Blue

-

Magenta

05

Quinaldine red (0.1% in methanol) Almost colorless

Table 04: Indicators used in non-aqueous titration 09. APPARATUS Non aqueous titration does not involved any expansive or specialized instrumentation, instead general laboratory glass wares (burette, burette stand, conical flask (Erlenmayer flask), beaker, dropper, glass rod etc) made up of borosilicate glass is self sufficient to perform overall phenomenon of titration accurately and precisely (see figure 01). It must be noted that all apparatus employed for non aqueous titration must be dried under strict condition of higher temperature in order to eliminate even a minute content of water, which may interfere with final titration result. However, a moisture content of less than 0.05% is permissible under certain condition of titration.

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Figure 01: Apparatus for Non aqueous titration 10. GENERAL PROCEDURE OF NON-AQUEOUS TITRATIONS Accurately weigh out sample to be analyzed, transfer it into conical flask, dissolve in appropriate quantity of non aqueous solvent, followed by addition of 2-3 drops of indicator and titrate resultant sample mixture with (continuous stirring) suitable titrant present in burette until end point obtained. End point detection in non-aqueous titration can be done either instrumentally with the help of potentiometer or physically by visual differentiation (indicator) of change in one to another color. 11.

PRECAUTIONS DURING TITRATION A. No addition of water during whole process of titration. B. All reagents must be of analytical grade. C. Avoid any rigorous change in temperature. D. Handle titrant mainly perchloric acid with suitable precaution. E. Dispose off post titration mixture slowly & properly into sink.

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F. Avoid adding acetic anhydride to concentrated perchloric acid. G. Always keep perchloric acid (concentrated/dilute) in ambient temperature, away from direct sunlight. 12. METHODS IN NON-AQUEOUS TITRATION Non aqueous titrations plays important role in assay of wide variety of chemicals including pharmaceutical active ingredient and dosage form which because of either limited solubility in aqueous solvent and/or weak acidic/basic property could not be assayed satisfactorily. For the sake of convenience, methods in non aqueous titrations is categorized into following two classes (also see table 04); 1. Acidimetric analysis in non aqueous titration

a. Titration of amines (Primary, secondary, & tertiary) b. Titration of halogen acid salt of bases. 2. Alkalimetric analysis in non aqueous titration. 12.A. Acidimetric analysis in non-aqueous titrations This methodology used for feasible titration of weak bases with the aid of suitable non aqueous solvent which enhance basic property of analytical sample with obsolescing hindrance cause by aqueous solvents. Standard acetous 0.1M perchloric acid is most commonly employed titrant in acidimetric analysis in non aqueous titrations.

12.A.1. Titrant for acidimetric non-aqueous analysis Perchloric acid dissolves in glacial acetic acid and dioxane (both are official too) is commonly employed as titrant in acidimetric analysis of numerous chemical compounds, including vast number of pharmaceuticals (official in I.P.) in non aqueous titration. Method of preparation as well as standardization of same is given below; 12.A.1.a. Preparation of acetous 0.1 M perchloric acid solution

Add 8.5 ml (72%) of perchloric acid to glacial acetic acid (900ml) with efficient mixing. Add acetic anhydride (30ml), adjust final volume to 1litre with glacial acetic acid, and stand for 24 hours before titration. Keep the mixture in an air tight container away from direct contact with light. Acetic anhydride reacts with water present in perchloric acid & acetic acid thereby making the mixture completely anhydrous. It has to be noted that excess of acetic anhydride is not always disadvantage but precaution should be taken when primary or secondary amines are titrated as they acetylated rapidly and giving non basic product. It is strictly recommended not to add acetic anhydride to concentrated perchloric acid which results in formation of explosive acetyl perchlorate.

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12.A.1.a.1. Standardization of acetous 0.1M perchloric acid

Accurately weigh out 0.5g of potassium hydrogen phthalate in 100ml round bottom flask attach to a reflux condenser & fitted with silica gel drying tube. Add 25ml of glacial acetic acid and warm the mixture until salt dissolves completely, cool down it to room temperature and titrate the resultant mixture with 0.1M perchloric acid using 2-3 drops of 0.5% w/v acetous crystal violet (end point –blue green) or alternatively Oracet blue B 0.5% w/v ( end point-pink). COOH

COOH

+ KCLO4

+ HCLO4 COOK

COOH

Potassium hydrogen phthalate Figure 02: Standardization of acetous 0.1M perchloric acid 12.A.1.b. Preparation of 0.1 M perchloric acid solution in dioxane

Add 8.5ml (72%) of perchloric acid to sufficient Dioxane (200-300ml) with efficient mixing and adjust final volume to 1litre. Keep the mixture in an air tight container away from direct contact with light. 12.A.1.b.1. Standardization of 0.1M perchloric acid solution in dioxane

Standardized 0.1M perchloric acid by same procedure as given in standardization of acetous 0.1M perchloric acid. 12.A.2. Types of Acidimetric analysis in non aqueous titrations: For ease of categorization, as acidimetric non aqueous titrations deals with large number of chemical compounds including therapeutic importance drugs, they are broadly classified into following two main groups as given below 12.A.2.1. Titration of amines and amines salts of organic acid

Primary, secondary, and tertiary amines are titrated with perchloric acid using non aqueous media. For example adrenaline, metronidazole, codeine, diazepam, ethionamide, nitrazepam etc. Also amino acids like glycine & aminocaproic acid. 12.A.2.2. Titration of halogen acid salt of bases -

-

-

Halide ions like Cl , Br , I , are too weakly basic to react quantitatively with acetous perchloric acid. This problem can be effectively overcome by use of mercuric acetate (undissociated in Chowrasia, Deepak



14

acetic acid solution) to halide salt which ultimately displace halide ion by equivalent quantity of acetate ion which itself is a strong base in acetic acid. 12.A.2.3. Assay of few basic chemicals by acidimetric non aqueous titration. 12.A.2.3.1. Assay of Ephedrine HCl:

Weigh out accurately 0.5g of ephedrine hydrochloride and dissolves it into sufficient amount of glacial acetic acid-mercuric acetate solution and titrate the resultant sample mixture with 0.1M perchloric acid (standardized before as per procedure given) using crystal violet as indicator. Each ml of 0.1M perchloric acid is equivalent to 20.17mg of ephedrine hydrochloride. 12.A.2.3.2. Assay of adrenaline:

Weigh out accurately 0.4g of ephedrine hydrochloride and dissolves it into sufficient amount of glacial acetic acid as solvent and titrate the resultant sample mixture with 0.1M perchloric acid (standardized before as per procedure given) using crystal violet as indicator. Each ml of 0.1M perchloric acid is equivalent to 0.01832g of adrenaline.

HO NH HO OH

adrenaline Figure 03: Adrenaline 12.A.2.3.3. Assay of Sodium Saccharine:

Weigh out accurately 0.3g of Sodium Saccharine, dissolves it into 20ml of glacial acetic acid as solvent and titrate the resultant sample mixture with 0.1M perchloric acid (standardized before as per procedure given) using crystal violet as indicator.

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Each ml of 0.1M perchloric acid is equivalent to 0.09842g of Sodium Saccharine. Non aqueous titrations

Titrant involved

Done for

Solvent employed

Indicator

Acidimetry

Acetous N/10 perchloric acid

Weakly basic substances & their salts like amines, heterocyclic nitrogen containing compounds.

Acidic solvent: acetic anhydride, glacial acetic acid. Neutral solvent: alcohols, benzene, chloroform.

Thymol blue, crystal violet, methyl rosaniline chloride, quinaldine red etc.

Alkalimetry

Sodium methoxide, lithium methoxide, potassium methoxide (rarely; due to formation of gelatinous precipitates), sodium aminomethoxide, and sodium triphenylmethane.

Weakly acidic compounds like pyrroles and phenols

Strong basic solvents: morpholine, nbutylamine, ethylene diamine, DMF etc.

Thymol blue and Azovoilet

Table 05: Non aqueous titration methods, their titrants and application 12.B. Alkalimetric analysis in non aqueous titration Alkalimetery is performed in non aqueous solvent for assay of chemicals that are weak acidic in nature with the help of suitable visual indicators or sometime by potentiometrically also. In this, analyte (weak acidic substance) is titrated with sodium, potassium (rarely used; form precipitate), lithium methoxide in toluene-methanol or tetrabutyl ammonium hydroxide in methanol

12.B.1. Titrant used in alkalimetric analysis in non aqueous titrations Various titrants used in alkalimetric assay of chemical compounds are given below; 12.B.1.a. Preparation of 0.1 M tetrabutylammonium hydroxide in Toluene-Methanol.

Dissolve 40g of tetrabutylammonium iodide in 30ml of absolute methanol followed by 20g of previously powdered purified silver oxide. Shake vigorously for 1 hour. Centrifuge small

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quantities from resultant mixture and test supernatant liquid for residual iodine, if get positive reaction for iodine then add additional 2g of silver oxide & shake for next 30 minutes. Repeat the mixture until the solution is get absolutely free from iodine. Filter off the solution with fine and clean sintered glass filter and rinse reaction vessel with three equal portion of 50ml dry toluene. Add washing to filtrate and dilute it to 1 litre with dry toluene & flush the solution with carbon dioxide free nitrogen for 5 minutes. Stored the resultant solution in a well closed tight container and precaution must be taken against moisture and carbon dioxide. 12.B.1.a.1 Standardization of 0.1 M tetrabutylammonium hydroxide

Accurately weigh out benzoic acid 60 mg in dimethlyformamide (DMF) 10ml followed by addition of thymol blue (0.2w/v in methanol; 3 drops) & titrate resultant mixture against 0.1 M tetrabutylammonium hydroxide in an atmosphere devoid of carbon dioxide free nitrogen. 12.B.1.b. Preparation of 0.1M Potassium methoxide in toluene-methanol

Add carefully with steady, but slow stirring, freshly cut pieces of potassium metal into flask containing mixture of methanol 40ml & dry toluene 50ml. once dissolution is completed, add few ml of additional methanol into resultant mixture to get a clear solution followed by addition of (50ml) toluene which turns mixture hazy. Repeat the process with alternate addition of methanol & toluene until a solution of 1 litre is obtained. It should be noted that the final solution must be visibly clear. Store so prepared solution in an air tight container, away from sunlight, at an ambient temperature. 12.B.1.b.1. Standardization of 0.1 M Potassium methoxide in toluene-methanol

Weigh out accurately 0.6g benzoic acid and transfer it into conical flask containing 10ml of dimethylformamide (DMF). Add 3-4 drops of thymol blue as indicator & titrate resultant solution with 0.1M potassium methoxide in toluene-methanol. 12.B.1.c.

Preparation of 0.1M lithium methoxide in toluene-methanol

Add carefully with steady, but slow stirring, freshly cut pieces of lithium metal into flask containing methanol 150ml followed by toluene 850ml. Remove cloudiness of resultant mixture by adding sufficient quantity of methanol (final volume should not exceed 1 liter). Stored prepared solution in an air tight container, away from sunlight, at an ambient temperature. 12.B.1.c.1. Standardization of 0.1 M lithium methoxide in toluene-methanol

Weigh out accurately 0.25g benzoic acid and transfer it into conical flask containing 25ml of dimethylformamide (DMF). Add 3-4 drops of quinaldine red as indicator & titrate resultant solution with 0.1M lithium methoxide in toluene-methanol.

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17

12.B.2. Assay of few acidic chemicals by alkalimetric non aqueous titration 12.B.2.a. Assay of Ethosuximide:

Dissolve 0.2g of Ethosuximide in 50ml of Dimethylformamide (DMF), subsequently add 2-3 drops of azo-violet and titrate the resultant solution with 0.1M sodium methoxide until colour change to blue. Each ml of 0.1M sodium methoxide is equivalent to 0.01471g of ethosuximide. 12.B.2.b. Assay of benzoic acid:

Dissolve appropriate quantity of benzoic acid in n-butylamine, followed by addition of 2-3 drops of thymol blue and titrate the resultant solution with 0.1M sodium methoxide until end point reached. 12.B.2.c. Assay of chlorthalidone:

Dissolve 0.3g of chlorthalidone in 50ml of anhydrous pyridine, titrate the resultant solution with 0.1M tetrabutylammonium hydroxide, and determine end point potentiometrically. Each ml of 0.1M tetrabutylammonium hydroxide is equivalent to 0.0338g of chlorthalidone. 13.

APPLICATIONS OF NON-AQUEOUS TITRATION

Analyte (X)

Calculation (each Solvent End point Amount ml of 0.1M titrant Titrant medium determination (gram) is equivalent to g of X.

Adrenaline

GAA

CV

0.3

PCA

0.01832

Amantadine HCl

GM

CV

0.12

PCA

0.01877

Acetozolamide

DMF

Pot.

0.4

TBAH

0.02222

Biscodyl

GAA

CV

0.5

PCA

0.03614

C

MR

0.5

PCA

0.02998

Clonidine HCl

GM

NB

1.4

PCA

0.01333

Cyproheptadine HCl

GM

CV

0.5

PCA

0.0323

Dehydroemetine

GM

CV

0.4

PCA

0.02758

Ephedrine HCl

GM

CV

0.5

PCA

0.02017

Ethambutol HCl

GAA

CV

0.2

PCA

0.01386

Codine phosphate

GAA

CV

0.4

PCA

0.03974

Ergotamine Maleate

GM

CV

0.1

PCA

0.04415

Isoprenaline sulphate

GAA

CV

0.4

PCA

0.0526

Chlordiazepoxide

Chowrasia, Deepak



Analyte (X)

Levo-dopa

18

Calculation (each Solvent End point Amount ml of 0.1M titrant Titrant medium determination (gram) is equivalent to g of X.

GM

OBB

0.6

PCA

0.01972

Nalidixic acid

DMF

Thymolpthalein

0.25

LMO

0.02322

Niclosamide

`DMF

0.3

LMO

0.02263

Diloxanide furoate

Py

Pt.

0.3

TBAH

0.03282

Hydrochlorothiazide

Py

Pt.

0.3

TBAH

0.01489

Allopurinol

DMF

Thymol blue

0.2

LMO

0.01361

Fenfluramine HCl

CAM

CV

0.3

PCA

0.02677

Mebendazole

GAA

Pt.

0.25

PCA

0.02953

Metronidazole

GAA

NB

0.45

PCA

0.01712

Nikathamide

GAH

CV

0.2

PCA

0.01782

Nicotinamide

GAH

CV

0.3

PCA

0.01221

Noscapine

GAA

CV

0.5

PCA

0.04134

Salbutamol sulphate

GAA

OBB

0.9

PCA

0.05767

Metformin HCl

GM

Pt.

0.25

PCA

0.008281

Phenformin HCl

GAH

CV/Pt.

0.25

PCA

0.0120

Lignocaine HCl

GM

CV

0.6

PCA

0.02708

Imipramine HCl

GM

CV

0.5

PCA

0.03169

Dequalinium chloride

GM

CV

0.7

PCA

0.02638

Cyproheptadine

GM

CV

0.6

PCA

0.3533

Dehydroemetine

GM

CV

0.4

PCA

0.02758

Ethylmorphine

GM

CV

0.3

PCA

0.03499

Tetramisole Hcl

GM

NB

0.5

PCA

0.02408

Verapamil HCl

GM

CV

0.5

PCA

0.04911

Oxyprenolol HCl

GM

NB

0.4

PCA

0.3018

Pentazoline HCl

GM

CV

0.65

PCA

0.03219

Imipramine HCl

GM

CV

0.5

PCA

0.02477

Pt.

0.6

PCA

0.04484

Propantheline Bromide GAA/GM

Table 06: Pharmaceutical applications of non aqueous titration (For elaborated list please check IP Vol I, II)

Chowrasia, Deepak



19

Key: PCA-0.1M perchoric acid; TBAH-0.1 M tetrabutylammonium hydroxide in TolueneMethanol; LMO-lithium methoxide; DMF-dimethylformamide; -Chloroform-acetonemercuric acetate; Py-pyridine; GAA-glacial acetic acid; GM-glacial acetic acid-mercuric acetate; GAH-glacial acetic acid & acetic anhydride; C-chloroform; CV-crystal violet; MRmethyl red; NB-1-nepththolbenzeine; OBB-Oracet blue-B; Pt-potentiometric determination. 14.

EXERCISE A. Explain various theories of acid & bases with special references to LoweryBronsted concept.

B.

What is non aqueous titration? Give their advantages and disadvantages over aqueous titrations.

C.

Discuss briefly “Ideal characteristics of non aqueous solvents” & comment their utilization, pros & cons over aqueous solvents.

D.

Classify solvents used in non aqueous titration with suitable example.

E.

What is leveling effect? Explain it in terms of protophilic solvents.

F.

Compare protophilic & protogenic solvents used in non aqueous titrations.

G.

Enumerate various methods used in determining end point in non aqueous titrations.

H.

Diagrammatically explain apparatus used in non aqueous titrations.

I.

Write an exhaustive note on methods used in non aqueous titrations with special emphasized on acidimetric analysis in non aqueous titrations.

J.

Give preparation & standardization of acetous 0.1 M perchloric acid? What are the various precaution should be taken during non aqueous titrations.

K.

Why mercuric acetate solution is added during titration of halogen acid salt of bases in acidimetric analysis in non aqueous titrations.

L

Why potassium methoxide used rarely as a titrant compare to sodium & lithium methoxide in alkalimetric analysis in non aqueous titrations.

M.

Write a short note on indicators used in non aqueous titrations.

N.

Explain briefly various applications of non aqueous titrations.

O.

“Non aqueous titrations are boon for pharmaceutical industries”. briefly with suitable examples.

Chowrasia, Deepak

Discuss



20

P.

Why non aqueous titration are performed under rigid control of temperature.

Q.

Give preparation & standardization of 0.1 M tetrabutylammonium hydroxide in Toluene-Methanol.

R.

Write a note on titrant used in alkalimetric assay in non aqueous titrations.

S.

How you will assay following class of chemical compounds a.

Adrenaline

b.

Benzoic acid

c.

Chlorthalidone

d.

Ephedrine

T. Give storage conditions of perchloric acid. 15.

MULTIPLE CHOICE QUESTIONS

Non aqueous titrations compounds which are a. b. c. d.

are

done

for

Water soluble Water insoluble Have higher value of ionization both b & c d

As per oldest definition of acids are a. b. c. d.

Proton donor Proton acceptor Bitter in taste None of above c

Arrhenius defined acid as _____ & base as ______ a. b. c. d.

Proton donor & proton acceptor Proton acceptor & proton donor Turn litmus red Turn litmus blue a

Electron pair acceptors are generally acids. This concept is a. Lewis concept b. Arrhenius concept c. Lux concept d. Usanovich concept a Advantages of non-aqueous titration includes a. Handling of poor water soluble drugs b. Titration of chemicals giving poor end point. c. Speedy analysis d. All the above d Moisture content titration should be a. >0.005% b. <0.05% c. <0.5% d. >0.005%

during

non-aqueous

b

Chowrasia, Deepak



Ideal characteristic of non-aqueous solvent include all except a. b. c. d.

Solubility Equalization High dielectric constant All the above

Protophilic solvents are a. Proton donor b. Proton acceptor c. Both d. None of above b

c

Protogenic solvents are a. Proton donor.

Thermal coefficient of expansion for nonaqueous solvent is

b. Proton acceptor

a. b. c. d.

High. Low Both None of above

a

a. Any container having wide opening. b. Container having narrow opening with well sealed mouth c. Both a and b d. None of the above b Non-aqueous solvents are classified into Two types. Three types Four types Five types c Aprotic solvents Donate proton Accept proton May donate or accept proton None of the above d

Chowrasia, Deepak

Amphiprotic solvents are a. Proton acceptor + proton donor b. Proton acceptor

Non-aqueous solvents can be stored in

a. b. c. d.

c. Both d. None of above

a

a. b. c. d.

21

c. Proton donor d. None of the above a Match following a. Protogenic solvent i. Perchloric acid b. Protophilic solvent ii. Acetic acid c. Amphiprotic solvent iii. Ethylenediamine d. Aprotic solvent iv. Carbon tetrachloride a-i, b-iii, c-ii, d-iv Dielectric constant for acetic acid is a. 6.15 b. 6.90 c. 7.15 d. 7.90 a



22

Dielectric constant for water is higher than most of the non-aqueous solvents a. True b. False c. Can’t be predicted d. May be true or false a

Leveling effect of protophilic solvent is mostly on a. Weak acidic substances b. Weak basic substances c. Strong acidic substances d. Strong basic substances a

Pick protophilic solvent a. Pyridine b. Ethylenediamine c. n-butylamine d. All of the above

Glacial ethanoic acid is also termed as a. Acetic acid b. Anhydrous acetic acid c. Both d. None of the above

d The correct sequence in acidic strength of protogenic non-aqueous solvents is a. HCL>H2SO4>HNO3>HCLO4 b. HCLHCL>H2SO4>HCLO4 d. HCLO4>H2SO4>HCL>HNO3 d Solvent effect is also termed as a. Leveling effect. b. Labor effect c. Lowering effect. d. All of the above a Leveling effect means a. Increasing acidic property of solvent b. Increasing basic property of solvent c. Equalizing acidic or basic property of substance with help of non aqueous solvent d. All the above c

Chowrasia, Deepak

a Water content of glacial ethanoic acid is acceptable within range of a. 0.1-1.0% b. 0.01-1.0% c. 0.01-0.001% d. None of the above a Acetic anhydride is added to ethanoic acid in order to a. Increase its acidity b. Remove if any water is present in solution c. Increase its reactivity d. None of the above b Pick out aqueous solvent among a. Acetonitrile b. Water c. Acetic acid d. Dioxan b



DMF is a a. Aprotic solvent b. Protophilic solvent c. both d. None of the above

Acidimetric analysis titration includes

b All are the non aqueous indicator except a. Crystal violet b. Nile blue A c. Quinaldine red d. Eriochrome black T

Crystal violet is commonly prepared in Glacial acetic acid Acetic acid DMF. None of the above

End point detection in non aqueous titration can be best done by

aqueous

a. Titration of amines (Primary, secondary, & tertiary) b. Titration of halogen acid salt of bases c. Both d. None of the above c

a. b. c. d.

Acidimetric non aqueous titration Alkalimetric non aqueous titration Both None of the above

Official solvent/s for perchloric preparations is/are a. Glacial acetic acid b. Dioxan c. Both d. None of the above

acid

c Strength of 0.1M perchloric acid is

Visual method Instrumental method Both None of the above c

Which type of instrumental method is used in end point detection in non aqueous titrations a. Amperometry b. Conductometry c. Potentiometry d. None of the above c

Chowrasia, Deepak

non

a

a

a. b. c. d.

in

Perchloric acid used in

d

a. b. c. d.

23

a. b. c. d.

72% 82% 92% 62% a

Acetic anhydride is usually added to solution of perchloric acid a. b. c. d.

Make it acidic in nature Make it anhydrous in nature Both None of the above b



Excess of acetic anhydride usually creates problem with primary & secondary amine by a phenomenon known as a. Acylation b. Acetylation. c. Alkylation d. None of the above b It is recommended not to add acetic anhydride to a concentrated solution of perchloric acid since a. It forms explosive acetyl perchlorate b. It forms explosive acetyl perchloric anhydride c. It makes reaction mixture non reactive

24

Acetous perchloric acid is used as a titrant in a. Acidimetric non aqueous titration b. Alkalimetric non aqueous titration c. Both d. None of the above a Acidimetric analysis titration is done for

in

non

aqueous

a. Strong acidic chemicals b. strong basic chemicals c. weak acidic chemicals d. weak basic chemicals including heterocyclic compounds containing nitrogen d

d. All are true a Solution of perchloric acid is standardized with a. Potassium hydrogen phthalate b. Sodium hydrogen phthalate

a. Strong acidic chemicals b. Strong basic chemicals c. Weak acidic chemicals d. Weak basic chemicals

c. Both of them

c

d. None of them a For acidimetric non aqueous titration ________solvents are employed a. Acidic b. Basic c. Neutral d. All the above a, c

Chowrasia, Deepak

Alkalimetric analysis in non aqueous titration is done for

Potassium methoxide is usually avoided as titrant in alkalimetric non aqueous titration since a. It forms gelatinous precipitate b. Make end point difficult to analyze c. Both of the above d. None of the above a



Tetrabutylammonium hydroxide Toluene-Methanol is used as titrant in

in

a. Alkalimetric non aqueous titration b. Acidimetric non aqueous titration c. Both

Indicator Quinaldine red prepared in a. Methanol b. Ethanol c. Butanol d. None of the above

25

is

usually

d. None of the above

a a

Solution of Tetrabutylammonium hydroxide is standardized with a. Potassium hydrogen phthalate b. Benzoic acid

c

c. Sodium hydrogen phthalate d. None of the above b Chlorthalidone is well titrated via a. Acidimetric non aqueous titration b. Alkalimetric non aqueous titration c. Both d. None of the above b Titration of sodium saccharine is done by a. Acidimetric non aqueous titration b. Alkalimetric non aqueous titration c. Both d. None of the above a levo-dopa can be suitably titrated via a. Acidimetric non aqueous titration b. Alkalimetric non aqueous titration c. Both d. None of the above a

Chowrasia, Deepak

Crystal violet gives blue green colour at a. Acidic pH b. Basic pH c. Neutral ph d. None of the above Problem associated with quantitative determination of halogen acid salts in acidimetric non-aqueous titration can be overcome by use of a. Mercuric acetate b. Sodium acetate c. Potassium acetate d. All of the above a Adrenaline and ephedrine can be well titrated by acidimetric non aqueous titration a. True b. False c. Only titrated by complexometric titration d. All the above are false a _____________ is used as indicator in titration of sodium saccharine by non aqueous titration a. Crystal violet b. Quinaldine red c. Methyl orange d. All the above a NON AQUEOUS TITRATIONS


Morpholine is an example of a. Protogenic solvent b. Aprotic solvent c. Protophilic solvent d. None of the above

c. Both method can be used d. None of the above b

c Indicator thymol blue is mostly used in a. Acidimetric non aqueous titration b. Alkalimetric non aqueous titration c. Both d. None of the above

Titrant used in Imipramine HCl is a. Perchloric acid b. Potassium methoxide c. Sodium methoxide d. None of the above a

b 0.1M Potassium methoxide solution is prepared in solvent a. Methanol-benzene b. Methanol-acetic acid c. Methanol-DMF d. Methanol-toluene d Formic acid is a. Protogenic solvent b. Aprotic solvent c. Protophilic solvent d. None of the above a Standardization of 0.1M Potassium methoxide is done by a. Potassium hydrogen phthalate b. Benzoic acid c. Both can be used d. None of the above b End point determination in propantheline and acetozolamide is done by a. Visual method (indicator) b. Instrumental method (potentiometrically)

Chowrasia, Deepak

26

Codine is titrated well via a. Acidimetric non aqueous titration b. Alkalimetric non aqueous titration c. Both d. None of the above a Solvent medium used for Diloxanide furoate and hydrochlorothiazide is a. Glacial acetic acid +pyridine b. Glacial acetic acid alone c. Pyridine alone d. Any one of the above a Perchloric acid, from analytical point of view, can be well prepared a. 24-hours before titration b. Right at time of titration c. Both a & b d. None of the above a Select non-aqueous solvent/s a. Sulphuric acid anhydrous b. Glacial acetic acid c. Dioxane d. All the above d


Chapter - 02 COMPLEXOMETRIC TITRATIONS - Deepak Chowrasia

Chowrasia, Deepak

COMPLEXOMETRIC TITRATIONS


29

COMPLEXOMETRIC TITRATIONS (Chapter Overview) 01. INTRODUCTION .................................... ...................................................... .................................... .................................... ................................... .......................... ......... 31 .................................................... .................................... .................................... .................................... ................................... .................... ... 31 02. PRINCIPLE ..................................

03. LIGAND ................................... ..................................................... .................................... .................................... .................................... ................................... .......................... ......... 31 04. TYPES OF LIGANDS .................................. .................................................... .................................... .................................... ................................... .................... ... 32 05. METAL IONS .................................. .................................................... .................................... .................................... .................................... ................................... .................33 ............................ 34 06. FACTORS GOVERNING COMPLEXOMETRIC TITRATIONS ............................

06.A. 06.B. 06.C. 06.D. 06.E. 06.F. 06.G. 06.H.

pH ................................... ..................................................... .................................... .................................... .................................... ................................... .......................... ......... 34 .................................................... .................................... .................................... ................................... .......................... ......... 34 pM indicator .................................. Quantity of metal ion concentration .................................. .................................................... ................................... .................... ... 35 Size and number of rings .................................... ...................................................... .................................... ................................... .................... ... 35 .................................................... .................................... .................................... ................................... .......................... ......... 35 Temperature .................................. Detection of colorimetric changes .................................... ...................................................... ................................... ....................... ...... 35 Solvents: .................................... ...................................................... .................................... .................................... .................................... ................................ ..............35 ...................................................... .................................... ................................... .................35 Types of functional groups ....................................

07. METALLOCHROMIC OR pM INDICATORS .................................... ..................................................... .......................... .........35 07.A. Ideal characteristics of pM indicators .................................. .................................................... ................................... .................36 08. MASKING AND DEMASKING AGENTS .................................... ...................................................... ................................... .................37 ...................................................... .................................... ................................... ....................... ...... 38 09. METHODS OF TITRATION ....................................

09.A. Direct titration .................................... ...................................................... .................................... .................................... ................................... .................... ... 38 09.A.I. Preparation of standard 0.05M disodium EDTA solution. .........................38 ................................... 38 09.A.II. Standardization of 0.05M disodium EDTA solution. ................................... 09.A.II.a. Method A: By granulated Zinc .................................. ................................................... .................... ... 38 ................................................. ..............39 09.A.II.b. Method B: By calcium carbonate carbonate ................................... ...................................................... ................................ ..............39 09.A.III. Preparation of ammonia buffer .................................... 09.B. Back titration .................................... ...................................................... .................................... .................................... ................................... ....................... ...... 39 09.C. Replacement of one complex by another or displacement/substitution titration .................................. .................................................... .................................... ............................. ........... 40 09.D. Alkalimetric titration of metal ions .................................. .................................................... ................................... ....................... ...... 40 10. END POINT DETECTION .................................. .................................................... .................................... .................................... ............................. ........... 40 10.A. Visual method .................................... ...................................................... .................................... .................................... ................................... ....................... ...... 40 ..................................................... ................................ ..............41 10.A.1. Metallochromic Metallochromic or pM indicators ................................... 10.A.2. PH indicators .................................... ...................................................... .................................... .................................... ............................. ........... 41

Chowrasia, Deepak



30

10.A.3. Redox indicators .................................... ...................................................... .................................... ................................... ....................... ...... 41 10.B. Instrumental methods .................................... ...................................................... .................................... ................................... .......................... ......... 41 .................................................... .................................... .................................... ................................... .................41 10.B.1. Photometry .................................. 10.B.2. Potentiometry ................................... ..................................................... .................................... .................................... ............................. ...........41 .................................................... .................................... ................................ ..............41 10.B.3. Miscellaneous methods .................................. ..................................... ... 42 11. APPLICATIONS OF COMPLEXOMETRIC TITRATIONS ..................................

12. EXERCISE .................................... ...................................................... .................................... .................................... .................................... ................................... .................... ... 43 13. MULTIPLE CHOICE QUESTIONS ................................... ..................................................... .................................... ............................. ........... 44

Chowrasia, Deepak



31

COMPLEXOMETRIC TITRATIONS 01. INTRODUCTION Gravimetry and oxalate permanganate titrations for detection of metal ions are now rarely important in chemical industries owing to their lengthy procedure and tedious methodology compare to complexometric titrations, which involves fewer steps, lesser time consumption, and economic in process. 02. PRINCIPLE Complexometric titrations plays dominant role in both chemical as well as pharmaceutical industries for determination of inorganic/organic compounds and pharmaceutical active ingredient including including dosage forms forms containing metal ions (cations (cations and anions). The titration is based on the simple principle of complexation, a process involving formation of complex between metal ions and motif containing electron donating groups, also called ligand by replacing solvent molecule from solvated metal ions. Complex formed by equal sharing of electrons between metal ion and ligand is termed as covalent complex while the complex form solely by sharing of electrons only from one of the species out of two participating in complex formation is known as coordinate complex. Undoubtedly, the reaction take place during the phenomenon of complexation can be simply be depicted as

Where, M=

Metal ion (Solvated)

H2O = Solvent bound to metal ion L=

Ligand

n=

Coordination Coordinati on number

03. LIGAND Essentially, ligand is any substance that contains one or more electron donating groups (EDG), which ultimately shares electrons fully or partially thus forming of stable metal-ligand complex. Chemically, Chemicall y, ligands are Lewis bases which are having capacity to bind with that of metal ions as well as protons, thereby forming stable complexes. pH of the solution therefore plays crucial role in complexation complexation phenomenon. phenomenon. If ligand only shares it’s it’s all electron to form complex with metal ion then such a complex is called coordinating complex on other hand if both ligand and metal shares electron equally then complex so formed is termed as covalent complex.

Chowrasia, Deepak



32

04. TYPES OF LIGANDS As said earlier, ligand are chemical agents that form bond with metal ions, depending upon the number of electron donating group a ligand is having they are classified as

a. Monodentate ligand. b. Bidentate ligand. c. Tridentate ligand. d. Quadridentate Quadridentate ligand.

Figure 01: Types of ligands Monodentate ligands are the simplest ligand that contain only single atom having a lone pair of electron which forms bond with central metal atom only at one point. Cyanide ion, ammonia, halide ion, and water are some of the best examples representing monodentate ligand. Any ligand having more more than one electron donating donating group group in its molecule is termed as polydentate ligand. Depending upon the availability of binding sites, a polydentate ligand is further being classified as bi, tri, quadra, or hexadentate ligand (See figure 01 & table 01). Ligand

Class

Cyanide ion

Monodentate ligand

Halide ion

Monodentate ligand

Ethylenediamine

Bidentate ligand

Oxalate ion

Bidentate ligand

Glycine

Bidentate ligand

N-hydroxyethylethylenediamine

Tridentate ligand

Diethylenetriamine

Tridentate ligand

Nitrilotriacetic Nitrilotriacetic acid

Quadridentate ligand

Triaminotriethylamine

Quadridentate ligand

Ethylenediaminetetraaceticacid

Hexadentate ligand

Table 01: Some common ligands & their classes

Chowrasia, Deepak



33

A chelate, chelator, chelants, chelating agent, sequestering agent (sometime) are intermingled terms which commonly & conjunctionally denotes “polydentate ligand”, binding metal ion at different sites (just like a crab claw) thereby forming a ring like structure. O

OH O

HO

N N

OH

OH O

O

EDTA

Figure 02: Structure of EDTA

For example, hexadentate ligand like ethylenediaminetetraaceticacid (EDTA) containing two amino & four carboxylic groups thereby make chemical bonds at different sites of a metal ion, thus forming a ring like structure. This phenomenon of emergence of cyclic ring like structure by a polydentate ligand via formation of multiple chemical bonds at different sites of a metal ion is termed as chelation and the agent which is responsible for this phenomenon of chelation is termed as chelating agent. Chelating agents that forms water soluble complexes is called as sequestering agents like EDTA, dimethylglyoxime, diethylenetriamine pentaacetic acid (DTPA, propylene diaminetetra acetic acid, and salicylaldoxime. 05. METAL IONS Inorganic metals (Refer table 02) and their salts are preliminary intend of complexometric titrations. Theoretically, it is considered that complexation phenomenon between a metal ion and a chelator should only contain mononuclear complex i.e. complex of single metal ion, but practically binuclear (contain two metal ions) and multinuclear (contains number of metal ions) complexes are formed provided under titrimetric condition high concentration of metal 2+ 2+ 2+ 3+ ions should be present. It is interesting to note that bivalent (Ca , Mg , Zn ), trivalent (Fe , 3+ 3+ 4+ 4+ Al , Cr ) and tetravalent (Sn , Ce ) metal ions forms 3, 4, and 5 ring complexes with metal ions. Group

Metal detected

Method of titration

Ia

Sodium, potassium

Indirect

Ib

Silver, gold

Indirect

Ib

Cupper

Direct

IIa

Magnesium, calcium, strontium, barium

Direct

Chowrasia, Deepak



IIb

Zinc, cadmium, mercury

Direct

IIIa

Aluminum, gallium, indium, thallium

Direct

IIIb

Scandium, yattrium, lanthanide metals

IVa

Tin, lead

Direct

IVa

Carbon

Indirect

Va

Antimony, bismuth

Direct

Va

Nitrogen, phosphorus, arsenic

Indirect

Vb

Vanadium

Direct

VIa

Sulphur, selenium

Indirect

VIb

Chromium, molybdenum, tungsten

Direct

Via

Halogens

Indirect

VIIb

manganese

Direct

VIII

Cobalt, nickel, palladium

Indirect

actinide

34

and Direct

Table 02: Metals ion and their titrating methods 06. FACTORS GOVERNING COMPLEXOMETRIC TITRATIONS There are numbers of factors that regulate complexometric titration in dual manner positive as well as negative. Some of them are enumerating below as; 06.A. pH: Control of pH is extremely necessary and critical for EDTA titrations. It is advisable to control overall pH of titration within a narrow range of ± 0.5 to ±1 unit and same can be simultaneously determined with aid of either a pH meter or an analytical grade pH paper. 06.B. pM indicator: The concentration of pM indicator (see next section) employed for detection of end point should be optimum and must be able to form weak bonds with metal ion compare to chelating agent, also the indicator must be able to show visually distinct colorimetric changes (only & sharply at end point) that can be easily identified by naked human eyes. The color change at equivalence point should be boldly different i.e. the indicator must be able to produce distinct color at bounded state compare to an unbounded state.

Chowrasia, Deepak



35

Figure 03: Diagrammatic representation of bonding between metal ion, indicator and chelator 06.C. Quantity of metal ion concentration: A good and satisfactory result of titration can be obtained at a metal ion concentration of 0.25 mmol in 50-150ml of solution to be titrated. 06.D. Size and number of rings: Commonly polydentate ligand ensures an easy and stable metal chelate formation compare to a monodentate ligand, but an unregulated increase in ring size ultimately reduces stability of metal complex due to ring strain. 06.E. Temperature: It acts as an important factor not only in complexometric titrations, but also in other volumetric analysis. An optimum temperature of 37-40 degree Celsius should be maintained during over all titration process in order to get a good, sharp and rapid end point. 06.F. Detection of colorimetric changes: Any visual error (human or pathological–color blindness) of reading color changes during titration may leads to error result which can be effectively reduces or totally overcome by use of photocell that are automatic and more sensitive color detection device compare to human eyes. 06.G. Solvents: Solvent in which the titration done shows variable effect in complexometric titrations. 06.H. Types of functional groups: Organic compounds containing functional groups with easily replaceable protons (–COOH, phenolic, enolic –OH groups) or neutral groups offering lone pair of electors (NH 2, CO, alcoholic –OH) forms strong and stable complexation. Nevertheless, presence of both acidic as well as basic group yields a complex soluble in wide range of pH. 07. METALLOCHROMIC OR pM INDICATORS pH sensitive indicators are generally used for detection of equivalence point (end point) in acid base (neutralization) titration owing to their highly selectivity towards rigorous color

Chowrasia, Deepak



36

change with respect to pH. Likewise, pM indicators or metal ion indicators or metal ion selective indicators (Refer table 03) are employed for determination of end point during complexometric titrations. A pM indicator is nothing, but metallochromic dye which itself act as chelating agent thus forming weak bonds with metal ions and able to show distinct visual color change at different state i.e. bound (complex) and free (unbound) state. However, in some complexometric procedures, such as determination of Zinc metal in phosphate solution or determination of Bismuth ion, where sudden changes in color at equivalence point cannot be obtained by use of pM indicator other appropriate automated or instrumental methods like amperometry, potentiometry, or spectrophotometry are preferred against pM indicators. 07.A. Ideal characteristics of pM indicators 1. Indicator must not adversely react with metal ions i.e. it should not degrade structure of analyte to be analyzed.

2.

It must form stable complex with metal ion but less stable than metal-ligand complex comparatively.

3.

It must be active within the given pH range of titration.

4.

Color formed must be rapid on accompanied of end point.

5.

Color must be distinct and visually identified during both bounded and unbounded state.

6.

The most important one “should be less or not harmful for analyst.

S. No 01

Indicator name Mordant BlackII

Other name

Color change

pH

Applications

Eriochrome Black-T or, Solochrome Black-T

Red to blue

6-7

Zn, Sr, Ba, Mg

Blue to orange

11-12

02

Catechol violet

Pyrocatechol

Red to yellow Yellow to violet Violet to red

1-2 6-7 8-10

Zn, Cd, Bi, Ni, Ca, Co.

03

Murexide

Ammonium purpurate

Red to violet Violet to blue violet

6-7 11

Ca

04

Xylenol orange

Yellow to red

5-7

Co, Bi, Hg, Cd, Zn

Chowrasia, Deepak

-



S. No 05

Indicator name Phthalein purple

Other name

Metalphthalein

Color change

Rose color to red Colorless to rose color

37

pH

Applications

10-11 6-7

Alkaline earth metals

Table 03: pM indicators and their applications 08. MASKING AND DEMASKING AGENTS The ideal characteristics of any titrimetric process depends upon following two factors

Specificity Selectivity EDTA itself acts as a nonselective ligand and able to forms complexes with wide variety of metals cations including di, tri, and tetravalent metal ions. Normally, an analyte containing bimetal ions (single solution contains two different metal ions) can be easily titrated with EDTA provided there is strict control of pH (titration of solution containing bismuth and lead at pH-2 and pH-5). However, a mixture containing polymetal ions (single solution contains numerous metal ions) cannot be titrated suitably and satisfactorily with EDTA due to interference caused by these metal ions during titrimetric procedure. In such a scenario masking agents are employed which render the entry of unwanted metal cations into chemical reaction without separating them physically and hence the masked metal ions no longer take part in titration process thus accurate end point can be obtained. This phenomenon of obscuring or hiding the unwanted metal ions without separating them physically from titration mixture is known as masking and the agent that are used to bring about this effect (masking) is termed as masking agents (refer table below). On other hand, the substance that itself are chelating agent, but can be effectively brings about phenomenon of masking if added into a solution containing multiple metal ions are called as auxiliary complexing agents. For example thiglycol, ascorbic acid, tartaric acid, and triethanolamine. One of the most commonly used masking agent is cyanide anion-A HIGHLY POISIONIOUS CHEMICAL MUST BE USED WITH FULL PRECAUTION forming stable metal complexes with mercury, zinc, copper, cadmium, cobalt, silver, platinum, and nickel, but unable to get complexed with lead, manganese, and alkaline earth metals.

Chowrasia, Deepak



S.Nos.

Masking agents

38

Metal masked

01

Potassium cyanide

Cu, Cd, Ni, Co, Zn, Ag

02

Thioglycols

Hg, Cu

03

Triethanolamine

Al

04

Ammonium fluoride

Al, Fe, Ti

05

Iodide

Hg

06

BAL (2,3-dimercapto-1-propan-1-ol)

Cd, Zn, Sn, Sb

07

Tiron (disodium catechol 3,5-disulfonate)

Al, Fe (III)

08

Ascorbic acid

Fe (III), Cu

Table 04: Masking agents and their application

Demasking is a process just opposite to that of masking i.e. the metals ion that are unphysically separated during titration process are again freed so that they can be titrated suitably. As stated previously, the cadmium and zinc ions that are masked by addition of potassium cyanide (KCN) can be effectively demasked by use of Formaldehyde: Acetic acid solution at a ratio of 1:3 or alternatively by chloral hydrate. In short, any chemical agents that reverse the phenomenon of masking are termed as demasking agents. 09. METHODS OF TITRATION The most important and effective method used for assay of metal ions is their titration with EDTA as a titrant. The methodology of complexometric titrations are broadly be classified as; 09.A. Direct titration The solution containing metal ions (Refer table 02) that has to be analyzed is first buffered to an optimum pH and then titrated directly with standard EDTA solution. Tartrate or citrate is added to the titrating mixture in order to overcome the problem of precipitation of metal hydroxide. The end point of titration is determined with the help of suitable visual or instrumental methods.

09.A.I. Preparation of standard 0.05M disodium EDTA solution Weigh out accurately 18.61g of disodium EDTA and dissolve in 1000mL of distilled water. 09.A.II. Standardization of 0.05M disodium EDTA solution 09.A.II.a. Method A: By granulated Zinc

Weigh out accurately 0.8g of granulated zinc (the Zinc must be 99.9% pure & must be pretreated with acid) and dissolve in 12mL of dilute hydrochloric acid by gentle heating, then

Chowrasia, Deepak



39

add 5 drops of bromine water and boil the resultant solution to remove any excess of bromine. Cool the solution and make up volume up to 200mL. Transfer carefully 20mL aliquot to Erlenmeyer flask and neutralized with sodium hydroxide (2N) solution; add 150mL of distilled water and sufficient ammonia buffer to a pH 10. Finally add 50mg of Mordant black II indicator and titrate the resultant solution with EDTA until solution turns green in color which confers end point establishment. Each 0.003269 g of granulated Zinc must be equivalent to 1mL of 0.05M disodium EDTA solution. 09.A.II.b. Method B: By calcium carbonate

Weigh out accurately 1.25g of pure and well dried calcium carbonate; transfer it into a standard flask (250ml), and make the solution clear by adding minimum quantity of hydrochloric acid (dilute). Now pipette out 20ml of solution and convey it into a clean conical flask (Erlenmeyer flask). To this add 5ml of ammonia-ammonium chloride buffer and titrate it against 0.05M disodium EDTA using Eriochrome black T as indicator until the color of solution changes from pink to blue. 09.A.III.Preparation of ammonia buffer Dissolve 13.5g of ammonium chloride in 130mL of strong ammonia solution and make a volume up to 200ml with distilled water. Calculation x (Each ml of Quantity 0.05M EDTA is equivalent to (grams) x grams of P

Pharmaceuticals (P)

Indicator

Calcium carbonate Dibasic calcium phosphate Magnesium chloride Magnesium trisilicate Zinc chloride Zinc stearate Zinc sulphate

Calcon mixture Hydroxy nephthol blue

0.1 0.2

0.005004 0.002004

Mordant black II Mordant black II Eriochrome black T Eriochrome black T Eriochrome black T

0.5 1.0 3.0 1.0 0.3

0.017017 0.002015 0.006815 0.004069 0.01438

Table 05: Pharmaceuticals titrated by direct complexometric titration 09.B. Back titration: The direct titration method have some of its limitations like unavailability of suitable metal ion indicator, insolubility of metal ions (lead sulphate and calcium oxalate), formation of unstable complex, slow formation of complex, compounds containing aluminum or bismuth

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40

metal ion, precipitation of metal ions under titrimetric condition. Thus a back titration is beneficial over direct titration under these circumstances. In this methodology (back titration) excess of disodium EDTA solution is added to a sample solution, pH is adjusted adequately with suitable buffering agent, indicator is added and the resultant solution is titrated back with suitable salt solution (magnesium sulphate or zinc sulphate solution is commonly used for this purpose). Pharmaceuticals (P)

Indicator

Quantity (grams)

Calculation x (Each ml of 0.05M EDTA is equivalent to x grams of P

Aluminium glycinate

Methyl red

0.25

0.002549

Dried Aluminium hydroxide

Methyl red

0.8

0.005098

Bismuth subcarbonate

Methyl red

0.5

0.02090

Aluminium sulphate

Methyl red

0.5

0.01711

Table 06: Pharmaceuticals titrated by Residual or back complexometric titration 09.C. Replacement of one complex by another or displacement/substitution titration: When direct or back titration is not able to give satisfactorily result due to unsatisfactorily reaction between metal ion and pM indicator, in such a case metal ion can be determined by titrating it with magnesium (Mg-EDTA) or zinc (Zn-EDTA) complex of EDTA. The amount 2+ 2+ of free Mg or Zn ions is equivalent to cations present and can be effectively titrated with the help of standard solution of disodium EDTA. The method is useful in determination of calcium, lead and mercury ions in the present of Mordent black II as indicator. 09.D. Alkalimetric titration of metal ions: The method based on the simple principle of displacement of protons from disodium EDTA by heavy metals. The so free protons are itself act as acid and can be effectively titrated with standard alkali solution using sensitive pH indicators. 10. END POINT DETECTION The end point detection in complexometric titration can be done by following two methods 10.A. Visual method It is one of the most commonly used methods for determination of end point owing to its simplicity, least cost, and accuracy. Following are some of the visual methods used for determining end point of a complexometric titration.

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10.A.1. Metallochromic or pM indicators Please refer section on pM indicator. 10.A.2. PH indicators: Titration of divalent or polyvalent metal ion with EDTA solution results in generation of protons. In an unbuffered system the acid so generated can be determined by titrating it with base using a pH selective or acid base indicator. Since, end point detection with this method has several limitations, hence method is rarely used. 10.A.3. Redox indicators: These indicators are useful for determining end point of those titration in which the metal ion exist itself in two different oxidation state. The method can be used for determining Fe metal with the help of Variamine blue-B indicator. 10.B. Instrumental methods Use of visual methods of determining end point is not free from limitations including inaccuracy in determination of end point or human visual errors. Thus an instrumentation technique could be satisfactorily employed to obscure limitation of visual methodology. Some of the instrumental techniques used in end point determination in complexometric titrations are given below;

10.B.1. Photometry: The method is suitable for detection of end point under following circumstances least intense color change at equivalence point, very dilute solution, unstable complex formation between metal ion and indicator, and requirement of large quantity of pM indicator. This method employs highly sensitive photocells capable of detecting even minute changes in color which usually not sensitized by normal human eyes. The solution to be analyzed is placed between photocell and source of light and any successive change in absorption during titration is thus recorded. 10.B.2. Potentiometry: This phenomenon is based on measurement of change in potential of an indicator electrode immersed in titration vessel along with reference electrode during titration process. The end point is analyzed by rapid and large change in potential. Commonly potentiometric titration or potentiometry employ platinum electrode or more commonly mercury electrode to measure change in potential. 10.B.3. Miscellaneous methods Beside above mentioned techniques for end point determination, conductometry, amperometry, and coulometry are some of the other techniques that are successfully used for determination of end point in complexometric titrations.

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42

11. APPLICATIONS OF COMPLEXOMETRIC TITRATIONS Complexometric titration pose advantages of accurate, economic, and rapid technique for determination of metal ions ranging from chemicals used for agriculture, metallurgical, food processing to pharmaceutical utility. The technique used effectively and conveniently for determination of total hardness of water (determination of calcium and magnesium) for industrial as well as house hold purpose by titrating water with standard solution of EDTA using Murexide as indicator.

The technique of complexometric titrations plays a pivot role in determination of total metal (ca, mg, Zn) content of biological fluid for biochemical assay. From the point of view of metallurgical process, complexometric titration can be used for determining presence of various metal ions in raw and finished material such as lead in mineral and zinc in light alloy by use of Eriochrome black T indicator at pH 9 and 10.

Figure 04: Structure of some ligands

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43

The method is equally effective in determination of inorganic pharmaceutical substance containing metal ions like zinc sulphate, calcium carbonate, dried aluminum hydroxide gel, Zinc chloride, magnesium chloride etc. 12.

EXERCISE 1. Explain why uses of gravimetry and oxalate permanganate titrations are obsolete now a day.

2.

Give a brief outline regarding basic principle of complexometric titrations.

3.

What are ligands? Give their brief classification along with suitable example representing each class.

4.

Explain following terms a.

Ligand

b.

Polydentate ligand

c.

Chelate

d.

Chelating agent

e.

Chelation

f.

Sequestering agent

5.

Write a short note on ligand and metal ions.

6.

Enumerate and explain various factors that affect complexometric titrations.

7.

What are pM indicators? Give their ideal characteristics.

8.

Tabulate various pM indicators with their effective pH range and applications.

9.

Write a short note on masking and demasking agents with suitable example.

10.

Give preparation and standardization of 0.05M disodium EDTA.

11.

Can a solution of EDTA be standardized by calcium carbonate? If yes, give procedure.

12.

Enumerate various methods used in complexometric titration and give a brief outline on replacement and alkalimetric complexometric titrations.

13.

Give a brief outline regarding applications of complexometric titrations.

Chowrasia, Deepak



13.

44


Gravimetry and oxalate permanganate titrations are obsolete due to a. Lengthy and tedious procedures b. More time consumption c. Both d. None of the above

In complexometric titration, a complex is formed between a. Two metal ions b. Two ligand c. A ligand and solvent molecule d. A ligand and metal ion c

d A covalent complex is formed by ______ sharing of electrons between metal & ligand molecules. a. Equal b. Unequal c. both d. None of the above

Complexometric titrations are based on principle of a. Complexation b. Reunion c. Replacement d. None of the above a Generally, titration of metal ions can be done by a. Non aqueous titration b. Acid base titration c. Redox titration d. Complexometric titration d Metal ions that can be analyze by complexometric titration are a. Cations b. Anions c. Both d. None of the above

a A co-ordinate complex is formed by ______ sharing of electrons between metal & ligand molecules. a. Equal b. Unequal c. both d. None of the above b A ligand contains a. Electron donating group b. Electron releasing group c. Both d. None of the above

c Phenomenon of Complexation involves a. Complex formation b. Dissociation of molecules c. Association of molecules d. None of the above

Chemically, a ligand is a. Lewis acid b. Lewis base c. Both ‘ d. None of the above d

Chowrasia, Deepak

a

b



Monodentate ligand contains a. Single binding sites b. Double binding sites c. Triple binding sites d. None of the above

c. Both d. None of the above a

a Bidentate ligand contains a. Single binding sites b. Double binding sites c. Triple binding sites d. None of the above A ligand having more than one electron donating groups is termed as a. Monodentate ligand b. Bidentate ligand c. Hexadentate ligand d. Polydentate ligand

Sequestering agents forms a. Water soluble complex b. Water insoluble complex c. Both d. None of the above a

d Cyanide ion, ammonia, halide ion, and water are example of a. Monodentate ligand b. Bidentate ligand c. Hexadentate ligand d. None of the above

EDTA is a. Monodentate ligand b. Bidentate ligand c. Tridentate ligand d. Hexadentate ligand d

a A chelate is an a. Metal ion b. Solvent molecule c. Motif containing electron donating group d. Motif containing electron withdrawing group

EDTA, dimethylglyoxime, and salicylaldoxime are examples of a. Sequestering agent b. Monodentate ligand c. Both d. None of the above a Cyanide ion is a. Monodentate ligand b. Bidentate ligand c. Tridentate ligand d. Hexadentate ligand a

c

Chowrasia, Deepak

Chelating agent is usually a. Monodentate ligand b. Bidentate ligand c. Polydentate ligand d. Hexadentate ligand c

b

Chelation is a phenomenon of a. Formation of multiple bond between atoms b. Formation of ring like structure between ligand and metal ions

45

Glycine represents a. Monodentate ligand b. Bidentate ligand c. Tridentate ligand d. Hexadentate ligand b



Diethylenetriamine is a. Monodentate ligand b. Bidentate ligand c. Tridentate ligand d. Hexadentate ligand c Match the following a. Halide ions i. Bidentatae ligand b. Nitrilotriacetic acid ii. Tridentate c. N-hydroxyethylethylenediamine iii. Quadradentate d. EDTA iv. Hexadentate e. Glycine v. Monodentate a-v, b-iii, c-ii, d-iv, e-i Monodentate ligand forms ring like complex with metal ion a. True b. False c. Can’t predicted d. Both a & b b Match the following Metal ions 2+ a. Ca 3+ b. Al 4+ c. Sn + d. Na

Ring complex form i. Five ii. Four iii. Three iv. No ring (a-iii, b-ii, c-i, d-iv) For EDTA titration, overall pH during titrimetric procedure should be within a. ±1 to ±2 b. ±0.5 to ±1 c. ±0.1 to ± 0.2 d. None of the above b

Chowrasia, Deepak

46

Bond formed between a chelating agent and indicator is a. Stronger than metal ion b. Weaker than metal ion c. Same in strength as that of metal ion d. None of the above a Does it is possible for a chelating agent to forms multinuclear complexes a. Yes it is possible b. No c. Can’t be predicted d. None of the above a Pick out optimum concentration of metal ion in following solutions for better results a. 0.75 mmol in 50-150 ml b. 0.25 mmol in 50-150 ml c. 0.55 mmol in 50-150 ml d. 0.65 mmol in 50-150 ml b Optimum temperature for complexometric titration is a. 27-40 degree Celsius b. 37-40 degree Celsius c. 17-40 degree Celsius d. 07-40 degree Celsius b Metal complex formed by a polydentate ligand is a. Unstable & difficult to form b. Stable & easy to form c. Can’t be predicted d. None of the above b A pM indicator is a. Metallochromic dye b. Metal selective indicator c. Metal ion indicator d. All the above d



Mordant black-II is also known as a. Eriochrome Black-T b. Solochrome Black-T c. Both d. None of the above c Ammonium purpurate is a chemical name of indicator a. Murexide b. Xylenol orange c. Phthalein purple d. None of the above a

Match the following Masking agent a. Thioglycols b. Tiron c. Ascorbic acid d. KCN

47

Metal masked i. Hg ii. Cu iii. Fe (III) iv. Al a-i, b-iv, c-iii, d-ii

Formaldehyde is an example of a. Masking agent b. Demasking agent c. Both d. None of the above b

Pyrocatechol is a chemical name of pM indicator a. Catechol violet b. Catechol green c. Catechol black-II d. None of the above a Masking agents provides suitable platform for a. Titration of solution containing no metal ion b. Titration of solution containing single metal ion c. Titration of solution containing multiple metal ions d. None of the above c Masking agents shows their action by a. Hiding the wanted metal ion b. Hiding unwanted metal ion c. Hiding both wanted and unwanted metal ions d. None of the above b

Chowrasia, Deepak

BAL, a masking agent has it chemical name as a. 3,4-dimercapto-1-propan-1-ol b. 2,3-dimercapto-2-propan-1-ol c. 3,3-dimercapto-1-propan-2-ol d. 2,3-dimercapto-1-propan-1-ol d Formaldehyde and acetic acid (1:3) used as a demasking agent for metal ions a. Cd, Zn b. Al, Na c. Hg, Cd d. None of the above a Masking agent Tiron is chemically known as a. Disodium catechol 2,3-disulfonate b. Disodium catechol 3,2-disulfonate c. Disodium catechol 3,5-disulfonate d. None of the above c



Tiron as a. b. c. d.

predominately masked metals such Ni, co Hg, Fe (III) Hg, Fe (II) Al, Fe (III) d

Chloral hydrate used as a. masking agent b. demasking agent c. both d. None of the above b Cyanide ion forms complex with large number of metal ions except a. Lead, manganese, and alkaline earth metals b. Copper, manganese, and alkaline earth metals c. Lead, manganese, and Aluminum d. None of the above a Metal ions detection in complexometric titration can be done by a. Direct titration b. Indirect titration c. Back titration d. All the above d Tartrate or citrate is added to the titrating mixture in ________ titration a Direct titration b. Indirect titration c. Back titration d. All the above e. a Function of citrate or tartrate addition in direct complexometric titration is a. Encourage metal hydroxide precipitation

Chowrasia, Deepak

b. Prevent metal precipitation c. As a buffering agent d. None of the above

48

hydroxide

b Amount of EDTA required to preparing its 0.05M solution is a. 18.4g b. 18.5g c. 18.6g d. 18.7g c Most common solvent used for preparation of EDTA solution is a. Anhydrous methanol b. Anhydrous pyridine c. Hydrous methanol d. Distilled water d 0.05M solution of EDTA can be standardized by a. Metallic zinc b. Calcium carbonate c. Both d. None of the above c What is the role of dil. HCl in EDTA standardization by calcium carbonate a. It aids in calcium carbonate solubility b. It act as masking agent c. It increase acidic strength of solution d. None of the above a Back titration is preferred over direct complexometric titration due to a. Unavailability of suitable metal ion indicator b. Insolubility of metal ions



d. None of the above

c. Slow formation of complex d. All the above

c d

Most commonly used salt solution in back complexometric titration is/are a. magnesium sulphate b. Zinc sulphate c. Both d. None of the above c End point detection in complexometric titration can be done by a. Visual method using pM indicator b. Instrumental method such as potentiometry or photometry c. Both

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49

A substance that can act as masking as well as chelating agent is termed as a. Masking agent b. Chelating agent c. Both d. Auxiliary chelating agent d Thiglycol, ascorbic acid, tartaric acid, and triethanolamine are examples of a. Masking agent b. Chelating agent c. Both d. Auxiliary chelating agent d


Chapter – 03 KARL FISCHER TITRATION - Dr. Nisha Sharma, Deepak Chowrasia

Chowrasia, Deepak

KARL FISCHER TITRATION


53

KARL FISCHER TITRATION (Chapter Overview) 01. INTRODUCTION .................................................................................................................... 55 02. PRINCIPLE .............................................................................................................................. 55 03. KARL FISCHER REAGENT-PROBLEM AND MEASURES. ................................... 55 04. PREPARATION OF KARL FISCHER REAGENT ....................................................... 56 05. STANDARDIZATION OF KARL FISCHER REAGENT ............................................ 56 06. OPERATING pH .....................................................................................................................57 07. PRECAUTIONS DURING TITRATION .......................................................................... 57 08. METHOD OF KARL FISCHER TITRATION ...............................................................58 08.A. Volumetric method.......................................................................................................58 08.B. Columetric method.......................................................................................................58 09. INSTRUMENTATION ...........................................................................................................58 10. APPLICATIONS .....................................................................................................................59 11. EXERCISE ................................................................................................................................ 59

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55

KARL FISCHER TITRATION 01. INTRODUCTION Aquametry is the measurement (qualitative or quantitative) of water content in inorganic and organic chemical compounds. Basically, there are numerous physical, chemical, and instrumental methods (thermal method, distillation, chromatographic determination, electrochemical techniques, U.V. spectroscopy, and nuclear magnetic resonance) are available for determination of water content in a sample, but undoubtedly Karl Fischer titration, although a classical chemical method of aquametry, but owing to its properties of specificity, sensitivity, & selectivity provides firm pillars for moisture content determination in most of the organic and inorganic compounds. The technique itself has been accepted as an official method for moisture content determination by most of the Pharmacopoeias (IP, USP, BP, NF). This technique of aquametry was first suggested by Karl Fischer (1935) and based on the philosophy of chemometric moisture content estimation by chemical reaction occurring between water molecule and Karl Fischer reagent. End point in Karl Fischer titration can be effectively determined by either volumetric (water content ≥1%) or coulometric (water content ≤1%) alternatively. 02. PRINCIPLE The overall reaction between water molecule and Karl Fischer reagent takes place in a manner as depicted by chemical reaction given at the end of this paragraph. Karl Fischer titration is a direct method of water estimation, based on the simple principle of “amount of iodine disappears during titration is directly proportional to net water or moisture content of sample” (1mole of iodine=1mole of water). Initially, iodine is getting reduced while sulphur dioxide is oxidized in presence of water to yield HI and sulphur trioxide. Later on, sulphur trioxide reacts with pyridine to yield an inert salt pyridine-sulphur trioxide complex, in turn react with methanol forming pyridinium methylsulphate or pyridine salt of methylsulfate. Hence each mole of iodine disappears in initial step ( chemical reaction 01) during titration is exactly equals to one mole of water present in the sample. Thus, overall chemical reaction involved in Karl Fischer titration is explained below as I2 + SO2 + H2O SO3 + C5H5N C4H5NSO3 + CH3OH

2HI + SO3……………………….

(1)

C5H5NSO3……………………

(2)

C5H5NCH3O-SO3H…...

(3)

03. KARL FISCHER REAGENT-PROBLEM AND MEASURES As per U.S.P, the Karl Fischer reagent is anhydrous mixture of iodine (125 g), pyridine (170 ml), methanol (670ml) & liquid sulphur dioxide (100ml). The reagent is well available

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56

commercially, but can be prepared freshly in laboratory with good success rate and stability of this freshly prepared Karl Fischer reagent can be increased by addition of sulphur dioxide to stock solution. It has been noted that original Karl Fischer reagent prepared with excess methanol was unstable and required standardization prior to use. This problem can be effectively overcome by use of methanol free Karl Fischer reagent containing substituted alcohols such as 2chlorethanol, 2-methoxy ethanol, 1-methoxy-2-propanol or trifluro ethanol in place of anhydrous methanol. Likewise, anhydrous pyridine, a weak base unable to counteract (neutralized) acid produced during titration thereby causing reversible reaction by reacting with sulphur dioxide giving sluggish end point. Nevertheless, replacement of pyridine with strong base such as imidazole ultimately screens out the problem associated with acid neutralization thus providing sharp end point. Hydranal reagents are commercially available & chemically modified Karl Fischer reagents employs imidazole or diethanolamine as a base rather than pyridine thereby making the aquametry procedure more effective, reliable and safe. 04. PREPARATION OF KARL FISCHER REAGENT Mix properly anhydrous methanol 400ml and anhydrous pyridine 80g in a clean combustible flask. Immerse flask in ice bath and slowly bubbled dried sulphur dioxide until the weight of flask increased by 20 g. Now add iodine 45g and shake the resultant solution until it dissolves completely. Keep the prepared solution in an air tight amber colored bottle for 24 hours and standardized prior to use 05. STANDARDIZATION OF KARL FISCHER REAGENT The Karl Fischer reagent can be standardized by titrating it either with water-methanol system or sodium tartrate as a reagent. Standardization of Karl Fischer reagent with water-methanol system: Prepare 0.2% v/v water-methanol solution by adding 2.0ml of water to 1000ml of anhydrous methanol. From this, accurately measure off 25ml of solution, transfer it into reaction vessel, and perform titration with Karl Fischer reagent. The net content of water mg per ml of water-methanol system can be calculated mathematically by using following formula;

Where, W= water content mg/ml Vk =Volume of Karl Fischer reagent required E f =Water equivalence factor determined against sodium tartrate (see below)

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57

Standardization of Karl Fischer reagent with sodium tartrate: Measure off 30ml of anhydrous methanol, transfer it into a clean water free reaction vessel, and slowly add (dropwise) Karl Fischer reagent till to get an end point. Once end point is obtained, immediately add 150350mg of sodium tartrate dehydrate and titrate to end point. The water equivalence factor E f (mg/ml) of reagent could be calculated by formula given below;

Where, E f =Water equivalence factor W=Weight of sodium tartarate in mg V=Volume of reagent required in ml 06. OPERATING pH Stoichiometrically, the ideal pH range for Karl Fischer titration is 5-7. Shifting of pH towards strongly basic side leads to an auto-initiation of side reaction, consuming iodine thus vanishing end point. On other hand, at acidic pH, the titration get slower due to reduction of reaction constant. Therefore, in order to minimize pH effect, it is highly recommended that during overall period of titration pH of titrating mixture must be at or in between 5-7. 07.

PRECAUTIONS DURING TITRATION 1. The titre value of Karl Fischer reagent when freshly prepared is about 5mg of water per ml of reagent, which gradually reduces with passage of time. Thus reagent must be standardized prior to an analytical procedure.

2.

All glass wares and apparatus used for making & determining end point in Karl Fischer titration must be absolutely free from even minute water content.

3.

Commercially available solution or freshly prepared Karl Fischer reagent must be placed in a tightly closed container away from moisture and direct light.

4.

Before titration, Karl Fischer reagent (freshly prepared or commercial grade) must be standardized by addition of 2ml of water to 100ml of methanol.

5.

All necessary precaution must be taken during titration so that reagent come in contact with moisture or light as less as possible.

6.

The analyte should not react adversely with any of ingredient of reagent or with hydrogen iodide yield during reaction.

Chowrasia, Deepak



7.

58

The sample must be free from following compounds as they interfere with end point: a.

Oxidizing agents

b.

Reducing agents

c.

Basic oxides and their salts

d.

Aldehydes and ketones

08. METHOD OF KARL FISCHER TITRATION Water determination in Karl Fischer titration is determined by two method volumetric and columetric. 08.A. Volumetric method The method is suitable for sample containing high amount of water (1%-2%) and is based on simple principle of total amount of water present in sample is equal to net amount of Karl Fischer reagent used during titration. 08.B. Columetric method The method is suitable for sample containing low quantity of water (1% or less) with the help of instrument called coulometer. Columetric determination of water is more accurate than volumetric method. 09. INSTRUMENTATION Karl Fischer titration apparatus or dead stop end-point assembly (see figure 01) is commonly used for determination of water content in sample. The apparatus consists of a reaction or titration vessel of approximately 60-100ml in capacity, fitted with two identical platinum electrodes (2cm apart having surface area 0.05sq.cm), in turn attached suitably with sensitive galvanometer, dry cells (1.5V), & a resistance (2000-ohms). Beside this, a nitrogen inlet tube (remove air bubble from solution), burette (for titration), drying tube, mechanical stirrer, and stopper are also assembled with apparatus so as to perform titration with an ease.

Initially, when no titrant is added into titration vessel, a little or no current flows thorough external circuit and any deflection in galvanometer if obtained is only due to absorbed layers of hydrogen and oxygen over respective electrodes. However, electrodes are gets depolarized thus current flows, during the course of titration with addition of Karl Fischer reagent and end point could be determined by noticing deflection in galvanometer remaining at least for 30 or more seconds.

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Figure 01: Instrumentation for Karl Fischer method 10. APPLICATIONS Karl Fischer titration is an efficient mean of determining moisture content in wide range of chemicals organic as well as inorganic in nature. 11.

EXERCISE 1. Explain principle of Karl Fischer titration by suitable chemical reaction.

2. What is Karl Fischer reagent? Briefly explain its constituents. 3. Give a method to prepare Karl Fischer reagent also mention quantity of ingredient utilized. 4. Outline various methods used for standardization of Karl Fischer reagent. 5. Enumerate various precautions that have to be taken during Karl Fischer titration. 6. Discuss in brief instrumentation of Karl Fischer titration by a well labeled sketch. 12.

FILL IN THE BLANKS

1. The terms aquametry is used for estimation of ___________ in sample ( Water). 2. Karl Fischer titration is a ______________ method of aquametry ( Chemical).

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3. The Pharmacopoeias accepted Karl Fischer titration as an official method are __________________ ( NF, USP, BP, IP). 4. Karl Fischer titration was first device in year ______________( 1935). 5. End point in Karl Fischer titration can be effectively determined by ________ or ______________ method. ( Volumetric (water content ≥1%) and Coulometric (water content ≤1%). 6. During a titration if 10 moles of iodine is disappears then amount of water present in sample is roughly equals to _____ moles ( 10 moles). 7. Anhydrous methanol and pyridine could be successfully replaced with _________ and _____________. ( Substituted alcohols and imidazole/diethanolamine) 8. Mention two names of substituted alcohols _______________ (2 -Chlorethanol, 2-methoxy ethanol). 9. Hydranal reagents contains ________in place of pyridine ( Imidazole). 10. Karl Fischer reagent can be standardized by _________ or __________ system (Water-methanol or sodium tartrate). 11. Ideal pH range for Karl Fischer titration is __________( 5-7). 12. For efficient Karl Fischer moisture determination, sample should be free from ________ (Oxidizing & reducing agents, basic oxides & their salts, and aldehydes & ketones). 13. Dead stop end point determination is mostly employed in ___________ procedure for water estimation. ( Karl Fischer titration).

Chowrasia, Deepak


Chapter - 04 DIAZOTIZATION TITRATION - Deepak Chowrasia, Ajay Kumar

Chowrasia, Deepak

DIAZOTIZATION TITRATION


63

DIAZOTIZATION TITRATION (Chapter Overview) 01. INTRODUCTION .................................................................................................................... 65 02. PRINCIPLE .............................................................................................................................. 65 03. FACTOR AFFECTING DIAZOTIZATION TITRATION ..........................................66 03.A. Types of amino groups ................................................................................................ 66 03.B. Overall temperature during titration process .......................................................66 03.C. pH of titration ................................................................................................................ 66 04. End point determination ........................................................................................................67 04.A. Visual method ................................................................................................................ 67 04.B. Electrometric method .................................................................................................. 67 05. ANALYTICAL PROCEDURE ............................................................................................ 67 05.A. Preparation of 0.1M Sodium nitrite solution ......................................................... 67 05.B. Standardization of 0.1M Sodium nitrite solution ................................................. 67 05.C. Assay of Isocarboxazide .............................................................................................. 67 06. APPLICATIONS .....................................................................................................................68 07. EXCERCISE .............................................................................................................................68

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DIAZOTIZATION TITRATION 01. INTRODUCTION Diazotization titration is one of the most renowned classical methodology used for determination of primary aromatic amino group presence in most of the chemical compounds including a well known second world war antibiotic-The sulfa drugs (also be termed as sulfonamide). Since diazotization titration makes use of sodium nitrite for determination of aromatic primary amino group thus sodium nitrite titration is their alternate name. 02. PRINCIPLE Diazotization titration is based on the reaction between aromatic primary amino group and nitrous acid in acidic medium resulting in the formation of diazonium compound. End point of titration is estimated by calculating excess of nitrous acid left either visually (using starchiodide paper or paste as an external indicator) or more accurately by dead stop end point method (electrometrically). The overall chemical reaction takes place during the whole titrimetric procedure can be summarized in a stepwise manner as below

1.

Initially, sodium nitrite (NaNO 2) reacts with hydrochloric acid (HCl) leads to formation of salt (NaCl) and nitrous acid (HNO 2) NaNO2+HCl

2.

NaCl + HNO2………………………………..

(1)

Nitrous acid then diazotizes primary aromatic amino group as depicted in chemical reaction given below Ar.NH2.HCl + HNO2

+

-

Ar.N2 Cl + H2O……………………

(2)

Once all aromatic amine is get consumed then excess of nitrous acid is determined visually by using starch iodide paper (prepared by impregnating filter paper with starch mucilage and potassium iodide solution) showing blue-green color at end point. The overall chemical reaction involved in visual end point detection can be summarized as below KI + HCl 2HI + 2HNO2 I2 + Starch

HI + KCl ………………………………………..

(3)

I2 + 2NO + 2H 2O…………………………..

(4)

Blue green color ……………………………….

(5)

Visual end point determination in diazotization titration can be well substituted by adopting the dead stop end point technique (electrometrically) which makes use of two platinum electrode immersed in sample solution for end point detection.

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03. FACTOR AFFECTING DIAZOTIZATION TITRATION The various factors that affect diazotization titration includes

1. Types of amino group 2. Overall temperature during titration process 3. pH of titration 03.A. Types of amino groups The rate of reaction or titration process occurs at a slower rate if the aromatic moiety containing amino group is highly substituted like anthranilic acid. While on other hand, less substituted aromatic amino molecule (Ethyl-4-aminobenzoate) easily and readily react with nitrous acid making the reaction process rapid and convenient. The overall rate of reaction of slow diazotized compound can be enhanced by addition of KBr (potassium bromide) along with providing sufficient time to a compound in order to get it react with nitrous acid.

Figure 01: Slow and fast diazotized compound 03.B. Overall temperature during titration process Temperature plays a key role in titration process. Usually the titration must be performed at lower temperature, since higher temperature result in decomposition of diazonium salts into phenolic compounds. It is advisable to maintain a temperate of 5-15 degree Celsius during overall reaction period. 03.C. pH of titration Diazotization titration must be performed at acidic pH which favors yield of nitrous acid thus end point detection using starch-iodide paper becomes more feasible, rapid and easy.

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04. End point determination In diazotization titration the end point can be easily and rapidly estimated by use of external visual indicator like starch-iodide paper or alternatively, but more accurately and precisely by electrometrical method. 04.A. Visual method: This method established end point visually by detecting change in color and employs starchiodide paper or its paste as an external indicator. Owing to its simplicity, ease of procedure, economical feasibility, and quantitative adoptability visual method uses widely and predominately for end point determination in diazotization titration. The method is based on simple phenomenon that excess of nitrous acid formed after exhaustion of whole aromatic amino group will liberate iodine which reacts with starch showing blue green color at end point. 04.B. Electrometric method It is an instrumental method utilizes pair of platinum electrode for detection of excess nitrous acid at end point. Basically under normal condition, the electrodes are in polarized state hence no current will flows through circuit thus no deflection has been observed in galvanometer. However, during the course of titration liberation of nitrous acid causes electrodes to get depolarized and a full deflection in galvanometer ultimately point out establishment of end point. 05.

ANALYTICAL PROCEDURE

05.A. Preparation of 0.1M Sodium nitrite solution Accurately weigh out 7.5g of sodium nitrite and make volume up to 1 liter with distilled water. 05.B. Standardization of 0.1M Sodium nitrite solution Weigh out accurately 0.5g of Sulphanilamide, add 20mL (11.5 M) of hydrochloric acid, dilute the resultant mixture with 50mL distilled water, stir to dissolve and cool it in ice bath. Titrate the resultant solution with 0.1M sodium nitrite & determine end point with starch-iodide paper till it give blue-green color. Each 0.01722g of Sulphanilamide is equivalent to 1 mL of 0.1 M sodium nitrite. 05.C. Assay of Isocarboxazide Weigh out 0.5g of Isocarboxazide, dissolve in 20ml glacial acetic acid, add 20ml of HCl, and 50ml of distilled water. Cool down resultant mixture in an ice bath and titrate it slowly with 0.1M sodium nitrite until appearance of distinct blue colour on starch-iodide paper. Each ml of 0.1M sodium nitrite is equivalent to 0.02313g of Isocarboxazide.

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06. APPLICATIONS Direct diazotization titrations are used for assay of drugs like dapsone, benzocaine, procaine, suramin, primaquine including all sulfa drugs containing free aromatic group. However, compounds devoid of free amino group must be first derivatized into a form suitable for their diazotization titrations like

a. Chloramphenicol & Metronidazole contains aromatic nitro group which has to be initially reduced with suitable reducing agent prior to their normal diazotization procedure. b. Paracetamol & succinyl sulfathiazole are suitably hydrolyzed in order to free their amino group for diazotization titrations. Indicator

Quantity (grams)

Calculation (x) (Each ml of 0.1M NaNO2 is equivalent to x grams of P

Sulphaphenazole

Starch iodide paper

0.5

0.03144

Sulphamethoxazole

Starch iodide paper

0.5

0.02533

Sulphamethizole

Starch iodide paper

0.5

0.02703

Sulphadimidine sodium

Starch iodide paper

0.5

0.03003

Sulphadimethoxine

Starch iodide paper

0.5

0.0313

Sulphadiazine

Starch iodide paper

0.5

0.02503

Succcinyl sulphathiazole

Starch iodide paper

0.5

0.03554

Dapsone

Starch iodide paper

0.3

0.01242

Sodium aminosalicylate

Starch iodide paper

2.5

0.01752

Procainamide

Starch iodide paper

0.5

0.02718

Primaquine phosphate

Starch iodide paper

1.0

0.04553

Pharmaceuticals (P)

Table 01: List of some drugs titrated via diazotization titration 07.

EXCERCISE 1. Explain briefly principle of diazotization titration

2. Enumerates various factors that affects diazotization titration

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3. What are the various methods that are used for end point determination in diazotization titration? Explain them briefly. 4. Give procedure for preparation and standardization of 0.1M sodium nitrite. 5. Give a brief outline regarding assay of isocarboxazide by diazotization titration. 6. Enumerate various applications of diazotization titration. 08.


Diazotization titration is also known as a. Sodium nitrite titration b. Non aqueous titration c. Sodium nitrile titration d. None of the above a Diazotization titration is primarily done for compounds containing a. Active methylene group b. Aromatic primary amino group c. Aromatic secondary amino group d. Aromatic tertiary amino group b End point determination in diazotization titration is done by a. Starch paper only b. Iodine and starch paper c. Protein and starch paper d. None of the above b Aromatic moiety containing highly substituted amino group will undergoes a. Fast reaction b. Slow reaction c. No reaction d. None of the above b

Chowrasia, Deepak

Slow diazotized reaction can be converted into fast by addition of a. Sodium nitrite b. Potassium bromide c. Potassium cyanide d. Potassium hydride b Temperature during diazotization titration should be ◦ a. 5-15 C ◦ b. 25-35 C ◦ c. 35-55 C d. None of the above a At higher temperature, feasibility of diazotization titration is difficult since higher temperature leads to a. Consumption of more amount of sodium nitrite b. Conversion of amino group into imino group c. Decomposition of diazonium salt into phenolic compound d. None of the above c



Following drug/s required prior reduction before proceeding for their diazotization titration a. Chloramphenicol & Metronidazole b. Sulphaphenazole & Sulphamethoxazole c. Both a & b d. None of the above a Paracetamol & succinyl sulfathiazole are suitably _____________ prior to their for diazotization titrations. a. Reduced b. Oxidized c. Hydrolyzed d. All of the above c

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70

Optimum pH for diazotization titration is a.

Acidic

b.

Basic

c.

Neutral

d.

None of the above a

End point determination in diazotization titration is done by a. Visual method b. Instrumental method c. Both d. None of the above c


Chapter – 05 KJELDAHL’S METHOD OF NITROGEN ESTIMATION - Dr. Nisha Sharma, Deepak Chowrasia

Chowrasia, Deepak

KJELDAHL’S METHOD OF NITROGEN ESTIMATION


73

KJELDAHL’S METHOD OF NITROGEN ESTIMATION (Chapter Overview)

01. INTRODUCTION .................................................................................................................... 75 02. PRINCIPLE .............................................................................................................................. 75 03. LIMITATION OF KJELDAHL METHOD ......................................................................77 04. MODIFICATION IN KJELDAHL METHOD ................................................................77 05. GENERAL PROCEDURE ....................................................................................................77 06. APPLICATIONS .....................................................................................................................77 07. EXERCISE ................................................................................................................................ 78

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KJELDAHL’S METHOD OF NITROGEN ESTIMATION 01. INTRODUCTION Nitrogen is an essential part of most of the organic compounds especially peptides & proteins and can be determined by various physical as well as chemical methods out of which Kjeldahl method has predominating significance owing to its simplicity and wider spectrum. The method named after its inventor Johann Kjeldahl who introduced this method in 1883 for nitrogen estimation, since from then Kjeldahl’s method either in its original or modified form holds its popularity not only in terms of nitrogen estimation in scientific or chemical laboratories, but also maintains its potential for quantitative nitrogen determination in allied branches of science dealing nitrogenous substances directly or indirectly. The method was initially designed in order to estimate nitrogen content of proteins along with some organic compounds, but due to its simplicity & practical acceptability, several modifications in original procedure has been done, which ultimately extended its spectrum from proteinous nitrogen estimation to nitrogen determination in vast number of organic & inorganic compounds. 02. PRINCIPLE Kjeldhal’s method is used strictly for nitrogen estimation in only those organic compounds in which nitrogen is converted into ammonium derivative mostly sulphate form, while the method looses its practical accessibility for compounds denied to transform their nitrogen into ammonium sulfate thus requiring either modification in original procedure or selection of another suitable method for their nitrogen determination. It is interesting to underline that compounds like oximes, nitrates, nitrites, azo, hydrazines, and osazones requires prior reduction with reducing agents such as glucose or thiosalicylic acid or hydriodic acid before proceeding for their nitrogen estimation by Kjeldahl method.

Principally, Kjeldhal’s method is based on fact that organic compounds containing nitrogen when heated with oxidizing agent (concentrated sulphuric acid) in the presence of catalyst (cupper sulfate) and boiling point enhancer (potassium sulphate) results in conversion of nitrogen present in compound into ammonium sulphate derivative. Nitrogen compound + H2SO4

(NH4) 2SO4 + H2O +CO2.............

(1)

The ammonium sulphate derivative of nitrogen is then treated with excess of alkali (sodium hydroxide solution) thus librating ammonia gas. (NH4) 2SO4 + 2NaOH

2NH3 + Na2SO4 +2H2O………………………

(2)

This ammonia gas is then used for estimation of nitrogen content in a sample by following one of the two ways;

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Directly distilling ammonia gas into standard solution of excess acid (sulphuric or hydrochloric acid) and unreacted acid so left is then back titrated with standardized alkali solution. Or,

Distilling ammonia gas into boric acid solution where the trapped ammonia is than directly titrated with standard solution of acid.

……………………

(3)

Chemical reaction involved Nitrogen compound + H2SO4 (NH4)2SO4 + 2NaOH

(NH4)2SO4 + H2O + CO2………

(4)

2NH3 + Na2SO4 + H2O + 2H2O…………

(5)

Titrated 2NH3

Nitrogen estimation…………………………………

(6)

Calculation for percentage of nitrogen in sample: Percentage of nitrogen can be calculated by following formula Wt of organic compound= W grams Volume of acid consumed=V milliliter Normality of acid=N V ml of N normal acid=V ml of N normal ammonia 1000ml of N normal ammonia contains=14 gram of nitrogen Then V ml of N normal ammonia contains 14/1000 x V x N=0.014NV Percentage of nitrogen= Weight of nitrogen x 100/weight of compound 0.014 x 100/W =1.4NV/W

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03. LIMITATION OF KJELDAHL METHOD The method is only applicable to organic compounds in which nitrogen is converted into its suitable derivative (ammonium sulphate). Nitrogen contained in compounds in form of nitro or azo can not determine by this method unless & until they are converted into suitable derivatives. Also the method looses its applicability in case of nitrogen present in cyclic compounds. 04. MODIFICATION IN KJELDAHL METHOD In its original form, Kjeldhal’s method suffers drawback of limited quantitative analysis of organic compounds for their nitrogen determination. However, proper choice of apparatus and modification in procedure widen its scope and reduces net sample quantity to micro to ultramicro in range. Addition of anhydrous sodium sulfate or potassium sulfate powder to sulphuric acid allows higher temperature for sample digestion. It has been found that addition of 500mg of potassium sulfate per ml of sulphuric acid will increase the boiling point approximately 40 to 330 degree Celsius making the overall reaction faster and efficient digestion of sample under test. 05. GENERAL PROCEDURE Weigh out accurately optimum quantity of organic sample containing at least 0.04 g of nitrogen and transfer it into a Kjeldahl flask. To the flask add 40ml of concentrated sulphuric acid, 15 grams of potassium sulphate and 0.7grams of mercury (II) oxide. Boil the content for at least 2-hours in a slight tilted position. Cool the reaction mixture to room temperature and then add 200 ml of water and 25 ml of 0.5 M sodium thiosulphate solution mix well. Add antibumping stones to the resultant mixture and carefully pour 11M sodium hydroxide sufficiently to make mixture strongly basic in nature. Connect the flask to distillation apparatus such that the delivery tube tip dives just below surface of measured volume of 0.1M hydrochloric acid. Boil the resultant mixture until 150ml of liquid from flask will distill into receiver. Titrate the hydrochloric acid solution with 0.1M sodium hydroxide using methyl red as an external indicator. Perform a blank titration on an equal volume of 0.1M HCL. 06. APPLICATIONS Kjeldhal’s method finds it application in determination of nitrogen content in wide varieties of organic compounds. The method is equally applicable for assay of fertilizers, assay of soil, nitrogen content determination of food material, milk & their product as well as waste water nitrogen estimation. Basically, nitrogen estimation for food or natural protein is not always true by Kjeldhal’s method and commonly requires a correction factor which when multiply with Kjeldahl percentage nitrogen provides average protein content of sample. For example a correction factor of 6.25 (maize, meat, and egg), 5.70 (wheat flour), and 5.46 (peanuts) has to be suitably induced in order to determine nitrogen content of respective food or natural protein.

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07.

EXERCISE 1. Give a suitable outline including principle on Kjeldhal’s method of nitrogen estimation. 2. Explain Kjeldhal’s method by suitable chemical reaction only. 3. What are the various limitations of Kjeldhal’s method of nitrogen estimation? 4. Give a brief outline regarding applications of Kjeldhal’s method of nitrogen estimation. 5. Enumerate general procedure for nitrogen estimation by Kjeldhal’s method.

08.


The Kjeldhal’s method was coined by a. Johann Kjeldhal in 1883 b. Joahnn Kjeldhal in 1884 c. John Kjeldhal in 1885 d. None of the above a Basically nitrogen content in form of oximes, nitrates, nitrites, azo, hydrazines, and osazones are not estimated by Kjeldhal’s method and has to be ___________ prior to nitrogen determination by same method. a. Oxidized b. Reduced c. Both d. None of the above b Glucose or thiosalicylic acid or hydriodic acid is used as _____________ agent a. Oxidizing b. Reducing c. Both d. None of the above b

Chowrasia, Deepak

In Kjeldhal’s method concentrated sulphuric acid, cupper sulfate, and potassium sulphate used as a. Reducing agent, catalyst & boiling point enhancer b. Oxidizing agent, reducing agent & boiling point stabilizer c. Oxidizing agent, catalyst, & boiling point enhancer. d. None of the above c For a compound to be determined by Kjeldhal’s method, its nitrogen must be suitably converted into a. Ammonium sulphate b. Ammonium hydrate c. Ammonium nitrate d. None of the above a Ammonia gas so liberated during procedure can be titrated via a. Sulphuric acid in excess b. Boric acid in excess c. Hydrochloric acid in excess d. All of the above d



Estimation for nitrogen content of cyclic compound containing nitrogen is done by Kjeldhal’s method a. True b. False c. Both d. None of the above b Addition of sodium or potassium sulphate to sulphuric acid a. Increases boiling point b. Decreases boiling point c. Stabilizes boiling point d. None of the above a

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What is the optimum quantity of potassium sulphate that has to be added to sulphuric acid for boiling point enhancement? a. 500mg b. 0.5g c. 0.05g d. 50mg a,b Kjeldhal’s method needs correction factor for estimation of nitrogen in natural protein a. True b. False c. Both d. None of the above a


Chapter – 06 PAPER CHROMATOGRAPHY - Deepak Chowrasia, Md Arshad

Chowrasia, Deepak

PAPER CHROMATOGRAPHY


83

PAPER CHROMATOGRAPHY (Chapter Overview) 01. INTRODUCTION .................................................................................................................... 85 02. PRINCIPLE .............................................................................................................................. 85 04. LIMITATIONS OF PAPER CHROMATOGRAPHY ...................................................86 05. COMPONENT OF PAPER CHROMATOGRAPHY .................................................... 86 05.A. Chromatographic paper ............................................................................................. 86 05.B. Mobile phase .................................................................................................................. 88 05.B.I. Important characteristic of solvent used in paper chromatography ..........89 06. TYPES OF PAPER CHROMATOGRAPHY ...................................................................89 06.A. Horizontal paper chromatography ..........................................................................89 06.B. Vertical paper chromatography ...............................................................................90 06.B.1. Ascending paper chromatography: ................................................................90 06.B.2. Descending paper chromatography: .............................................................. 90 06.C. Hybrid paper chromatography .................................................................................90 06.D. 2-Dimentional paper chromatography ....................................................................90 07. Rf or R VALUE .........................................................................................................................91 08. FACTORS AFFECTING Rf VALUE .................................................................................92 09. IMPORTANCE of Rf VALUE .............................................................................................. 92 10. APPARATUS & PROCEDURE ........................................................................................... 92 10.A. Apparatus .......................................................................................................................92 10.B. Procedure .......................................................................................................................93 11. COMPONENT DETERMINATION ..................................................................................94 12. APPLICATION OF PAPER CHROMATOGRAPHY ...................................................95 13. EXERCISE ................................................................................................................................ 95 14. MULTIPLE CHOICE QUESTIONS ..................................................................................96

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PAPER CHROMATOGRAPHY 01. INTRODUCTION The phenomenon of paper chromatography was first given by Consden, Goiden, & Martin in 1944 and Martin along with Synge shares Noble prize (1952) in Chemistry for their contribution towards development of partition chromatographic technique. Although, this technique of chromatography is superseded by most of the advance chromatographic procedures, but still it remains as an effective & official tool for separation and identification of various chemical compounds including pharmaceutical active ingredients like Ergometrine, Liothyronine, Methotrexate, Phenformin, vitamin A and many more. 02. PRINCIPLE Paper chromatography is the simplest, economical, and highly convenient form of chromatographic technique serving as a foundation for separation of mixture into their respective components along with establishing their identification as well as purity justification by two possible mechanisms viz. capillary action & solute solubility pattern“like dissolve like”. Technically, paper chromatography is a planner, open bedded chromatographic technique based on the phenomenon of partitioning of solute between two layers of liquid, one is a stationary liquid layer held in pores of chromatographic paper (paper mostly made up of cellulose) and other is mobile liquid layer runs over the stationary liquid phase. Since, partitioning of solute molecules occur between these two liquid phases (stationary & mobile phase) thus paper chromatography, in broad sense sometime also termed as liquid-liquid partition chromatography. However, to some an extent, adsorption also plays a crucial role during separation process, but partition predominate it in terms of practical separation. Fractionation of compound into their respective components in paper chromatography depends upon their affinity towards stationary and mobile liquid phases. Thus, components of mixture having high affinity towards stationary liquid phase will adhere to it and runs at a velocity lesser than a components having higher affinity for mobile phase hence get separated out. 03.

ADVANTAGES OF PAPER CHROMATOGRAPHY

a.

Most simplest and rapid method for fractionation of chemicals as well as biological compounds.

b.

Easily used for remote analysis of compounds.

c.

Compatible with wide range of components.

d.

Required small amount of sample.

Chowrasia, Deepak



04.

e.

Usually no special skills are required.

f.

Comparatively, cheapest form of separation technique.

g.

Does not require sophisticated instrumentation or apparatus.

h.

Used for both analytical as well as educational purposes.

i.

Less solvent consumption compare to column chromatography.

86

LIMITATIONS OF PAPER CHROMATOGRAPHY a. Most of the chromatographic papers are not amenable for reuse.

b.

Only able to deal smaller quantity of sample.

c.

Hardly used as a preparative chromatography.

d.

Quantitative analysis with paper chromatography is tough to establish.

e.

Useless while dealing with compounds which are highly volatile in nature.

05. COMPONENT OF PAPER CHROMATOGRAPHY As other chromatographic techniques paper chromatography consist of following two components

Chromatographic paper Mobile phase 05.A. Chromatographic paper A chromatographic paper is a stable, planner surfaced, highly porous, larger surface area backing medium ( but not the stationary phase since stationary phase is liquid entrapped into pores of chromatographic paper ) provides support for separation of solute particles. Paper may range from a simple filter paper (for demonstration or educational purposes) to highly sophisticated chromatographic papers ( Refer table 02) of different grade and texture used for various analytical works. Whatman paper No-1 ( Refer table 01) is the paper of choice for most of analytical procedures associated with paper chromatography. Nature of solute to be separated, solvent employed for separation, and flow rate of mobile phase that has to be maintained during fractionation process are some of the factors that affects the selection criteria of chromatographic paper. Basically, thin & smooth textured light to medium density chromatographic paper is preferred for separation of wide varieties of solute irrespective of their nature or physiochemical properties (except some exceptions) under fast to medium flow rate. On other hand, rough surface thick papers are used for small scale preparative chromatographic procedure including electrophoresis, separation of protein, peptides, amino acids, and inorganic substances at medium to slow flow rate.

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Figure 01: Various moldings of chromatographic paper (A-rectangular, B-arrow shape, C-circular, & D-drum shape) S. No.

Paper grade

01

1

02

2

03

3

04

4

05

7

06

20

07

54

08

542

Paper characteristic

Solvent flow rate

Applications

(mm/30min.)

Plain & smooth texture with medium weight. (t-0.18-0.20mm) Slight more weight, but somewhat similar in texture with grade1(t0.18mm) Thick with rough textured surface (t-0.36mm) Medium weigh & open texture(t0.21mm) Rough surface & thick (slightly)

++; 130

All purpose paper

+; 115

Electrophoresis, separation of amino acid & proteins

++; 130

Tight texture with uniform pattern (t-0.17mm) Single acid washed & hardened with greater wetting strength (t-0.18mm) Double acid washed

+; 85

Electrophoresis & inorganic applications Amino acid & carbohydrate separations Electrophoresis, amino acid, proteins, carbohydrate and general separation procedures Genius results with most of compounds separated Excellent for 2D chromatography Organic inorganic separation

+++ ++

+++; 180 +

Table 01: Some Whatman papers their grades, characteristic, & applications (Key: +++fast, ++medium, +slow;t-thickness)

Chowrasia, Deepak



S.NO

Sophisticated chr. papers

88

Applications

A

Acetylated paper

Reverse phase chromatography

B

Ion exchange paper

Exchange of ions

C

Stannic antimonite paper

Cation separation

D

Kieselguhr paper

Separation of fine semi-colloidal system

E

Silica paper

Pesticide & vitamins separation

Table 02: Sophisticated chromatographic papers & their applications 05.B. Mobile phase Mobile phase used in paper chromatographic procedure is a free flowing low viscosity mono or multi-component solvent system (Refer table 03) aiding fractionation of mixture into individual components during chromatographic procedure. For an ideal separation, two or more solvents are used in a predetermined proportion in such a way that one component predominantly dissolves greater than other component for better separation.

Since as per the principle of “like dissolves like” thus, polar solvents are employed for separation of polar components while non-polar solvents used for lipophilic components separation. For better result it should be boldly under lined that what so ever the mobile phase selected for paper chromatography it should not cause under any circumstances self interference with any of the components of mixture to be separated or with supporting medium i.e. chromatographic paper and must perform its two vital functions i.e. dissolution of solute & acting as carrier medium for dissolved solute for their efficient separation. Compound

Mobile phase

Ratio

Phenformin hydrochloride

Ethyl acetate-Ethanol

95:5

Vitamin A

Dioxan-methanol-water

70:15:5

Capreomycin Sulphate

1-propanol-water-glacial acetic acid-triethylamine

75:33:8:8

Vancomycin Hydrochloride

2-methylbutan-2-olacetone-water

2:1:2

Table 03: Mobile phases & their ratio for separation of different components

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05.B.I. Important characteristic of solvent used in paper chromatography a. Inert-no interference with sample, chromatographic phases, including techniques used for sample detection. b.

Good dissolution.

c.

Miscibility with other solvents in a sufficient extent.

d.

Ability to resolve components of mixture.

e.

Composition remains constant throughout the chromatographic procedure.

f.

if possible, recycled.

g.

Non toxic and least environmentally hazardous.

06. TYPES OF PAPER CHROMATOGRAPHY On the basis of mobile phase direction of flow through chromatographic paper, paper chromatography can be broadly categorized into following four types 06.A. Horizontal paper chromatography 06.B. Vertical paper chromatography 06.C. Hybrid paper chromatography 06.D. 2-dimentional paper chromatography 06.A. Horizontal paper chromatography In this technique, paper is kept horizontally over suitable support such as Petri disc or special glassware containing mobile phase and the solvent (mobile phase) with the help of wick (placed at centre of paper) runs from middle of paper towards periphery resulting in formation of circular band of separation. This technique is also known as radial or circular paper chromatography.

Figure 02: Circular paper chromatography (Small circle at middle indicate place for wick or cotton immersed in solvent system while arrow point-out radially outward movement of mobile phase during chromatographic procedure)

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06.B. Vertical paper chromatography Vertical paper chromatography is further differentiated into two types;

06.B.1. Ascending paper chromatography: In involves mobile phase movement from bottom towards upper side of paper i.e. against gravity. 06.B.2. Descending paper chromatography: Mobile phase in this type of chromatography runs from upper end of paper towards lower end i.e. towards gravity. 06.C. Hybrid paper chromatography It is a combination sub-chromatographic techniques blanket under vertical paper chromatography. In this technique, paper impregnated with spot is exposed to ascending and descending chromatography in a sequential manner.

Figure 03: A-Ascending, B-descending, & C-hybrid (ascending-descending) paper chromatography 06.D. 2-Dimentional paper chromatography In this type of paper chromatography, the solvent system initially runs over the chromatographic paper in a manner analogues to that of ascending paper chromatography. After drying, another solvent of differential polarity runs on the same paper, but in a direction right angle to first. This ultimately increases efficiency of paper chromatographic by providing larger surface area for separation.

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Figure 04: 2-Dimentional paper chromatography (Note fractionation of sample spot into different components on subsequent chromatographic development) 07. Rf or R VALUE During chromatographic procedures, it is observed that components present in mixture does not travels with same instead different velocities i.e. some component travels with a velocity equal to the velocity of mobile phase thus moves along the solvent front while other component runs at a speed slightly or largely lesser than mobile phase velocity hence either remains at middle or near the baseline (origin of spot, commonly 1-2cm from bottom of chromatographic paper). This relative & differential distance traveled by individual component in a chromatographic system under same experimental condition is unique, constant, and can be used to establish their identification by comparing their R f value with Rf value of known component or standard. In terms of planner chromatography (paper chromatography) R f is also known as retention factor (retardation factor in column chromatography) which is defined as the ratio of distance traveled by centre of solute spot to distance traveled by solvent front from baseline. Sometime it is also expressed as R (somehow confusing also) and denotes overall resolution of mixture into their respective components in terms of their chromatographic movement.

Mathematically, Rf = Distance traveled by component from initial point/distance traveled by solvent front from same point.

Universally, Retention factor will never be less than 0 and more than 1. Solute particles having Rf value of 0 (zero) will have higher affinity for stationary phase, remains adhere to baseline while the solute particles having R f value of 1 will have highest affinity for solvent system and runs along with solvent front.

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08. FACTORS AFFECTING Rf VALUE Generally retention factor depends upon nature of stationary phase, thickness of stationary phase, mobile phase, its composition & viscosity, quantity of sample apply, method of spot application, size of spot, type of sample used, condition during experimentation, time run for experiment, temperature, and quality of paper employed. ( Note-Stationary phase here is denoting to chromatographic paper while in case of TLC it indicate adsorbent ). 09. IMPORTANCE of Rf VALUE Retention factor plays key role in identification of unknown compounds by matching their R f values with known one, also it can be used for determining structure of certain groups of compounds.

Chemicals

Rf value

Amobarbital

0.35

Butobarbital

0.18

Pentobarbital

0.47

Secobarbital

0.56

Chrysophenol

0.9

Rhein

0.0

Emodin

0.5 Table 04: Rf value of some synthetic & natural compounds

10.

APPARATUS & PROCEDURE

10.A. Apparatus Apparatus used for paper chromatography is rather simple and inexpensive comparatively with other chromatographic techniques. General laboratory glass wares made up of borosilicate glass are sufficient for performing most of the chromatographic procedures. However, under some circumstances bulky chromatographic chamber could be replaced (as per need) suitably with portable borosilicate beakers covered with air tight lid- may be a Petri disc. A simple paper chromatography experimentation (see figure 05) consist of a chromatographic chamber having an air tight lid, Whatman paper or other chromatographic paper as per requirement of procedure, paper holder, solvent system, and an spot analysing agent or instrument.

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Figure 05: Instrumentation of paper chromatography 10.B. Procedure Mold a suitable chromatographic paper into appropriate size or use commercially available pre-molded chromatographic paper, mark a line on it appropriately 1.5-2.0cm with a pencil from bottom and apply fine (2-10microlitre) uniform spot of sample along with reference compound adjacently (suitably spaced). Ideally, size of each spot should not be more than 45mm in diameter and should be at least 5-2cm apart depending upon number of spot applied on paper. Simultaneously, prepare a solvent system and transfer it into chromatographic chamber. Carefully placed spotted chromatographic paper into the chamber, supported with fine, but strong thread such that bottom of paper just immersed into the solvent system but spotted baseline should not. Allow solvent system to run at least ¾ though paper for better separation of individual components. Remove paper carefully, dry it appropriately, and locate the spot by suitable visualizing mean & analyse it quantitatively by different techniques ( Refer table 05). NOTE: Solvent system must be placed in chromatographic chamber several hours prior to analytical procedure so as to saturate the chamber with vapors of mobile phase.

A

Techniques for spot quantification Planimetric measurement

B

Excise spot weighing

Comparing developed dried spot with blank chromatographic paper of same area.

C

Square counting method

Trace spot over ruled graph paper & count number of square covered by spot; tedious method; accuracy up to 4-5%.

S.No.

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Remarks

Measure spot area with planimeter (accuracy-2%)



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D

Spot visual comparison

Determine concentration of unknown spot by comparing its color & intensity with known spot.

E

Spot length measurement

Log. Of drug concentration is directly proportional to logarithm of spot length.

Table 05: Methods in quantitative determination of spot 11. COMPONENT DETERMINATION Method used for determining or locating spot depends upon the nature of component to be analyzed. Colorimetric analysis is done either for self colored compounds or compound forms color when react with some specific chemical reagents (Refer table 05) such as ninhydrin or Ehrlich reagent and thus can be located by naked eyes. On other hand colorless compounds absorbing ultraviolet (U.V.) light are reveled in UV region of electromagnetic spectrum by irradiating them with light of 254nm. Likewise, fluorescence compounds are analyzed by irradiating them at a wavelength of 365nm, while radioactive compounds are suitably analyzed either by Geiger-Muller counter or autoradiography. Reagent

Chemical Constituent

Compound analyzed

Dragaendorff’s reagent

Potassium bismuth iodide

Alkaloids

Ehrlich’s reagent

0.5% dimethylaminobenzaldehyde in butanol

Intense yellow color with amines

Bratton-Marshall’s reagent

2.5% Sodium nitrite in 0.5% N-sulfuric acid

Aromatic amines gives reddish purple colour

Ninhydrin

Ninhydrin 0.1% saturated butanol

Amino acid gives purple color on heating

Bromocresol green

Bromocresol green 5%

Pauly’s reagent

Alcoholic solution diazotized sulfanilic acid

Ammonical silver nitrate

Equal volumes of 0.3N silver nitrate and 5N ammonium hydroxide

in

water

Fatty acid of

Phenols and polyphenols Carbohydrate brown black spot.

gives

Table 06: Chemical based colorimetric determination of components

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12. APPLICATION OF PAPER CHROMATOGRAPHY Paper chromatography is one of the most simplest and efficient technique available for qualitative determination of mixture for individual components. The technique can be used for separation of wide varieties of organic and inorganic compound (polar as well as non polar) irrespective of their origin like pigments, amino acid, protein, carbohydrate, nucleic acid, fatty acid, alkaloids, tannins, vitamins, saponins etc.

Owing to its simplicity and practically accessibility, paper chromatography still holds a valid position for separation of various pharmaceuticals product such as Capreomycin sulphate, Vancomycin hydrochloride, Vitamin A, sodium pertechnetate, Methotrexate injection by descending paper chromatography while ascending paper chromatography is done for sodium iodohippurate, sodium phosphate injection, brilliant green and crystal violet paints, sodium iodide preparations, and Ergometrine maleate. On other hand, Liothyronine sodium utilizes circular chromatographic technique. To a certain extent, technique of paper chromatography could be useful in forensic scene investigation & presence of various metal ions like mercury, cupper, cobalt, iron and silver etc. 13.

EXERCISE a. Give principle & application of paper chromatography.

b.

What are the various factors that have to be considered during selecting a solvent for paper chromatographic separation?

c.

Explain apparatus used in paper chromatography.

d.

Enumerate various types of paper chromatography. Give advantages of 2D paper chromatography over other paper chromatography techniques.

e.

Write a short note of sophisticated chromatographic paper and their applications.

f.

Explain components of paper chromatography with suitable examples.

g.

What is Rf value? Enlist various factors that affect R f value & give its importance in term of analysis.

h.

Enumerate various advantages and disadvantages of paper chromatography.

i.

Explain characteristic and application of Whatman paper grade 1, 2, 3, & 54.

j.

Write a brief note on spot detection methods used in paper chromatography.

k.

Enlist various method used in quantitative determination of chromatographic spot.

Chowrasia, Deepak



14. MULTIPLE CHOICE QUESTIONS Martin & Synge shares Noble prize in Chemistry for their contribution towards development of partition chromatographic technique in a. 1952 b. 1953 c. 1954 d. 1955 a Paper chromatography is based on the principle of a. Adsorption b. Absorption c. Partition d. None of the above c

In paper chromatography partitioning of solute takes place between two layers of a. Stationary phase b. Mobile phase c. Stationary phase & mobile phase d. None of the above b

c Generally, a chromatographic paper is made up of a. Amylase b. Cellulose c. Polymer d. None of the above b Choice of chromatographic paper in paper chromatography depends upon a. Nature of solute to be separated b. Solvent employed for separation. c. Flow rate of mobile phase. d. All of the above d

a

c

Chowrasia, Deepak

b. Liquid-liquid adsorption chromatography c. Liquid-liquid partition chromatography d. Solid-liquid partition chromatography

All purpose Whatman paper is a. Whatman paper number 01 b. Whatman paper number 02 c. Whatman paper number 15 d. Whatman paper number 17

Stationary phase in case of paper chromatography is a. Chromatographic paper b. Solvent system c. Layer of mobile phase entrapped into pores of chromatographic paper d. None of the above

Paper chromatography is a type of a. Solid liquid adsorption chromatography

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Acetylated chromatographic paper is used in a. Normal phase chromatography b. Reverse phase chromatography c. Both d. None of the above b



Radial paper chromatography is also termed as a. Vertical paper chromatography b. Horizontal paper chromatography c. Both d. None of the above b In ascending paper chromatography, mobile phase runs a. Towards gravity b. Against gravity c. Both d. None of the above b Most commonly used technique of paper chromatography is a. Ascending b. Descending c. Circular d. None of the above

In 2-dimentional chromatography, solvent used for second development is usually have polarity a. Same as that of initial development b. Different than that of initial development c. Can’t be predicted d. None of the above b Second development in 2-dimentional paper chromatography is done at an angle of _________ Degree. a. 70 b. 80 c. 90 d. 100 c

a In 2-dimentional chromatography, mobile phase runs in a direction from a. Periphery to centre b. Against gravity c. Towards gravity d. Centre to periphery

Rf in case of paper chromatography is also known as a. Relation factor b. Retardation factor c. Retention factor d. None of the above c

d Hybrid paper chromatography technique blanket under a. Vertical paper chromatographay b. Horizontal paper chromatography c. Circular paper chromatography d. None of the above a

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Rf ranges from a. 0 to 1 b. Less than 0 c. 1-10 d. None of the above a A component having R f value of 0.9 is found ________ in chromatographic paper chromatography a. Right on base line b. Slightly above base line c. Near about solvent front d. At middle of paper chromatography c



Can Rf is used for structure determination of a chemical compound? a. Yes b. No c. May be d. None of the above A Factor that affect R f value is/are a. Spot size b. Temperature c. Nature of mobile phase d. All the above

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c. Fat d. Carbohydrate a,b Liothyronine sodium can be analyze by _________ type of paper chromatography a. Horizontal b. Vertical c. Circular d. None of the above c

D Ninhydrin solution is used for spot determination of a. Amino acid b. Proteins

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Ammonical silver nitrate solution used for spot determination of a. Amino acid b. Proteins c. Fat d. Carbohydrate d


Chapter – 07 THIN LAYER CHROMATOGRAPHY - Deepak Chowrasia

Chowrasia, Deepak

THIN LAYER CHROMATOGRAPHY


101

THIN LAYER CHROMATOGRAPHY (Chapter Overview) 01. INTRODUCTION .................................................................................................................. 103 02. PRINCIPLE ............................................................................................................................ 103 03. ADVANTAGES OF THIN LAYER CHROMATOGRAPHY .................................... 103 04. COMPONENT OF THIN LAYER CHROMATOGRAPHY ..................................... 104 04.A. Stationary phase .........................................................................................................104 04.A. Mobile phase ................................................................................................................ 105 05. PREPARATION & MONITORING OF TLC PLATE ................................................106 05.A. Slurry preparation and spreading ........................................................................... 106 05.B. Activation of TLC plates ............................................................................................ 106 05.C. Spotting on TLC plate or sample application ....................................................... 107 05.D. Chromatogram development ....................................................................................107 05.E. Post chromatographic evaluation of TLC-plate ...................................................107 05.e.i. Spot detection ..................................................................................................107 05.e.ii. Evaluation of chromatogram ......................................................................... 108 05.e.ii.A. Qualitative analysis ....................................................................... 109 05.e.ii.B. Quantitative analysis ....................................................................109 06. APPLICATIONS ...................................................................................................................109 07. EXERCISE .............................................................................................................................. 110 08. MULTIPLE CHOICE QUESTIONS ................................................................................111

Chowrasia, Deepak


THIN LAYER CHROMATOGRAPHY 01. INTRODUCTION Advancement in the field thin layer chromatography (TLC) has leveled it up in the quantitative and qualitative analytical procedures. The technique of thin layer chromatography was initially demonstrated by Kirchner in 1950, but fundamental and elaborated work of E. Stahl on natural product ultimately established universal acceptance of this technique. Further in 1958, Stahl introduced an instrument for preparing TLC plate of uniform thickness thus establishing it importance further as a novel analytical tool. Thin layer chromatography provides a low cost, effective analytical technique for separation of wide varieties of substance without expense on any sophisticated instrumentation. Modernization in the field of automation and improvement in quality of adsorbents leads to emergence of highly improved version of TLC known as “high performance thin layer chromatography” or HPTLC which is a fully automated and highly versatile technique for separation of not only synthetic chemicals, but also vast range phytochemicals obtained from natural resources. 02. PRINCIPLE Principle involved in thin layer & paper chromatography is somewhat same since both are planner open bedded technique based on the phenomenon of partitioning as well as adsorption (partition predominate in paper chromatography while adsorption ruled out in TLC) of solute between stationary & mobile phase. In paper chromatography separation of solute takes place on piece of chromatographic or Whatman paper while in case of thin layer chromatography solid adsorbent (Silica or alumina) of unvarying particle size coated uniformly (0.1-2mm thickness) over a rigid support such as glass slide or Aluminium foil acting as stationary phase. Separation of mixture into its individual components, in this type of chromatographic technique, depends upon their differential affinity towards stationary as well as and mobile phase. Component of mixture having high affinity for stationary phase get retained on TLCplate and moves at a velocity comparatively lesser than a component having high affinity for mobile phase thus get separated out. 03. ADVANTAGES OF THIN LAYER CHROMATOGRAPHY TLC poses numerous benefits over conventional paper & column chromatographic technique as;

a. It is simple and elegant technique. b. Easy sample preparation and its application. c. Sample can be run multiple times without causing any damage to TLC plate. d. Less time consumption in terms of development of plate.


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e. Simultaneous detection of sample during processing. f. Required less quantity of sample as well as solvent system. g. Sample preparation is somewhat easier. h. TLC plates can be used numerous times after efficient washing. i.

Wide varieties of samples are easily handled irrespective of their physiochemical properties.

j.

Comparatively good resolution.

k. Detection of plates (TLC) is easier, simple, and multiple detections can be applied to single plate. l.

Less costlier, efficient and simplest technique required no elaborated instruments.

04. COMPONENT OF THIN LAYER CHROMATOGRAPHY Basically it consist of following two components

04.a. Stationary phase 04.b. Mobile phase 04.a. Stationary phase Physically, stationary phase in case of TLC is a solid (liquid phase-paper chromatography) of smooth texture, containing adsorbent particles of small and uniform dimensions (1-5mµ), thus providing larger surface area for efficient separation of mixture into their respective components. Selection of stationary phase depends upon nature of compound to be separated, their physiochemical properties such as solubility, nature of functional group, reactivity towards adsorbent (stationary phase) & solvent system (mobile phase), including compatibility with binder (gypsum-rarely or calcium sulphate-commonly, or starch). Stationary phase ( Refer table 01), usually consist of a thin layer of suitable adsorbent of uniform particle size (10-40 micrometer) and ranges from normal Silica gel-G (bounder in case of reverse phase TLC), alumina-G, Kieselguhr-G (G-indicate presence of binder mostly calcium sulphate 10-12%) to most recent and highly advance microcrystalline cellulose mixed with calcium sulphate or starch coated on a rigid & inert backing material like glass slide, plastic plates, or aluminum foil. The coating material sometime may contain fluorescent (zinc silicate) material which fluoresces on exposure to radiation of suitable wavelength. Acidic sorbents like silica gel (activated at 105-110ºC) is used for separation of basic compounds while Alumina (dried at 200-500ºC), a basic sorbent employed for fractionation of acidic substances. 04.b. Mobile phase There is no thumb rule for selecting mobile phase for thin layer chromatograph as there are wide varieties of solvent are available, ranging from single pure solvent to mixture of solvents

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in some definite proportion. Selection of an ideal solvent system mostly adopted on the basis of trial and error method which in turns depends upon nature of substance used for separation and ability of a solvent to fractionate mixture into their individual constituents. It should however be noted that what so ever the solvent system is opt for separation, it must not cause any sort of chemical degradation of analyte under examination. It is better to avoid solvents which are hazardous to environment and human system.

Adsorbent Inorganic adsorbent Aluminum silicate Aluminum oxide ore (Bauxite) Bentonites Calcium carbonate Calcium hydroxide Calcium oxalate Calcium silicate Calcium sulphate Dicalcium phosphate Fuller earth Hydroxyl apatite Magnesium silicate Silica gel Tricalcium phosphate Anhydrous calcium sulphate Organic adsorbent Dextran gel Charcoal & activated carbon Sucrose Polyethylene powder polyamide

Analytical Use Sterols and glycosides Ergot 2,4-dinitrophenyl hydrazone of aldehyde and ketones, vitamins, sterols Napthaquinones and Xanthophylls Carotenoids Anthraquinones Carbohydrates and their phenylosazones Lipids and steroids Carotenes Amino acids Glycerides and proteins Esters, steroids, glycosides, lactones Fatty acids, sterols, glycerides, amino acids, sugars etc Enzymes Azobenzenes and their derivatives Protein and nucleic acid Sugar amino acid Chlorophylls and xanthophylls Esters and fatty acid Flavanoids

Table 01: List of adsorbents used in thin layer chromatography

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TLC can act as a better medium for separation of non-ionic molecules soluble in organic solvents. On other hand, polar non-ionic compounds are suitably separated either by reverse phase or bonded phase chromatographic technique. As per rule, normal phase TLC utilizes polar solvents while non-polar organic solvents plays pivot role in reverse phase TLC. Petroleum ether, ethanol, methanol, diethyl ether, chloroform, acetone, dimethylformamide (DMF), water, pyridine, carbon tetrachloride, n-hexane are some of most commonly employed solvents use either singly or in combination of specific proportion with each other in separation process during TLC. 05.

PREPARATION & MONITORING OF TLC PLATE

05.a. Slurry preparation and spreading High quality pre-coated plates are available commercially and can be satisfactorily used for experimentation alternatively plates can easily be prepared manually in laboratory with no aid of external cost. Basically, laboratory manual method of TLC preparation requires mixing of adsorbent and solvent in a ratio of 1:3 (quantity can vary as per requirement and procedure) in a pestle-mortar till a slurry of optimum consistency is form (very thin or thick slurry is problematic) which is then either transfer to spreader/applicator (Stahl’s applicator) for spreading over glass slide or suitable rigid material.

However, alternatively, the same can be poured manually over supporting material by moving hand backward and forward direction till a uniform layer of adsorbent forms over whole plate. Besides above mentioned techniques, TLC plate can also be formed by following any one of the following methods I.

Immersing plate in a suspension containing adsorbent.

II.

Spraying adsorbent over the plate.

III.

Spreading adsorbent over plate with the aid of glass slide.

Anyhow, what so ever the methodology opted, fine layer of adsorbent (250m µ) must be formed for accurate & precise fractionation of solute during procedure. For manual method (hand pour method) it is recommended that solvent content of slurry is slightly higher than any other spreading methodology. Often water is used as solvent for making Silica gel slurry while acetone replaces water for preparing slurry of cellulose. 05.b. Activation of TLC plates It is essential to remove even a minute quantity of water from TLC plate as it causes interference with normal chromatographic procedure. Activation of TLC plates can be conveniently done by keeping freshly prepared air dried plate in hot air oven for 1-hour (vary literature to literature) at 100-110 degree Celsius. Not only prepared but commercially

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available pre-coated plates are also recommended to get activated in the same manner for better result prior to their use. In order to get better result plates can be activated at higher temperature 150-170 degree Celsius for 3-5 hours strictly depending upon the type of adsorbent used. Once the activation process is over, removes plate and kept in desiccator. 05.c. Spotting on TLC plate or sample application Sample (2-20µL) must be spotted on origin line located approximately 2-2.5cm from bottom and distance between two spots must be 2-3 cm depending upon dimension and size of plate. For quantitative analysis accuracy and precision of sample spot is very essential.

Solvent employed for preparing sample and standard must be clean, pure and volatile so that it can be easily evaporated after application of spot. Spotting can be done with any suitable instrument like fine needle of syringe, micropipette, fused capillary or any other suitable device with maximum emphasis on smaller spot area for getting sharp and accurate result. It is highly recommended that second spotting (if required) should be done only when first one is dried properly. 05.d. Chromatogram development Ascending techniques (sometime or rarely descending technique) are mostly used for development of TLC plate in which plate is immersed in solvent system such that origin line (base line) containing spot should not comes in direct contact with mobile phase. Spotted plate is then kept in a closed chromatographic chamber (at 60º angle) of suitable size, depends upon size and number of plate used for development. In order to saturate chromatographic chamber with solvent system a rolled or planner piece of filter paper impregnated with solvent system is lined up with the wall of chamber prior to running the plate. Solvent system or mobile phase is allowed to run on the TLC plate for sufficient th distance to ensure fine resolution of components (3/4 or more of total length of TLC plate, but not out of plate). Once plate is get developed, it is removed from chamber, solvent front is marked carefully with pencil, and developed plate is then allowed to dry either at room temperature if component to be analyzed is head liable or in hot air oven for thermostable components. 05.e. Post chromatographic evaluation of TLC-plate Post chromatographic evaluation of TLC plate involves two main procedures

05.e.i. Spot detection. 05.e.ii. Evaluation of chromatogram 05.e.i. Spot detection Visual or colored component like carotenes, xanthophylls, chlorophylls can be easily detected with naked eye without aid of any external visualizing agent while invisible (colorless)

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substances such as amino acid, alkaloids, fatty acid can be detected visually either by irradiating them with UV-light of suitable wavelength or alternatively by imposing a chemical reaction between colorless component and visualizing agent thus enabling their detection. (Please refer table 06 given in chapter on paper chromatography).

Figure 01: Instrumentation of thin layer chromatography

TLC plate containing radioactive or isotopic compounds can be detected either by autoradiography or by Geiger Muller chamber.

Figure 02: Diagrammatic illustration of pre & post developed TLC plates

05.e.ii. Evaluation of chromatogram Chromatogram can be evaluated for both qualitative and quantitative purpose as

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05.e.ii.A. Qualitative analysis

It includes determination of quantities like R f value and Rst value and comparing their data with standard. A. Rf value or retention factor

In terms of TLC Retention factor depends upon various parameter like thickness of adsorbent layer, quality of adsorbent, type of solvent system, temperature during process, maintain of equilibrium in chromatographic chamber, type of chromatographic technique employed, area of spotting including quantity of sample applied, presence of impurities etc. R f value of test spot (unknown) can be compared with R f value of standard for its identification (for mathematical expression and other discussion please refer chapter on paper chromatography) B. Rst value: It is a newer relation; ultimately obsolete all the possibilities or factors affecting normal chromatographic process. R st can be defined as distance traveled by substance to distance traveled by standard. Unlike R f value, Rst may be more than 1.

Mathematically Rst=Rf of sample/Rf of standard 05.e.ii.B. Quantitative analysis

Direct quantitative analysis of TLC plate includes measurement of spot area, color intensity determination with an aid of densitometer, and characterization of individual spot by chromatogram spectrometer. On other hand, indirect quantitative analysis includes elution of spot followed by eluate analysis with any one or combined instrumental method like colorimetry, radiometry, UVspectroscopy, polarography, fluorimetry, vapour phase chromatography etc. 06. APPLICATIONS Thin layer chromatography plays an important role not in the field of chemistry but also in other specialized area of science including molecular biology, forensic study and pharmaceutical sciences. Owing to the availability of wide variety of adsorbents TLC can be effectively used for separation of compounds ranging from pure synthetic products to biologically derived molecules, and metabolites. Some of the renowned facet of TLC includes separation of synthetic products including isomers, carbohydrate, proteins, amino acid, steroids, vitamins, nucleic acid, alkaloids, fixed oils etc. Relative comparison of Rf values of unknown component with known (standard) component aids up in former identification. It is highly recommended that before final judgment, confirmatory test must be performed for the same.

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TLC plays an important role in quality control of pharmaceutical substance for determination of impurities in variety of therapeutic importance substance like presence of hydrazine in carbidopa a drug used for Parkinsonism disease or detection of morphine in apomorphine hydrochloride.

Compounds

Cascara tablets Pentagastrin Dichlorophen Chlorpropam ide

Solvent system

Ethyl acetate;methanol;water Ether:glacial acetic acid:water Toluene Chloroform:methanol:cyclohex ane:ammonia

Ratio of solvent system 100:17:13

10:2:1 100 100:50:30:11.5

Emetine

Chloroform:methoxyethanol:me 100:20:5:2:0.5 thanol:water:diethylamine Amitriptyline Carbon tetrachloride:toluene 3:7

Impurity detected

Frangula Foreign substances p-chlorophenol 4-chlorobenzene sulphonamide and NN-dipropylurea Other alkaloids Ketone

Table 02: Some compounds and their solvent system

It is a well adopted technique for optimization of solvent system for other chromatographic procedures, determination of completeness of synthetic chemical reactions, monitoring of column chromatography, and determining effectiveness of purification process. 07.

EXERCISE a. Explain briefly principle involved in thin layer chromatography.

b.

Differentiate between paper and thin layer chromatography.

c.

Write a short note on stationary phase used in TLC.

d.

Enumerates various advantages of TLC.

e.

Enlist various adsorbent and tabulate their analytical uses.

f.

Give a brief account on preparation and development of TLC plates.

g.

Diagrammatically, explain instrumentation of thin layer chromatography.

h.

Write a short note on qualitative & quantitative evaluation of developed chromatographic plates.

Chowrasia, Deepak



i.

08.

111

Explain briefly a).

Rf value

b).

Rst value

j.

Enumerate various direct and indirect method used for quantitative analysis of post developed TLC plates

k.

Give a brief outline chromatography.

regarding

various

applications

of

thin

layer


The technique of thin layer chromatography was initially demonstrated by a. Kirchner in 1950 b. Kirchner in 1951 c. Kirchner in 1952 d. All are correct a Popularity of TLC plate among scientific community enhanced due to a. Work of Kirchner b. Work of Stahl c. Both d. None of the above b An advanced version of TLC in terms of stationary phase particle size is a. HPLC b. HPTLC c. TLC itself d. None of the above b Expand TLC and HPTLC a. Thin layer chromatography & high performance thin layer chromatography

Chowrasia, Deepak

b. Thick layer chromatography & high performance thick layer chromatography c. Thin layer chromatogram & high performance thin layer chromatogram d. None of the above a In paper & TLC chromatography stationary phase is a. Solid phase & liquid phase b. Solid phase & solid phase c. Liquid phase & liquid phase d. Liquid phase & solid phase d Paper & TLC chromatographic techniques are based on principle of a. Adsorption & absorption b. Absorption & partition c. Partition & adsorption d. Adsorption & partition c Most commonly used adsorbent in TLC is a. Silica gel b. Calcium carbonate c. Alumina d. All the above a



Particle size of adsorbent used in TLC is a. 1-5mµ or 10-40 micrometer b. 1-5mµ or 40-80 micrometer c. 10-50mµ or 10-40 micrometer d. None of the above a

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Alumina is _____ in nature thus used for fractionation of _________ substances. a. Acidic, Acidic b. Basic , Basic c. Acidic, basic d. Basic, acidic d

Silica gel G, alumina G, & Kieselguhr G are some of the adsorbents used in TLC, here G indicate a. Gravity b. Granularity c. Binder d. None of the above c

Sometime, coating material may contain fluorescent like a. Zinc silicate b. Zinc sulphate c. Zinc phosphate d. Zinc carbonate a

Most commonly used binder used along with stationary phase in TLC is a. Alumina b. Calcium carbonate c. Calcium sulphate d. Starch c In the above question, pick out stationary phase a,b At what percentage, calcium sulphate is act as an ideal binder in TLC a. 8-10% b. 10-12% c. 12-14% d. 14-16% b Silica gel is _____ in nature thus used for fractionation of _________ substances. a. Acidic, Acidic b. Basic , Basic c. Acidic, basic d. Basic, acidic c

Chowrasia, Deepak

Activation of TLC plate means a. Making smooth surface b. Placing plate at an angle of 80 degrees c. Removing moisture from plate d. None of the above c Silica and alumina plates are activated as temperature a. 105-110ºC, 200-500ºC b. 105-110ºC, 10-110ºC c. 90-110ºC, 100-110ºC d. None of the above a Silica gel is an a. Organic adsorbent b. Organic + inorganic adsorbent c. Can’t be predicted d. Inorganic adsorbent d



Solvent selection in TLC can be done on the basis of a. Nature of component to be handled b. Their physiochemical properties c. Both d. None of the above c Normal phase TLC, makes use of a. Polar solvent b. Non polar solvent c. Both d. None of the above a Stahl’s applicator is a device use in a. HPLC b. UPLC c. TLC d. None of the above c Function of Stahl’s applicator is a. As a drying instrument b. As a TLC plate preparation instrument c. Both d. None of the above b What is the size of adsorbent layer in TLC plate a. 150mµ b. 250mµ c. 350mµ d. 450mµ b

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TLC plates can be activated at 105-110ºC for a time period of a. 60 minutes b. 40 minutes c. 20 minutes d. None of the above a Amount of sample to be spotted over TLC plate is a. 20-200µL b. 0.2-20µL c. 0.02-0.2µL d. 2.0-20µL d Pick out correct sentence a. Rf value range from 0-1 while Rst can be more than 01. b. Rf value range from 0-1 while Rst can be less than 01. c. Rf value can be less than 01 while Rst be always be more than 01 d. None of the above a Densitometer used in post TLC development as a. Visualizing agent b. Color intensity determining agent c. Both d. None of the above b


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COLUMN CHROMATOGRAPHY (Chapter Overview) 01. INTRODUCTION .................................................................................................................. 119 02. PRINCIPLE ............................................................................................................................ 119 03. COMPONENTS OF COLUMN CHROMATOGRAPHY ...........................................120 03.A. Chromatographic column ........................................................................................ 121 03.B. Stationary phase .........................................................................................................121 03.B.I. Characteristics of a stationery phase ............................................................ 121 03.B.II. Types of adsorbents ........................................................................................ 121 03.C. Mobile phase ................................................................................................................ 122 04. FACTORS AFFECTING RESOLUTION IN COLUMN CHROMATOGRAPHY ..................................................................................123 04.A. Dimensions of column................................................................................................ 123 04.B. Adsorbent particle size .............................................................................................. 123 04.C. Solvent system ............................................................................................................. 124 04.D. Temperature ................................................................................................................. 124 04.E. Solvent flow rate .......................................................................................................... 124 04.F. Column Packing ........................................................................................................... 124 04.G. Time to run ...................................................................................................................124 04.H. Concentration of sample ............................................................................................ 124 05. INSTRUMENTATION & PROCEDURE ....................................................................... 125 05.A. Column preparation ...................................................................................................125 05.B. Sample addition .......................................................................................................... 125 05.C. Chromatogram development ....................................................................................126 05.D. Component collection .................................................................................................126 05.E. Component detection ..................................................................................................126 05.E.1. Determining number of components in sample: .........................................126 05.E.2. Detection of individual components: ............................................................ 126 06. APPLICATIONS ...................................................................................................................126 07. ADVANTAGES & DISADVANTAGES OF COLUMN CHROMATOGRAPHY ..................................................................................127 08. EXERCISE .............................................................................................................................. 127

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“Separation Under Tunnel” 01. INTRODUCTION Serendipitous, phenomenon of column chromatography was initially gripped by Russian Botanist Mikhail Semyonovich Tsvet also known as Mikhail Semyonovich Tsvett in 1906, during his experimentation on filtering petroleum ether extract of chlorophyll in a column containing calcium carbonate as a matrix. He observed, with increase in quantity of petroleum ether added to column, a series of color bands separate out and descend down the column at different rate thereby separating chlorophyll pigment into distinct zones. He names the column as chromatogram and technique as chromatography. Since from then, chromatographic techniques upgraded not only in terms of analyte handling but also adopted ever changing technology in the field of instrumentation, and present itself as ascendant analytical tool. Column chromatography provides good mean for efficient separation and purification of sample both at small as well as large scale without any elaborated instrumentation. 02. PRINCIPLE The principle applied in column chromatography is analogous to that of thin layer chromatography (TLC) since in both of them “ adsorption” plays an dominating role in separation of mixture into their individual components. However, adsorption in case of TLC is perceived on an open planner surface composed of silica gel or any other suitable adsorbent (for adsorbent list, please refer table 01 of thin layer chromatography) rested against strong backing material (glass or Aluminum foil), while the same phenomenon i.e. adsorption in case of column chromatography occurred inside a column packed with uniform sized matrix acting as a stationary phase. Since overall process of separation in later case takes place interior of a column that why this technique of chromatography is called as column chromatography. The rate at which mixture separates out into their respective components depends upon relative affinity of mixture’s components towards stationary as well as mobile phase. Component having higher affinity for stationary phase moves at a slower rate along the length of column comparatively to a fast moving component having lower affinity against stationary, but higher for mobile phase thus get separated out. It is highly recommended that before developing column, adsorbent must be well saturated with solvent of lower polarity and during fractionation, polarity of solvent increases slowly in a stepwise manner for better separation of mixture into their respective components. Rate of component movement inside the chromatographic column can be denoted as “ R” which express as ratio of rate of movement of component to rate of movement of mobile phase. This can alternatively also be expressed as ratio of distance traveled by solute molecules to the ratio of distance traveled by solvent molecules. However, in presence of liquid mobile phase, mathematically, the value of “ R” could be expressed as

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Where, R=Rate of movement of component inside chromatographic column Am=Average cross section of mobile phase =Partition coefficient

α

As=Average cross section of stationary phase

Figure: 01 Principle of column chromatography (A-mixture; B-initial column development with less polar solvent; C-increase in solvent polarity, enhanced separation of polar components; D-more polar compounds (grey color) get separated out, followed by lesser polar (brown & green color), and finally non polar compounds-blue color) 03.

COMPONENTS OF COLUMN CHROMATOGRAPHY 03.A. Chromatographic column

03.B. Stationary phase 03.C. Mobile phase

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03.A. Chromatographic column Chromatographic column is a hollow, transparent (inert glass) or opaque (stainless steel), tubular shaped structure of variable length (5cm to 1 meter) and diameter (5mm to 50mm) used for handling of sample during separation or purification process. On the basis of availability & suitability of process (quantity of analyte to be handle), a chromatographic column can be ranges from laboratory pasture or calibrated glass pipette to lengthy industrial columns used for purification of chemical on a large scale. Make of column should be such that its one end is wider for ease of sample & solvent entry while other end is sufficiently narrower for uninterrupted, but controlled exit of purified product. As per general rule, length to diameter ratio of separating column should ideally be 10:1 for preparative work while slightly more than this for analytical purposes. Normally, longer columns are preferred over shorter one for separation of multi-component mixture or a mixture containing components with nearby relative affinity against column matrix. However, shorter column favors separation of mixture having components with larger differential affinity against adsorbent. Generally, wider column gives bad resolution compare to narrower one. 03.B. Stationary phase

03.B.I. Characteristics of a stationery phase A wide range of adsorbents are used as solid stationary phase (matrix) in column chromatography. Ideal adsorbent is still a need for present era, some of the most acceptable & dominating qualities including characteristics a stationary phase reckon is their chemically inertness, optimum mechanical strength, insolubility in eluting medium, uniform particle size, optimum activeness, colorless, easy availability, cost effectiveness and suitability for allowing optimum flow rate through its bed. 03.B.II. Types of adsorbents Basically, wide varieties of adsorbent are commercially available ranging from weak adsorbent (starch, talc, sucrose, inulin) to medium (calcium hydroxide, magnesium oxide, magnesium carbonate, calcium carbonate) up to strongest one (activated silica also known as magnesium silicate-commonly used, alumina, charcoal, fuller’s earth, magnesia) having highest adsorbing capacity. Careful study must be done prior to selection of a stationary phase for separation, since an incompatible stationary phase may cause degradation, decomposition, hydrolysis, isomerization, or even excessive binding with component leading to their poor resolution and separation. Silica and alumina, if employed as stationary phase, must be used only after their activation by exposing them to a temperature of 150-200 degree Celsius for satisfactorily results. Commonly used adsorbents in column chromatography includes silicic acid (silica gel used for adsorbing unsaturated and polar compounds), alumina, bentonite, powdered charcoal, sodium carbonate, magnesium carbonate, lime, inulin, talc, sucrose, and fuller earth.

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Particle size (Refer table 01) of adsorbent or stationary phase plays an important role in separation process since smaller particle size enhance surface area and thus provides better resolution. It must be noted that any rigorous reduction in particle size of adsorbent ultimately reduces net flow rate of mobile phase through column bed and sometime even cease solvent flow thereby interpreting fractionation process. Anyhow, this problem can be effectively tackled by use of suitable filter aids like Celite 503, Celite 545, or alternatively by Hyflow Super-Cel which ultimately makes column more permeable for mobile phase. Adsorbent

Particle size in micron

Fuller’s earth

3.0

Alumina

7.0

Magnesium oxide

1.5

Hydrated calcium sulfate

10.5

Calcium carbonate precipitated

1.5

Table 01: Particle size of adsorbent commonly used in column chromatography 03.C. Mobile phase Mobile phase or eluent used in column chromatography must be of analytical grade and free from impurities that may interfere with normal chromatogram development. Either a single or binary solvent system with different polarities (gradient) is a choice. However, non-polar solvents like benzene or petroleum ether gives strongest adsorption and can be used singly (alone) for developing chromatogram. The phenomenon of separation can be accelerated by increasing the polarity of eluent by slow and steady addition of second polar solvent to first non-polar solvent. In most of the laboratory columns, solvent or eluent runs under the influence of gravity which ultimately extends separation time. However, attaching solvent pump or a gas compressor pushes the solvent against adsorbent bed thereby reducing considerable separation time. It is necessary to maintain constant flow rate of solvent through column bed during whole of the process without any disruption. However, any interruption in solvent flow thus causes column cracking (entrapment of air) leading to bad resolution. Both polar as well as non-polar solvents are used in column chromatography. Nevertheless, petroleum ether, carbon tetrachloride, cyclohexane, ether are preferred as non-polar solvents while water, acetonitrile, and alcohols are front lined in case of polar solvents.

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Solvent (lower to higher polarity)

Petroleum ether Carbon tetrachloride Cyclohexane Carbon disulfide Diethyl ether Acetone Benzene Toluene Esters Chloroform Alcohols Water Pyridine Organic acid Table 02: Common solvent system for column chromatography 04. FACTORS AFFECTING RESOLUTION IN COLUMN CHROMATOGRAPHY There are various factors that affect efficiency of column like 04.A. Dimensions of column Numerous types of columns are available commercially depending upon the quantity of sample to be handled and process adopted for separation. Before selecting a column, optimization should be done. As per general rule,

a.

A narrow bored column gives far better separation then wider bored columns.

b.

A longer column gives better resolution than shorter column, but this is none of a hard and fast rule. Optimum length of column should be selected as per the nature of component to be separated. For example a longer column should be selected for mixture containing multiple components or for separation of mixture containing components having same or nearby relative affinity towards adsorbent, while a short column should be selected for component having greater difference in their affinity towards adsorbent.

04.B. Adsorbent particle size Particle size (refer table 02) plays an essential role in separation. A uniformly packed homogeneous particle in column gives better separation than irregularly packed

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heterogeneous particles. Lesser the particle size better is the separation, but excessive reduction in particle size beyond a limit creates chocking problem due to higher resistance offered by smaller particle of matrix system. 04.C. Solvent system The solvent system employed for elution should be pure and does not cause any short of chemical degradation or decomposition with any components of sample along with stationary phase. As per the requirement, homogenous or heterogeneous mobile phase of suitable low viscosity, good flow-ability, and optimum polarity must be selected for elution. Since, viscosity alters solvent flow ability thus resolution therefore a low viscous solvent must be preferred for better elution. 04.D. Temperature Usually most of the column chromatographic procedures give good resolution at room temperature itself and mostly any minute changes in temperature does not cause any hindrance with normal separation process. As per need, under some circumstances, slight increase temperature can be maintained, which ultimately enhance separation process. However, an excessive temperature enhancement is contraindicated while dealing with heat labile components such as proteins and vitamins. 04.E. Solvent flow rate During overall process of separation an optimum flow rate of solvent through adsorbent bed must be maintained without any interruption or gap. Cracking of column during fractionation procedure gives poor results which could be minimized by maintaining a solvent head over the matrix bed. For good resolution, it is recommended to maintain medium flow rate which ultimately separates mixture into individual components at a good distance apart, but at a cost of greater time consumption. 04.F. Column Packing Regular packed column gives good resolution than an irregularly packed one. Effective degassing prior to development must be done to ensure better resolution Also column must be run blank with at least 50-100ml of eluent before developing chromatogram. 04.G. Time to run A shorter run time will gives poor separation than longer one as time of contact of sample between stationary and mobile phase reduces. 04.H. Concentration of sample Concentrated sample or large quantity sample need a long column and takes more time to separate than diluted sample or sample containing fewer numbers of components.

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INSTRUMENTATION & PROCEDURE

05.A. Column preparation A suitable sized clean chromatographic column is selected mounted tightly & vertically with column stand. A plug of cotton or glass wool is pressed down suitably inside column till its reaches the point few centimeters above the bottom, some quantity of previously washed and dried sand (if available) is then passes over plug forming a thin film over it. Slurry of adsorbent (prepared by mixing adsorbent with solvent) is then poured into the column (previously containing solvent) and allows to get settled. Once slurry is settled a disc of filter paper or cotton plug is placed over it to prevent any disturbance caused by addition of fresh solvent during chromatogram development. Drain out excess solvent if present, but always maintain a solvent head over the adsorbent. Before developing a chromatogram at least run 50-100ml of solvent at a rate of 10-15ml per minute.

Figure 02: Typical instrument for column chromatography 05.B. Sample addition Sample can be added into column either in liquid or solid form. Liquid sample can be drained directly into column while solid sample in small amount introduced into the column by placing it over previously placed filter paper or cotton plug and covering it with another set of filter paper or cotton plug.

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05.C. Chromatogram development Chromatographic column can then be developed by slowly, but continuous adding of mobile phase initially of lower polarity and then increasing its polarity in a stepwise steady manner. During whole of the procedure “solvent head” of at least 1-2cm must be maintained over the adsorbent bed and under no circumstances cracking of column is allowed. 05.D. Component collection Collect eluate in an appropriate receiver and analyze the component as per appropriate methodology. From the purity stand point of view, it is desired to collect column eluate in smaller fractions (5-10ml) and analyze each of them for desired components 05.E. Component detection

05.E.1. Determining number of components in sample: 05.E.1.a. Frontal & displacement analysis: This is done in order to determine number of components present in mixture & their affinity towards stationary phase. In frontal analysis, mixture to be analyzed is eluted through adsorbent bed of column. The mixture component having lesser affinity towards stationary phase comes out first while greater affinity component eluted lastly. On other hand, component emerging in between first and last will have intermediate affinity for stationary phase. Displacement analysis involved column development in a manner same as that of frontal analysis instead mobile phase used for elution in this case contains an additional substance having higher value of affinity towards stationary phase compare to components present in the sample. 05.E.2. Detection of individual components: Visual detection, rather than a simplest method, helps in detection of only those components which are itself colored in nature or form suitable visible color when treated with visualizing agent. For example sugars form color when treated with p-phenylazobenzoyl chloride. Addition of colored indicator can also be used to study the down flow pattern of components moving through chromatographic column. Instrumental methods like UV irradiation, change in conductivity, pH, or refractometry is used for analysis of components which are colorless thus can’t be monitored or determined by any of visual methods mentioned above. 06.

APPLICATIONS 1. A versatile method for separation and purification of large number of synthetic as well as biologically origin chemicals such as Phenacetin, Phenobarbital, diphenylhydantion, amino acid, alkaloids, glycosides etc.

2. As a part of routine purification process in synthetic organic laboratories.

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3. To study pharmacokinetic pattern of drug in human system & isolation of various metabolites from body fluids. 4. Cold extraction of various phytochemicals like alkaloids & components of chlorophyll. 5. Determination of quinine and strychnine from elixirs. 6. Estimation of active ingredient in various formulations like, phytomenadione in tables & injections, flucinolone actinide in creams etc. 7. Separation of tautomers, diasteriomers, and racemates of chemical compounds. 07. ADVANTAGES & DISADVANTAGES OF COLUMN CHROMATOGRAPHY Column chromatography is an efficient mean of separating varieties of mixtures irrespective of their quantities (microgram to gram). Selection of wider range of mobile phase, efficient handling of sample, recycling of solvent (may or may not be achieved) excellent purity of end product, availability of wider range of adsorbent, least space consumption, requirement of non-costlier instrumentations are some of the advantages of this technique. However, automation in column chromatography ultimately enhances its utility but at also make the technique expansive & complicated. Time consumption, requirement of large quantity of organic solvents, and greater cost of separated components are some of its limitations. 08.

EXERCISE 1. Elaborate with principle method of column chromatography

2.

Write a short note on components of column chromatography

3.

What are the various factors that affects separation process in column chromatography.

4.

List the use of filter aid in column chromatography.

5.

Outline briefly along with labeled diagram instrumentation of column chromatography

6.

Write a note on component detection in case of column chromatography

7.

Explain briefly Frontal & displacement analysis.

8.

Give applications of column chromatography.

9.

Enumerate various advantages and disadvantages of column chromatography.

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09. MULTIPLE CHOICE QUESTIONS

Mikhail Semyonovich Tsvett in ________ describes phenomenon of column chromatography a. 1906 b. 1907 c. 1908 d. 1909 a In above question, what extract did Mikhail Semyonovich Tsvett is dealing with a. Carotene b. Alfa- carotene c. Chlorophyll d. None of the above c Why this technique of chromatography is termed as column chromatography a. Since it deals with tunnel digging b. It handle column shaped compound for purification c. Separation of chemicals occurred inside column chromatography d. None of the above c Principle of column chromatography is a. Partition b. Adsorption c. Absorption d. None of the above b Stationary phase in case of column chromatography is a. Chromatographic column b. Solvent system c. Adsorbent bed d. None of the above c

Chowrasia, Deepak

Pick out correct option a. There is no need to saturate a column before its development b. Column must be saturated prior to development c. Column development initially started with low polarity solvent d. None of the above b,c A chromatographic column is developed with polar solvent, pick out the correct sequence of component elute out first to last a. Polar; non polar; less polar b. Non polar, polar, less polar c. Less polar; non polar; polar d. Polar; less polar; non-polar d R=Am/Am+αAs, in this equation, α denotes to a. Flow rate of solvent b. Partition coefficient c. Both a & b d. None of the above b The above equation indicates for what a. Movement of component inside column b. Chromatography feature of column c. Both d. None of the above a Length to diameter ratio of separating column should ideally be ____ for preparative work. a. 10:1 b. 10:10 c. 0.1:100 d. 10:100 a



Longer columns are preferred over shorter one for separation of a. Multi-component mixture b. Mixture containing components with nearby relative affinity c. Both d. None of the above c Bad resolution generally observed in

a. b. c. d.

Longer column Shorter column Narrower column Wider column d

All are the ideal properties of a stationary phase used in case of column chromatography except a. Uniform particle size b. Larger surface area c. Good resistance towards solvent flow rate d. Activeness including better mechanical strength c Starch, talc, sucrose, inulin are example of a. Strong adsorbent b. Very strong adsorbent c. Medium adsorbent d. Weak adsorbent d Silica, a type of adsorbent is also known as a. Magnesium silicate b. Magnesium sulphate c. Magnesium salicylate d. Magnesium carbonate a

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Pick out medium adsorbent from list given below a. Calcium hydroxide b. Magnesium oxide c. Magnesium carbonate d. Calcium carbonate (abcd) Silica is a. Strong adsorbent b. Very strong adsorbent c. Medium adsorbent d. Weak adsorbent a Silicic acid is another name of a. silica gel b. Alumina c. Calcium carbonate d. None of the above a Flow rate of mobile phase through column bed can be enhanced by use of a. Filter aid b. Floater aid c. Filtering plate d. All the above a Pick out filter aid from list given below a. Celite 503 b. Celite 545, c. Hyflow Super-Cel d. All the above d Extremely reduction in particle size ultimately a. Increases separation process b. Choked the column c. No effect on separation d. None of the above b



Column cracking means a. Breakage of column b. Degradation of component c. Entrapment of air d. None of the above

c. Any of the above d. None of the above a

c For separation of concentrated product, a column required is a. Longer b. Shorter

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Column chromatography cannot handle a a. Liquid sample b. Gaseous sample c. Solid sample d. All the above b


Chapter – 09 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - Deepak Chowrasia

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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Or,

HIGH PRESSURE LIQUID CHROMATOGRAPHY (Chapter Overview) 01. INTRODUCTION .................................................................................................................. 135 02. ADVANTAGES OF HPLC ..................................................................................................135 03. HPLC Vs GC (GAS CHROMATOGRAPHY) ...............................................................135 04. COMPONENTS OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ......................................................................................136 04.A. Stationary phase .......................................................................................................... 136 04.A.I. Characteristics of stationary phase .............................................................136 04.A.II. Classification of stationary phase ..............................................................136 04.A.II.a. Stationary phase for Adsorption HPLC ..................................... 136 04.A.II.b. Stationary phase for partition HPLC .........................................137 04.A.II.C. Stationary phase for ion exchange HPLC ................................ 137 04.B. Mobile phase ................................................................................................................. 138 04.B.I. Criteria for mobile phase selection ...............................................................138 04.B.II. Mobile phase for various HPLC techniques ...............................................138 05. INSTRUMENTAL SCHEME ............................................................................................. 139 05.A. Solvent delivery system ............................................................................................. 140 05.A.I. Reservoir, degassing system & gradient devices ......................................... 140 05.A.II. Pumps ............................................................................................................... 140 05.A.II.a. Characteristic of HPLC pumps...................................................140 05.A.II.b. Types of HPLC pumps ..................................................................140 05.A.II.b.1. Reciprocating pumps ............................................... 140 05.A.II.b.2. Displacement pumps or Syringe type pump .......... 141 05.A.II.b.3. Constant pressure pumps ........................................ 143 05.B. Sample injection system or sample injection port ..............................................143 05.B.1. Septum injectors ............................................................................................. 143 05.B.2. Stop-flow septum-less injectors ....................................................................143 05.B.3. Micro-volume sampling valves .....................................................................143 05.C. Columns ........................................................................................................................143 05.C.1. Separating columns ........................................................................................ 143 05.C.1.1. Construction & dimensions of separating columns: ................ 143 05.C.1.2. Column Preparation & packing .................................144

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05.C.2. Guard column:. ............................................................................................... 144 05.C.1.3. Types of HPLC columns ...............................................................144 05.D. Detectors .......................................................................................................................145 05.D.1. Characteristics of HPLC detectors ..............................................................145 05.D.2. Classification of HPLC detectors .................................................................145 05.D.2.a. Bulk property detectors ................................................................146 05.D.2.b. Solute property detectors .............................................................146 05.D.2.b.I. UV visible Spectrophotometer ................................. 146 05.D.2.b.II. Fluorescence detector ............................................. 146 05.E.2.b.III. Electrochemical detectors. .....................................147 06. DERIVATIZATION ............................................................................................................. 147 06.A. Types of derivatization ............................................................................................... 147 06.A.I. Pre-column off line derivatization (done before separation) .................... 147 06.A.II. Post column derivatization (done after separation) .................................. 147 07. APPLICATIONS ...................................................................................................................148 08. EXERCISE .............................................................................................................................. 148 09. MULTIPLE CHOICE QUESTIONS ................................................................................149

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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Or, HIGH PRESSURE LIQUID CHROMATOGRAPHY 01. INTRODUCTION High performance liquid chromatography (HPLC) was first described by Csaba Horvath in 1964 at Yale University. The phenomenon of HPLC is somewhat analogous to column chromatography except later chromatographic process involves wide diameter glass columns packed with finely divided adsorbent through which mobile phase percolate under positive effect of gravity making the overall process tedious and lengthy, while on other hand, the former process i.e. HPLC system utilizes narrower bored short columns through which mobile phase forces under high pressure 1000-300psi (thus termed as higher pressure liquid chromatography) enhancing overall efficiency of the system in term of separation, identification, quantification, accuracy, precision, sensitivity, and time consumption. In some respect versatility of HPLC is greater than gas chromatography owing to its inherent capacity of handling high molecular weight, polar, and thermolabile compounds. 02.

ADVANTAGES OF HPLC a. A fast and efficient separation process comparatively.

b. High resolving power. c. Better and efficient handling of multi-component mixture. d. Tackle both polar and non-polar substances. e. Overall automation reduces tedious sample handling procedures, components detection, and data handling. f. Easily manage non-volatile and thermolabile compounds. g. Utilizes only small quantity of sample & solvent. h. A non-destructive process, can endure wide varieties of organic and inorganic compounds without changing its physiochemical properties. i.

Provides a suitable platform for rapid, accurate, reproducible and adaptable quantitative analysis.

j.

Continuous monitoring of column effluent.

k. Handle macromolecules. 03. HPLC Vs GC (GAS CHROMATOGRAPHY) High performance liquid chromatography and gas chromatography, both are highly advance automated chromatographic techniques that are analogues to each other in terms of selectivity, applicability, accuracy, precession, reproducibility, sample quantity, quantitative analysis, and

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non-destructive procedures, but differs in terms of sample handling, component detection, instrumentation cost, and overall time required for analysis. Some of major distinction between HPLC and GC is tabulated as below High performance liquid chromatography A type of liquid chromatography Handle thermolabile, non volatile and inorganic components Instrumentation required is complicated and expansive Yet seeking an universal detection Analysis time is more than GC Free from inflammability and other problem associated with handling if gas as mobile phase

Gas chromatography

Gas liquid chromatography Not handle Simple and somewhat less expansive instrument FID is used as universal detector An rapid technique. Greater care must be taken

Table 01: Difference between HPLC & GC 04.

COMPONENTS OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

04.A. Stationary phase High performance liquid chromatography makes use of same packing material as utilized by column chromatography, but is of lesser dimension in terms of size (3-10 micrometer). A larger size (30-70 micrometer) packing material is rarely used, except in packing guard columns (fitted before separating column for purpose of its protection)

04.A.I. Characteristics of stationary phase Stationary phase used in high performance liquid chromatography must be stable, chemically unreactive, free from impurities, and must be uniformly sizes small rigid particles able to withhold high pressure of solvent (generated by pump) during separation process. The choice of packing material for HPLC depends upon need of chromatographic procedure and mobile phase employed. 04.A.II. Classification of stationary phase 04.A.II.a. Stationary phase for Adsorption HPLC

HPLC grade silica offers advantage of large surface area, porous micro-size irregular or spherical particles of dimension ranging from 3-10 micrometer. Plane silica used in column chromatography can also be used efficiently due to its porosity thus allowing the system to run at normal to medium pressure (2000-2500 psi). Basically, silanol group (Si-OH) of silica is responsible for separation mainly of polar components by a well known phenomenon of adsorption thereby retaining them predominately in contrast to their non-polar counter Chowrasia, Deepak



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analogues. Alumina is rarely used except under some special circumstances such as separation of structural isomer or aromatic compounds. 04.A.II.b. Stationary phase for partition HPLC Normal phase partition HPLC utilizes more polar stationary phase compare to mobile phase. Stationary phase used in this type of HPLC technique is prepared by bonding polar functionalities such as amino>diol>cyno (order of polarity) over silica thereby enhancing its polarity (non polar solute elute first) over mobile phase, which is a less polar solvent system composed of single or binary solvent mixture pre-saturated with stationary phase to prevent dissolution assisted stationary phase loss during chromatographic procedure. On other hand, reversed phase partition HPLC technique rely upon less polar stationary phase compare to mobile phase (polar solute elute first), by chemically bonding silica with lesser polar functional groups via siloxane linkage ( Si-O-Si-C). Commercially, they are prepared by heating silica in the presence of dilute acid for 1-2 days, subsequently treating with Organochlorosilane. Adsorption property of untreated silanol groups can be effectively rendered by reacting them with trimethylchlorosilane. These bonded phase stationary phase pose advantages of tackling nagging problem mostly encountered during adsorption chromatographic procedure along with enhancing their stability over wider pH range (2-9), and temperature (80-90 degree Celsius). Octadecysilane (ODS) is one of the most popular and commonly used bonded phases containing a linear chain of C-18 hydrocarbon. A typical chemical reaction yielding bounded phase silica is described briefly as below;

Where, a=Exposed –OH group of silica (Silanol group) b=Organochlorosilane c=Bounded phase silica d=Byproduct R=Length of carbon chain (R=18 then compound c is known as ODS-Octadecyl silane). 04.A.II.C. Stationary phase for ion exchange HPLC

For ion exchange HPLC, cross linked polystyrene divinylbenzene (DVB) resin or any suitable ion exchanger bonded chemically with silica can be used. Likewise, in size exclusion HPLC techniques, may either employ a cross linked polystyrene divinylbenzene resin or alternatively silica microspheres for separating macromolecular sized solute particles.

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04.B. Mobile phase Since particle size of stationary phase used in HPLC is very small thus mobile phase must be pushed under high pressure through stationary bed of adsorbent packed interior of column for better resolution. Ordinary but highly pure, particulate matter & UV impurities free, costlier solvents commercially known as HPLC grade solvents are generally employed as mobile phase. Generally HPLC grade mobile phase includes wide varieties of both polar as well as non-polar solvents such as HPLC grade water, methanol, acetonitrile, chloroform, ethyl acetate etc. These solvents are used directly in separation process without any further purification, but require an immediate degassing prior to use. Although, solvent are costlier but efficient recycling (if possible) ultimately reduces overall capital cost of system.

04.B.I. Criteria for mobile phase selection Selection of suitable mobile phase in HPLC is of paramount important and depends upon type of sample and procedure required for separation, stationary phase employed, eluting power and polarity of solvent, its viscosity, boiling point, volatility, toxicity, compatibility, and flammability. Dispersion, hydrogen bonding, dipolar and dielectric interaction are some of the major forces act between solute and mobile phase and must be taken into consideration both for selection of mobile phase & designing a separation methodology. An ideal solvent ( ?) for HPLC can be selected either by trial and error method or by exhaustive literature survey. 04.B.II. Mobile phase for various HPLC techniques Adsorption HPLC mostly make use of organic solvents either singly or a suitable combination of miscible solvent of optimum polarity. While on other hand, water and a less polar organic solvent modifier like Acetonitrile or methanol is of preliminary choice for reversed phase HPLC. Solvent

Water Methanol Ethanol Chloroform Carbon tetrachloride Diethyl ether 1,4 dioxane Acetonitrile

Eluent strength High

Polarity index

Boiling point

0.95 0.88 0.40 0.18

10.2 5.1 4.3 4.1 1.6

100 64.7 78.5 61.2 76.8

0.38 0.56 0.65

2.8 4.8 5.8

35 101.3 82

UV cut off (nm) 190

210 207 245 265 205 216 190

Table 02: HPLC Solvents

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HPLC procedure Adsorption

Stationary phase Plane silica and alumina (rarely except with few cases)

Mobile phase Organic solvent optimum polarity

Partition Normal phase

Polar stationary phase mostly bonded silica with polar functional groups (cyno, thiol, amino)

Less polar mobile phase compare to stationary phase; pre-saturated with stationary phase to prevent its dissolution

Reverse phase

Non-polar stationary phase; bonded silica with hydrophobic functional groups

Binary mixture of polar solvent with differential polarity (water:acetonitrile)

Miscellaneous Size exclusion

Silica microsphere or silica bonded with cross linked polystyrene divinylbenzene resin

Single aqueous or organic solvents

Ion exchange

Silica bonded with cross linked polystyrene divinylbenzene resin or any analogues resin depending upon ion to be separated.

Aqueous solvents or aqueous buffer with organic solvent for poor water soluble compounds.

with

Table 03: Overview of various HPLC techniques 05.

INSTRUMENTAL SCHEME

Figure 01: Layout of HPLC system

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A HPLC instrument comprises of following basic components 05.A. Solvent delivery system Solvent delivery system composed of following two assemblies viz.

05.A.I. Reservoir, degassing system & gradient devices 05.A.II. Pumps 05.A.I. Reservoir, degassing system & gradient devices Solvent reservoir consist of tightly sealed glass or plastic bottles of suitable size imbedded with solvent carrying tubing which are further be connected with HPLC pump. Degassing of mobile phase is done by subjecting them under vacuum distillation and spurging with fine spray of inert gas like argon or helium. Gradient devices are used to maintain pressure sufficient to transport mobile phase to pump. Sometime gradient devices are also used for prefiltration of mobile phase before it reaches to pumps. 05.A.II. Pumps Pumps in HPLC system plays dual role of passing mobile phase through column bed under high pressure and maintaining a constant pulse free flow rate throughout the separation process. 05.A.II.a. Characteristic of HPLC pumps

Salient feature of HPLC pumps includes maintenance of variable but pulse free flow rate as desired by analytical procedure. Components and accessories used in pump manufacturing must be non corrosive in nature and must be compatible with both man and machine including mobile phase and mixture to be separated. Long life, economical, easy to dismantle and repair are some of the other needful characteristic of pumps. 05.A.II.b. Types of HPLC pumps

Ordinarily, any one of the below mentioned HPLC pumps can be used with HPLC systems; 05.A.II.b.1. Reciprocating pumps

It is an inexpensive and widely used pump, able to maintain wide range of flow rate. Reciprocating pump creates pressure onto a flexible diaphragm via hydraulic fluid by moving its piston (mounted on eccentric cam or gear) forward and backward direction (see figure 02). Dual piston reciprocating pumps are on the side of superiority over single piston pumps by producing almost pulse free flow rate due to simultaneously phased movement of pistons, but are more expansive than single piston reciprocating pumps.

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Figure 02: Systematic sketch of reciprocating pump Working principle: A single piston reciprocating pumps consist of a motor driven piston moving backward and forward direction in an hydraulic chamber of capacity 30-400 microlitres. During backward stroke, inlet valve opens (V1) and solvent from mobile phase reservoir moves into collecting chamber. At this time, outlet valve (V2) to separating column is closed. In forward stroke, piston pushes mobile phase into the separating column via opening of outlet valve, while the backward flow of solvent into reservoir is check by closure of inlet valve simultaneously. Advantages of reciprocating pump: Reciprocating pumps are able to maintain a wide range of flow rate without causing any cross contamination (sometime may be) of mobile phase during separation process. They are easy to operate, non corrosive, deals with large volume of mobile phase and are continuous in operation. Since, flow of solvent in this type of pump is guided by piston movement thus creating pulsating phenomenon, which however can be effectively minimized by use of damping devices. Leakage of hydraulic fluid into solvent system and fatigability of flexible diaphragm are some rare circumstance faces during its operations. 05.A.II.b.2. Displacement pumps or Syringe type pump: These pump pose an advantage over reciprocating pump of maintaining pulse free delivery of mobile phase, requiring no damping devices, but suffers drawback of limited reservoir capacity and non continuous in operation. Working principle: The pump works on the principle of positive displacement of solvent system by a piston mounted on screw feed drive through a gear box and run by a digital motor. The rate of solvent delivery by this type of pump is controlled by changing voltage on

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digital motor. Capacity of solvent chamber of displacement pump is adequate for proper working with small bore columns. A pulse free high pressure (200-475 atm) flow rate can be easily achieved by this type of pump.

Figure 03: Outline of displacement pump Advantage of displacement pumps: Efficient working with small bore column, pulse free flow rate, easily to operate and control flow rate are some of its up points while non-continuous working and limited reservoir capacity are its limitations.

Figure 04: Constant pressure pump

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05.A.II.b.3. Constant pressure pumps: Construction and working of these types of pump are analogues to reciprocating pumps instead of using hydraulic fluid they uses air under pressure for pushing solvent system through separating column. These pumps are capable of producing a pulse free slow solvent delivery (1-2ml/min.), having a limited reservoir capacity, and generate a pressure of 100-200 atm. As constant pressure pumps are free from use of hydraulic fluid chance of cross contamination of mobile phase is very rare. 05.B. Sample injection system or sample injection port

They are used to inject sample into chromatographic column either directly or indirectly. An ideal sample injection system must be free from void space. Following are the three different types of sample injection system employed in HPLC. 05.B.1. Septum injectors They rely on high pressure syringe for delivery of sample via self sealing elastomer septum. These injectors pose a problem of leaching effect of mobile phase causing ghost or pseudo peak. 05.B.2. Stop-flow septum-less injectors They are much better as compare to septum injector. As it name indicates flow of mobile phase via column stopped for a moment and when an optimum pressure is reached by column, sample is injected. 05.B.3. Micro-volume sampling valves This type of injector system is employed with sophisticated HPLC instruments and is costlier compare to other two injector mention above. This type of injector poses advantages of automatic injection with fairly better precision and reproducibility 05.C. Columns

05.C.1. Separating columns They are also known as HPLC separating columns as separation of mixture into individual component takes place interior of column onto the bed of adsorbent (plane or bonded phase silica) by the phenomenon of adsorption/partition. They are an essential part of HPLC based chromatographic system and serve the purpose of holding stationary phase. 05.C.1.1. Construction & dimensions of separating columns: Basically, HPLC columns are made up of high quality stainless steel having precise bore and mirrored polished internal finishing. Commercially, wide ranges of HPLC columns are available with differential dimensions. A typical HPLC column has an internal diameter of 4-5mm and a length of 10-30 cm (3-6cm for short column). Stainless steel frits with mesh of size 2 micrometer or less

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guard stationary phase from washing away during separation process. Length of column not only affects separation but also resolution of components. Standard columns are longer than their counterpart shorter columns and utilize a long duration, but good resolution comparatively. A narrow bore column is more expansive than its analogues wider bore column. 05.C.1.2. Column Preparation & packing: Commercially ready to use HPLC columns are available as per need of analyst, but they can also be prepared inland with an aid of suitable pressurizing filling device. Usually, dry packing is suitable for particles of diameter more than 20-30 micrometer, while wet packing methodology is ideal for particles of dimension less than 10-20 micrometer.

Column packing can be done by two modes, a superficial packing (less efficient) which consist of porous stationary phase of larger particles coated over solid core usually a glass bead or alternatively total porous packing (more efficient) which include wide range of small sized (3-20 micrometer) packing material of high surface area thereby providing good resolution. 05.C.2. Guard column: These are basically non-analytical shorter column place between HPLC separating column and sample injection system where they serve the function of protecting separating column from damaging or clogging due to particulate matter. Also they shade up main HPLC column from getting in contact with strongly adsorbed chemicals present in mobile phase or analyte. 05.C.1.3. Types of HPLC columns Internal diameter (mm) 4-5mm

HPLC column

Standard column

Radial compression column Narrow bored column Short column

1-2

Length (cm)

Remark

10-30

Longer column; large sample capacity; required large amount of packing material (3-5 micrometer) and solvent for elution; good efficiency Wide separation range and good efficiency

10-25

Elution done with non conventional solvents Highly efficient fast column; required less amount of sample and solvents. Packing material dimension 3 micrometer

3-6

Table 03: Types of column & their characteristics

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05.D. Detectors Detector serves the function of monitoring mobile phase loaded with substance of interest to be analyzed. Compare to gas chromatography, detection process in liquid chromatography is somewhat tedious and problematic; still a universal detector for HPLC is awaited .

05.D.1. Characteristics of HPLC detectors An ideal detector for HPLC must be highly sensitive, linear in response, sense a wider range of constituents present in sample (but in a selective manner), good limit of detection, and have shorter response time. Selection of suitable detector is done on the basis of sample to be handled and sometime even multiple detectors are employed for detection process. 05.D.2. Classification of HPLC detectors For the sake of simplicity HPLC detectors are classified into following two types 05.D.2.a. Bulk property detectors 05.D.2.b. Solute property detectors.

Figure 05: Outline of HPLC detectors

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05.D.2.a. Bulk property detectors: These detectors ultimately measure some of the physical properties of analyte present in mobile phase against blank mobile phase for example detection of conductivity or refractive index. These detectors are somewhat lesser sensitive and limited in range than solute property detectors and are easily get affected by even a minute change in mobile phase thus are not suitable with gradient elution. For efficient working, bulk property detectors required a good control over temperature change. Christiansen and Fresnel detectors are some of its major types Christiansen detector measures degree of deflection of monochromatic light by a blank mobile phase against mobile phase containing analyte. On other hand Fresnel detector or refractometer measures changes in fraction of reflected and transmitted light from glass-liquid interface due to change in refractive index of liquid when it contains analyte. 05.D.2.b. Solute property detectors: These detectors measures physical or chemical properties of analyte irrespective of mobile phase and can be used effectively with gradient elution. They are 1000 times more sensitive to bulk property detector & insensitive towards temperature or flow rate change. Spectrophotomeric, fluorescence and electrochemical detectors are some of most commonly employed detectors of this class. 05.D.2.b.I. UV visible Spectrophotometer: They are one of the most widely used sensitive, specific, and low cost detectors and account for about 70% against all other. They are based on the simple phenomenon of concentration of analyte in post column eluent is directly proportional to amount of UV light absorbed. For the sake of accuracy post column eluent must be free from air bubble which causes interference with detection process by creating spikes on chromatogram. However, this problem can be effectively reduced by degassing the system prior to analysis. Both single and double beam UV spectrophotometer are used for accurate and precise detection. Range of detection in this type of detector system can be efficiently increase by employing a variable wavelength detector which covers a range of 210nm to 800nm. 05.D.2.b.II. Fluorescence detector: They are one of the most highly sensitive (detection range-nanogram to picogram) and selective, but less applicable detectors due to its limited applicability either toward fluorescence compounds or their derivative that show fluorescence phenomenon. Besides this swamping of detector signal due to presence of any fluorescence related impurities in sample or mobile phase also limit its further application. To overcome these problems, chemiluminescent detectors are developed, which ultimately make use of chemically derived excitation energy rather then spectroscopic means for efficient detection process. Mostly peroxyoxalate or luminol is mixed with post column eluent before analysis with fluorescence detectors. Less expansive fluorescence detectors make use of filter while monochromators are used by expensive fluorescence instruments.

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05.E.2.b.III. Electrochemical detectors: Electrochemical detectors ultimately measures net electron transfer during a chemical reaction i.e. the current produced from oxidation or reduction phenomenon of an analyte. As the current produced is directly proportional to concentration of solute/analyte present in eluent, thus it can be utilized for quantification. Although a number of electrochemical detection process (polarograhy, amperometry, coulometry, and potentiometry) are available for detection, but none of them are popular except polarographic detectors. Purity of eluent in this type of detector system is utmost important, as presence of oxygen, halides or metal contaminant cause a false current generation, thereby interfering with normal detection process. 06.

DERIVATIZATION

Derivatization is a technique of improving sensitivity and selectivity of analyte containing polar functional group with an aid of suitable chemical reagent known as derivatizing agents. Fluorotags and chromatags are some of nearby classes of derivating agent which enhances the detection phenomenon of compound in fluorescence range in former case while in ultra-violet range in later one. 06.A. Types of derivatization

06.A.I. Pre-column off line derivatization (done before separation) When the process of derivatization is done before chromatographic separation then it is known as pre-column off line derivatization. Pre-column off-line derivatization done either to improve resolution or stability or alter the retention time of analyte without modifying instrument. This technique of derivatization encounter problem associated with presence of excess reagent or byproduct which ultimately interferes with normal separation process. Beside this, the functional group introduced into the analyte for increasing its detection may cause change in chromatographic properties of analyte. 06.A.II. Post column derivatization (done after separation) It is done after chromatographic separation for enhancing sensitivity of solute detection. Advantage of this type of derivatization includes separation and analyte detection simultaneously Chromatas are the reagents that forms derivative which are strongly absorbs UV/visible radiation for example ninhydrin while fluorotages (dansyl chloride) are non fluorescent reagents when treated with analyte convert it into fluorescence molecule that can easily be detected by fluorescence detectors.

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07. APPLICATIONS There hardly any sphere of synthetic, semisynthetic or bioactive compounds (except volatile compounds & few exceptions) that cannot be separated or analyzed by HPLC.

a. Detection of psychoactive drug (benzodiazepines, phenothiazine, tricyclic antidepressant, and neuroleptic) in body fluid including blood, urine, CSF can be done with an aid of C-18 reverse phase HPLC column. b. Analysis of cardiac glycosides, separation of bioactive alkaloids, anthocynides, xanthines, isofavones, tannin etc can be done with highest grade of resolution with reverse phase HPLC technique using a C-18 column. c. HPLC can be used for study of various microbiological process of industrial importance like HPLC controlled analysis of penicillin production. d. Assay of human insulin by using a Vydac C-18 column. e. A combination of HPLC with suitable detection technique allows an accurate and precise identification of analyte like oestradiol, catecholamines, opium alkaloids, aspirin, paracetamol tablets, verapamil and its metabolites. f. Analysis of vitamins both water and fat soluble via ion exchange HPLC. 08.

EXERCISE a. Give a brief account on HPLC and mention some of its advantages.

b.

Differentiate HPLC with GC (gas chromatography).

c.

Write a short note on stationary phase used in HPLC.

d.

Differentiate normal and reverse phase partition HPLC techniques.

e.

Give an outline on procedure required for preparation of bonded phase stationary phase for reversed phase partition chromatography.

f.

Write a note of HPLC grade mobile phase. Enumerate various criteria for their selection.

g.

Diagrammatically explain various components of HPLC.

h.

Write a brief note on HPLC instrumentation with special emphasis on detection process.

g.

Enumerate various pumps used in HPLC with their pros & cons.

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h.

Differentiate bulk and solute property detector.

i.

Write a note on

a.

Separating column

b.

Displacement pump

c.

HPLC spectrophotometric detectors

j.

What is guard column? How it protect main column from deterioration.

k.

Classify HPLC detectors.

l.

What is derivatization? How it improves separation process in HPLC.

m.

Write a note on pre and post column derivatization technique.

n.

Give application of HPLC.

149


HPLC was first described by ______in _______ at Yale University a. Csaba Horvath, 1963 b. Csaba Horvath, 1964 c. Csaba Horvath, 1965 d. Csaba Horvath, 1966

Did HPLC have a universal detector? a. Yes b. No c. Can’t say d. All are false b gas

HPLC, when compared to chromatography can handle a. High molecular weight substances b. Polar substances c. Thermolabile compounds d. All the above

c Gas chromatography differs from HPLC in a. Being a automated technique b. Handle volatile components c. Both d. None of the above b

Chowrasia, Deepak

b HPLC is a type of a. Gas chromatography b. Solid-gas chromatography c. Liquid chromatography d. None of the above c “Analysis time for HPLC is more than GC” statement is a. True b. False c. Can’t predicted d. None of the above a



FID (flame ionization detector) is a universal detector for GC a. True b. False c. Can’t predicted d. None of the above a Packing material for HPLC must have following characteristics a. Stable & inert. b. Free from impurities. c. Uniformly small sized rigid particles withstanding high pressure d. All the above d Particle size for HPLC grade silica is a. 0.3-10 micrometer b. 30-10 micrometer c. 3-100 micrometer d. 3-10 micrometer d Silanol group (Si-OH) for _____ bond with _____ compounds a. Covalent bond, hydrophilic b. Coordinate bond, hydrophobic c. Hydrogen bond, hydrophilic d. None of the above c Silanol group (Si-OH) is responsible for a. absorption of solute b. adsorption of solute c. both d. None of the above b A mixture containing polar as well as non polar components is passes through HPLC column containing silica gel as packing material. Pick out the correct sentence from options given below

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a. Polar component elute first followed by non polar b. Non-Polar component elute first followed by polar c. Both components elute out at same time d. All are false b Normal phase HPLC denotes a. Polar stationary phase; non polar mobile phase b. Non-polar stationary phase; polar mobile phase c. Both are incorrect d. Both are correct a A bounded silica gel means a. Silica bounded on rigid stationary phase b. Silica bounded with HPLC column c. Silica bounded with different functional groups d. None of the above c Bounding of silica gel with different functional group ultimately a. Enhance it utility b. Increase polarity c. Both are possible d. None of the above c In normal phase HPLC, bounded silica gel used as stationary phase must be a. Polar with respect to mobile phase b. Non-polar with respect to mobile phase c. May be polar or non-polar depending upon sample to be separated d. None of the above a



Pick correct arrangement of functional group attach with silica according to their polarity a. Methyl>Amino>Diol>Cyno

151

Solvent used in HPLC are a. Only polar in nature b. Only non-polar in nature c. May be any of the two; depending upon necessity of separation procedure d. All are true

b. Amino>Diol>Methyl>Cyno c. Amino>Methyl >Diol>Cyno d. Amino>Diol>Cyno>Methyl

c d

Reversed phase HPLC denotes a. Polar stationary phase; non polar mobile phase b. Non-polar stationary phase; polar mobile phase c. Both are incorrect d. Both are correct b Siloxane linkage can be denoted as a. Si-O-Si-C b. Si-H-Si-C c. Si-OH-Si-C d. Si-O-Si-C-OH a The problem of nagging is commonly encountered by using a. Plane silica gel b. Bounded phase silica gel c. Both are suitable d. None of the above b Octadecy silane (ODS) is a type of bounded non polar silica gel containing a linear chain of ________hydrocarbon a. C-17 b. C-18 c. C-19 d. C-15 b

Chowrasia, Deepak

Pick out correct statement a. HPLC grade solvent are costlier and can be used directly without undergoing any purification procedure. b. Degassing of solvent prior to use is recommended c. Both are correct d. Both are incorrect c In HPLC, role of pump is/are a. Introduction of solvent into the column b. Maintenance of constant flow rate c. Both d. None of the above c Pick out false statement regarding reciprocating pump a. Maintain wide range of flow rate b. No pulsation during operation c. Easy to operate d. All are false b Syringe type pump is another name of a. Displacement pump b. Reciprocating pump c. Constant pressure pump d. None of the above a



Damping device is usually used in reciprocating pump to overcome problem of a. Solvent contamination b. Pulsation c. Solvent delivery d. None of the above

d. None of the above c,a

b Pick out correct statement regarding reciprocating pumps a. Maintain wide flow rate b. Continuous in operation c. Large volume of mobile phase can be handle d. All are true Limitation of syringe type pumps are a. Limited reservoir capacity b. Non continuous in operation c. Both d. None of the above

A guard column in HPLC installed between a. Sample injector and solvent reservoir b. Sample injector and detector c. Detector and plotter d. Sample injector and main column d

c Pick out the pump suitable for working with small bored column. a. Displacement pump b. Reciprocating pump c. Constant pressure pump d. None of the above a Pick out the pump having highest possibility of cross contamination a. Displacement pump b. Reciprocating pump c. Constant pressure pump d. None of the above b

Chowrasia, Deepak

A typical HPLC column has an internal diameter of ___mm and a length of __cm a. 0.4-5; 10-30 b. 4-50; 10-300 c. 4-5; 100-300 d. 4-5; 10-30 d

d

Pick out the pump having lowest possibility of cross contamination a. Displacement pump b. Reciprocating pump c. Constant pressure pump

152

A bulk property detector is a. Highly sensitive than solute property detector b. Less sensitive than solute property detector c. Can be used with gradient elution d. None of the above b Consider the following statement “For efficient working, bulk property detectors required a good control over temperature change”. a. Correct b. Incorrect c. Can’t be predicted d. None of the above c Christiansen and Fresnel detectors are types of a. Bulk property detector b. Solute property detector c. Both d. None of the above a



Pick out correct statement regarding solute property detectors a. They are 1000 times more sensitive to bulk property detector b. They are insensitive towards temperature or flow rate change. c. They can be used with gradient elution d. All are correct d A solute property detector measures a. Physical property of analyte b. Chemical property of analyte c. Physiochemical property of analyte d. Physiochemical property of mobile phase c Pick out solute property detector/s a. Spectrophotomeric detector b. Fluorescence detector c. Electrochemical detector d. All the above d Most commonly used solute property detector in HPLC is/are a. UV detector b. Amperometric detector c. Fluorescence detector d. All the above a Least commonly used solute property detector in HPLC is/are a. UV detector b. Amperometric detector c. Fluorescence detector d. All the above c Limited application of fluorescence detector is due to a. Selectivity only towards fluorescence component of mixture

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b. Swamping of signal c. Both d. None of the above c Peroxyoxalate or luminol is mixed with post column eluent before analysis in which type of detectors a. UV detector b. Amperometric detector c. Fluorescence detector d. All the above c Pick out a commonly used electrochemical detector/s a. Amperometric b. Coulometric c. Polarographic d. Potentiometric c Contamination of eluent with oxygen, halides or metal contaminant cause a false ________ in a. UV detector b. Amperometric detector c. Fluorescence detector d. Electrochemical detector d Derivatization in HPLC used for increasing a. Selectivity b. Sensitivity c. Both d. None of the above c Chemical agents used in derivatization phenomenon is known as a. Derivatizing agent b. Digitizing agent c. Daring agent d. None of the above a



A Pre-column off line derivatization is done _______ chromatographic separation a. After b. Before c. Anytime d. None of the above b

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Pre-column off-line derivatization done to improve_____ of analyte a. Resolution b. Stability c. Alter the retention time of analyte d. All are correct d


Chapter - 10 GAS CHROMATOGRAPHY - Deepak Chowrasia, Dr. Nisha Sharma

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GAS CHROMATOGRAPHY (Chapter Overview) 01. INTRODUCTION .................................... ...................................................... .................................... .................................... ................................... ........................ ....... 159 02. TYPES OF GAS CHROMATOGRAPHY CHROMATOGRAPHY ................................... ..................................................... ................................... ................... 159 ..................................................... ................................... ..................... ....159 02A. Gas solid chromatography (GSC) ................................... 02.B. Gas liquid chromatography chromatography (GLC) .................................... ...................................................... ................................... ................... 159 ............................................ ......... 160 03. FACTORS AFFECTING GAS CHROMATOGRAPHY ...................................

03.A. 03.B. 03.C. 03.D. 03.E

Particle size .................................... ...................................................... .................................... .................................... ................................... ........................ ....... 160 ...................................................... .................................... ................................... ........................ ....... 160 Dimensions of column.................................... Carrier gas flow rate ................................... ..................................................... .................................... .................................... ........................... .........160 Column temperature .................................. .................................................... .................................... .................................... ........................... .........160 ................................................... ................... 160 Nature and concentration of stationary phase ..................................

............................161 04. INSTRUMENTATION SCHEME-GAS CHROMATOGRAPHY CHROMATOGRAPHY ............................

04.A Gas container, carrier gas, and pressure regulating units. ............................... ............................... 161 04.A.I. Gas container .................................... ...................................................... .................................... .................................... ........................ ...... 161 ...................................................... .................................... .................................... .............................. ............162 04.A.II. Carrier gas .................................... 04.A.II.a. Ideal characteristic of carrier gas .................................. .............................................. ............162 04.A.II. b. Types of carrier gas.................................... ...................................................... ................................. ...............163 04.B Sample injection port.................................. .................................................... .................................... .................................... ........................... .........163 ...................................................... ................................... ..................... .... 164 04.B.I. Sample handling techniques .................................... 04.B.I.a. Sample pyrolysis ................................... ..................................................... ................................... ........................ ....... 164 ..................................................... ................................. ...............164 04.B.I.b. Sample derivatization ................................... 04.B.I.c. Metal complexation .................................... ...................................................... ................................... ................... 164 .................................................... .................................... .................................... .............................. ............165 04.C. Column thermostat .................................. 04.D. Separating column ................................... ..................................................... .................................... .................................... .............................. ............165 ...................................................... ................................... ..................... .... 165 04.D.I. Types of separating column .................................... 04.D.I.a. Packed column .................................. .................................................... .................................... ........................... .........165 .................................................. ...............166 1 66 04.D.I.a.1. Column dimensions: ................................... 04.D.I.a.2. Components of packed column ................................ ................................ 166 04.D.I.a.1. Backing or solid support material. .......................... ..........................166 1 66 04.D.I.a.2. Ideal characteristic characteristic of solid support support material ......167 04.D.I.a.3. Problem & measures of peak tailing .....................167 ................................................... ..................... .... 168 04.D.I.a.2. Stationary phase .................................. 04.D.I.a.2.i. Liquid Liquid stationary stationary phase .................................. ........................................... .........168 1 68 .........................168 Ideal characteristic characteristic of liquid stationary phase .........................

.................................... 168 Classification of liquid stationary phase ..................................

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04.D.I.b. Capillary column .................................... ...................................................... ................................... ..................... .... 168 ............................................... ............169 04.D.I.b.1. Micropacked column: ................................... 04.D.I.b.2. Open tubular capillary column (OTC): ..................169 04.D.I.b.2.1. 04.D.I.b.2.1. Wall coated Open tubular capillary column ....................................... .... 169 (WCOT)/wide bore capillary columns ................................... 04.D.I.b.2.2 Support coated open tubular column (SCOT) ..... 170 04.D.I.b.2.3. Porous layer Open tubular .................................................... ........................... .........170 capillary column (PLOT) .................................. 04.E. Detectors ................................... ..................................................... .................................... .................................... .................................... .............................. ............170 04.E.1. Ideal requirement requirement of detectors detectors includes .................................... ..................................................... ................... 171 04.E.2. Types of gas chromatographic chromatographic detectors .................................. ................................................... ...................171 04.E.2.I. Thermal conductivity detectors (TCD)/Hot (TCD)/Hot wire detectors/ Katherometer .................................... ................................................... ...............171 04.E.2.II. Flame ionization detectors (FID) ................................... ............................................... ............ 172 .............................................. ............174 04.E.2.III. 04.E.2.III. Electron capture detector (ECD) .................................. 04.E.2.IV. Photoionization detectors (PID).................................... ................................................ ............174 05. CHROMATOGRAPHIC CHROMATOGRAPHIC BEHAVIOR OF SOLUTE .................................... ................................................... ...............174 05.A. Retention time .................................. .................................................... .................................... .................................... ................................... ..................... .... 174 .................................................... ................................. ...............175 05.A.I. Factors affecting retention time .................................. 05.B. Adjusted retention time ................................... ..................................................... .................................... ................................... ..................... .... 176 .................................................... .................................... .................................... .............................. ............176 05.C. Capacity factor (K) .................................. 05.D. Retention volume ................................... ..................................................... .................................... .................................... ................................. ...............177 05.E. Adjusted retention volume .................................... ...................................................... .................................... ................................. ...............177 .................................................... .................................... .................................... ........................... .........177 05.F. Net retention volume .................................. 05.G. Partition coefficient .................................. .................................................... .................................... .................................... .............................. ............178 05.H. Partition ratio ................................... ..................................................... .................................... .................................... ................................... ..................... .... 178 .................................................... .................................... .................................... ................................. ...............178 05.I. Relative retention .................................. 06. COLUMN EFFICIENCY .................................. .................................................... .................................... .................................... .............................. ............179 06.A. Height of effective theoretical plate (HETP) ................................... .................................................... ..................... .... 179 ..................................................... ................................. ...............179 06.B Number of plate or plate number (N) ................................... 06.C. Resolution or degree of separation ................................... ..................................................... ................................... ..................... .... 180 ..................................................... ...................180 07. APPLICATION OF GAS CHROMATOGRAPHY CHROMATOGRAPHY ....................................

08. EXERCISE .................................... ...................................................... .................................... .................................... .................................... ................................... ................... 182 09. MULTIPLE CHOICE QUESTIONS ................................... ..................................................... .................................... ........................... .........183

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GAS CHROMATOGRAPHY 01. INTRODUCTION Gas chromatography is a highly advanced and sophisticated version of chromatographic chromatographic based analytical technique, which is used for analysis ( qualitative & quantitative) and separation primarily of volatile, low molecular weight, thermostable components present in mixture with an aid of inert and highly pure gas acting as mobile phase. Although, the technique was suitably advanced and refined efficiently to deals with wide varieties of compounds with an aid of suitable methodology and reagent (derivatizing agents) yet the technique pose limitations of handling highly polar, non-volatile, ionic and thermolabile substances which however can be easily and efficiently deals with HPLC. Martin & Synge first introduced this analytical technique in 1941 and true experimentation on gas chromatography was initially done by Martin & James in 1954 with lower fatty acid. Essentially, the technique requires vaporization of sample to be analyze which is then mixed up with mobile phase (gas) and passes through a separating s eparating column packed with suitable size uniformly distributed stationary phase. The separation of the component occurs due to their differential affinity against mobile & stationary phase. 02. TYPES OF GAS CHROMATOGRAPHY On the basis of stationary phase, gas chromatography chromatography can be of following two types 02A. Gas solid chromatography (GSC) In this technique, the stationary or fixed or non-mobile phase consists of solid material (granular silica particles or alumina of carbon) packed interior of main column and the phenomenon of separation takes place between solid stationary phase and gaseous mobile phase with an renowned physiochemical process termed as adsorption. The technique of GSC however has limited application due to following reasons

a.

Retention of active gas over stationary stationar y phase ultimately ultimatel y reduces overall surface area thus affects resolution as well as separation of components.

b.

Problem of tailing due to non linear adsorption isotherms.

02.B. Gas liquid chromatography (GLC) In this technique, stationary or fixed phase is a non-volatile liquid or suspension coated as a thin layer over a chemically inert and thermally resisted solid backing material or support like Kieselguhr. The phenomenon of separation based on the process of partitioning of component between thin layer of liquid coated over solid support and gaseous mobile phase GLC is one of the most widely applicable gas chromatographic technique not only over the GSC but also comparably superior over other column chromatographic techniques.

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160

FACTORS AFFECTING GAS CHROMATOGRAPHY

03.A. Particle size As with other chromatographic techniques, any reduction in stationery phase particle size, increase overall surface area and hence resolution & efficiency. In concerned with column chromatographic technique, it is worth to noted that decreases in particle size (up to certain limit), increase the number of theoretical plate resulting in better separation. 03.B. Dimensions of column Length and internal diameter of column rigorously affects separation process not only in terms of resolution, but also in terms of its cost. Usually, longer but lesser diameter columns are preferred for improved separation. Generally, 1-10 meter long and 0.25 inches diameter columns are commonly employed with gas chromatography.

Length of column is directly proportional to its resolving power i.e. longer columns provides better separation, but at an expense of cost and time. At the time of writing, longest column so far available is 1.3 miles or 2100 meter in length with an approximate plate count of 2,000,000 approximately. On other hand, diameter of column is inversely proportional to efficiency i.e. lesser is diameter higher is efficiency, but again severe limitation are imposed on whole system ranging from sample injection to its detection. 03.C. Carrier gas flow rate An optimum flow rate of gas is maintained throughout the chromatographic process, in order to achieve better separation efficiency. A very low flow rate results in broadening of peak while extremely fast leads to peak clumping. 03.D. Column temperature For an efficient separation, column must be maintained at sufficient higher temperature so that analyte will remains in vapor phase without undergoing any degradation. However, it must also be noted that an excessive increased in temperature results in volatilization or bleeding of liquid stationary phase affecting accuracy and sensitivity of gas chromatographic technique. Basically, as per rule of thumb, separation process accelerates with upward adjustment of temperature, better resolution with longer retention at downward temperature adjustment. 03.E Nature and concentration of stationary phase Efficiency of column is maximally affected by amount of stationary phase. “Like retains like” philosophy holds true while dealing with liquid stationary phase. An increase in concentration of liquid stationary phase ultimately increases number of theoretical plate and thus resolution, but beyond certain limit peak tailing ultimately results.

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INSTRUMENTATION SCHEME-GAS CHROMATOGRAPHY Gas container, carrier gas, and pressure regulating units

04.B

Sample injection port

161

04.C. Column thermostat 04.D. separating column 04.E

Detector

Figure 01: Overview of gas chromatography instrumentation 04.A

Gas container, carrier gas, and pressure regulating units

04.A.I. Gas container It is a metallic cylinder also known as Gas Tank or Gas Cylinder filled with carrier (under optimum pressure) gas acting as mobile phase for gas chromatography. Purity of carrier gas inside the system is maintained by molecular sieves, which help in removing impurities including water if contained by carrier gas. The cylinder is boldly fitted with pressure

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regulating valves, electronic or mechanical pressure gauge, and flow regulators which meticulously controls overall flow rate of gas from gas container to chromatographic system. The gas container must be kept in upright position, maintained at an ambient temperature, and must be placed away from any direct or indirect contact with inflammable source. For safety purpose, it must be supported with proper adhering clamps and chains. 04.A.II. Carrier gas Carrier gas is act as mobile phase in both GLC and GSC. Pure air, hydrogen, nitrogen, helium, carbon dioxide, and argon are some of the gases normally used as mobile phase in gas chromatography. The selection of carrier gas is done on the basis of its physiochemical and economical parameters including type of methodology used for separation, nature of sample to be handled, type of separating column and detector employed for analysis. For example helium is choice of gas while dealing with thermal conductivity detectors however hydrogen can also be used with equal efficiency with same detector but generally avoided owing to its higher inflammability. 04.A.II.a. Ideal characteristic of carrier gas

a. Inertness The gas employed as mobile phase must be chemically inert, dry, stable, and do not react with any of the component of sample, instrumentation, and analyst. b. Availability It must be easily & reasonably available without an expense of high cost. c. Purity It is an important parameter that affects both accuracy and sensitivity of chromatographic procedure. A gas with purity markup of 99.995% to 99.9995% pure is suitable for most of the chromatographic procedure. d. Viscosity and flow rate Optimum viscous gas with an optimum flow rate of approximately 300-1 900ml/min is ideally employed for gas chromatography. e. No water no oxygen Ideally, the gas should be free from traces of water and oxygen. f.

Density and thermal conductivity A least dense gas with good thermal conductivity is preferable for gas chromatography.

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g. Hazardous free The gas must be friendly from the point of view of analyte, analyst, and instrumentation mainly detectors. It should also be free from risk of explosion. 04.A.II. b. Types of carrier gas

Helium is one of the most commonly used carrier gas having all the desired characteristic including safety and can be used efficiently with flame conductivity detectors, thermal conductivity detectors, and electron capture detectors, but is expansive comparatively. Hydrogen is yet an another choice of mobile phase is gas chromatography due to its availability, cost effectiveness, low density, high flow rate, good thermal conductivity, and high sensitivity with detectors, but it react with most of the unsaturated compounds and highly explosive in nature. Like helium, hydrogen is also compatible with flame conductivity detectors, thermal conductivity detectors (mostly), and electron capture detectors thus can be employed without any problem.Nitrogen & Argon are invariably used, but former pose limitation of expansiveness and insensitivity while later is used under some special circumstances. 04.B Sample injection port It is meant for introducing sample directly at the head of separating gas column thus built near to them and maintained at suitable higher temperature (50 degree Celsius higher than lowest boiling point of sample) which ensures quick vaporization without causing any thermal decomposition of sample. The port is made up of heavy mass material containing a pliable septum via which samples are injected and mixed up with carrier gas before passing through separating column. It must be noted that only those samples that are easily and efficiently vaporizes are considered for injection else are converted or derivatized into suitable form before their introduction into the separating column. Capillary column st I II Mix choice choice up He H* H, He N Ar/M H He N H, He N He N st

Packed column st I II choice Mix choice up He H, N, Ar N Ar Ar/M N He H, He N He N st

Detectors FI EC FP

TC + -

+ -

+ -

+ -

NP +

Table 01: Carrier gases & their applications Symbols have their usual meaning;*-may be first choice but used under high caution and strict safety regulation owing to its explosive nature; M-methane; TC-thermal conductivity

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detector, FI-flame ionization detector; EC-electron capture detector; FP-flame photometric detector; NP-nitrogen phosphorous detector.

04.B.I. Sample handling techniques Liquid samples must be judge for presence of any air bubble prior to their injection into the system. Usually they are injected suitably by means of micro-syringe (0.1-100 µL) with hypodermic needle. Gas tight syringe (capacity 0.5-10ml) withstanding back pressure at column head is used for injection of gaseous sample. Solid samples must be dissolved in suitable solvent before their injection. Insoluble, non volatile, extensive polar, high molecular weight and thermally unstable sample must not be injected directly as they clog and damage the chromatographic column hence they must be suitably converted into soluble, volatile, non-polar or less polar, lower molecular weight, and thermostable form for efficient separation and accurate detection. Following methodologies can be adopted for efficient handling of such type of samples; 04.B.I.a. Sample pyrolysis

The technique converts high molecular weight non-volatile compounds into lower molecular weight volatile fragments by breaking them in the presence of heat in controlled oxygen less atmosphere. As the breaking of high molecular weight compound is done in the presence of heat for improving its separation for gas chromatography it is also known as pyrolysis gas chromatography (PGC). The technique is used mainly for separation and analysis of polymers including analyzing some molecules by GC/MS which are not chromatographed by conventional methodology. 04.B.I.b. Sample derivatization

Certain compounds with high polarity, thermal instability and low volatility induces problem for their separation and analysis via gas chromatography. This problem can be efficiently overcome by treating these compounds with chemical reagent which replace active hydrogen atom from parent compound with trimethylsilyl -Si(CH 3)3 or similar group. This process of replacing active hydrogen with trimethylsilyl group is known as Silylation and reagent employed is called as silylation reagent (see figure 08) such as N-trimethyl silylimidazole (TSIM), N-O-bis-trimethyl silyl trifluoro acetamide (BSTFA), and N-O-bis-trimethylsilyl acetamide (BSA). 04.B.I.c. Metal complexation

Separation and quantitative analysis of metals are quite tedious by gas chromatography due to their thermal stability and low volatility. The phenomenon of complexation is better suited for metal ions that form neutral metal complexes with β-diketone ligands thereby making them easy to handle. A wide varieties of metals like beryllium, aluminum, chromium-(III) can be separated and quantitatively analyzed by forming complexes with either unsubstituted

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acetylacetone or their halogenated (fluorinated) derivatives like trifluoroacetylacetone or hexafluoroacetylacetone (see figure 08). 04.C. Column thermostat For efficient working and better resolution, temperature of column must be controlled to few tenths of a degree. The optimum temperature at which column has to be maintained is solely depends upon sample boiling point and degree of resolution. A reasonable elution interval of 2-30 minutes is achieved if column temperature is maintained either slightly greater or equal to average boiling point of the sample. Electrically heated metal blocks, circulating air baths, and jacket fed with vapors from constant boiling liquid are some of important methodology used for controlling column temperature. Minimal temperature provides good resolution but at cost of greater elution time. Wide boiling range samples are easily handled by automated temperature programming systems. As per need the temperature of column is increased or decreased in a steady stepwise pattern or continuously. 04.D. Separating column Specially designed chemically inert and thermally resisted cylindrical shaped rolled columns, packed with suitable stationary phase and enclosed in a thermostatically controlled chamber (oven) are used for separation and analysis in gas chromatography. Column dimensions, temperature, mode of packing, nature of stationary phase & its concentration, type of backing material are some of the important factors that affect net resolution of components.

04.D.I. Types of separating column On the basis of column dimension and type of stationary phase used for packing gas chromatographic columns are of following two types 04.D.I.a. 04.D.I.b.

Packed column Capillary column 04.D.I.b.1. 04.D.I.b.2.

04.D.I.a.

Micropacked column Open tubular column 04.D.I.b.2.1. Wall coated open tubular column (WCOT ) 04.D.I.b.2.2. Support coated open tubular column (SCOT ) 04.D.I.b.2.3. Porous layer open tubular column ( PLOT )

Packed column

They are one of the oldest columns used initially in gas chromatographic experimentation. These column exhibit following characteristics

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04.D.I.a.1. Column dimensions: Length: 3-5 meters Internal diameter : 1.6-9.5 mm (ideally 2-4mm) Shape: cylindrical Material of construction: Metal (stainless steel, copper, aluminum), thermal resisted glass & plastic (polytetrafluoroethylene or Teflon) 04.D.I.a.2. Components of packed column

Essentially, packed column consist of following two components; 04.D.I.a.1. Backing or solid support material 04.D.I.a.2. Stationary phase

Figure 02: Gas chromatographic columns & their types 04.D.I.a.1. Backing or solid support material

Solid support material meant for providing support to thin uniform layer of liquid stationary phase over it. Acid washed and activated diatomaceous earth or Kieselguhr (commercially known as Dicalite, Chromosorb P, Sterchamol, Celit, Chromosorb W) is most commonly used

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support material with an average internal pore diameter of 2 µm if derived from fire brick and 9 µm for filter aid derived material. For better resolution, it is advisable that internal diameter of column should be 8 times more than that of support material. Carborundum, Teflon, microglass beads, and alumina are some of other but less widely used supporting material used in gas chromatography. 04.D.I.a.2. Ideal characteristic of solid support material a. Porous with large surface area b. Chemically inert, thermally resisted, and mechanically stable. c. Consist of microsized uniform spherical particles. d. Good wet-ability with liquid phase 04.D.I.a.3. Problem & measures of peak tailing

Although a most commonly used backing material, diatomaceous earth is itself not free from negative head. One of the major drawbacks associated with it is presence of numerous hydroxyl groups (Silanol group) over their surface causing adsorption mainly of polar compound thereby leading to a well known phenomenon of peak tailing. This problem of peak tailing of polar compounds can however be overcome by following any one of the two mentioned methodologies: Employing a highly polar liquid stationary phase which adsorbed firmly over the solid supporting material and hide up the effect of surface silanol group. Or, alternatively and more efficiently by chemically converting silanol group (Si-O-H) into silyl ether (Si-O-Si) by use of suitable silanizing reagent like demethyldichlorosilane, or hexamethyldisilazane. Stationary phase

Carbowax 200 Apiezon-L

Squalance Silicon rubber gum

Carbowax20M/polyethylene glycol

Application

Withstanding ◦ temperature( C) Aldehyde and ketones 150 Aldehyde, ketones, aromatic 250-300 compounds, alcohols, pesticides, fatty acids Hydrocarbons 150 Aromatic compounds, alcohols, 300-400 vitamins, pharmaceuticals, sugars, alkaloids, gases, pesticides, urinary and bile compounds Gases, alcohols, pesticides, aromatic 200-250 compounds

Table 02: Various stationary phases of gas chromatography & their characteristics

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04.D.I.a.2. Stationary phase

Stationary phase can either be solid or liquid, later is advantageous in terms of efficiency, resolution, & application. Thus discussed here briefly; 04.D.I.a.2.i.

Liquid stationary phase

Liquid stationary phase is the principle site of separating mixture of compound by partition phenomenon between mobile gas phase and stationary liquid phase. Till date now, numerous liquid phases with differential degree of polarity are available as per the requirement of procedure and sample to be handled. Generally for better resolution liquid phase is coated over solid support material (see backing or solid support material section) in a uniformly distributed thin layer pattern (1-15%) providing a large surface area for efficient separation. Ideal characteristic of liquid stationary phase a. It should be an ideal solvent for ideal separation b. It should be chemically inert. c. It must have better separation power. d. It must be compatible with wide range of material to be handled. e. It should be non-volatile in nature and must able to resist high temperature during operation. Classification of liquid stationary phase

Liquid phases are classified on the basis of it degree of polarity which is as follows A.

Polar liquid stationery phases Glycol, hydroxy acid, glycerol

B.

Moderately polar liquid stationery phases Dinonyl phthalate

C.

Highly polar liquid stationery phase Polyglycols or carbowaxes

D.

Hydrophobic liquid stationery phase Silicone gum rubber (for high temperature), paraffin oil (Nujol), Apiezon-L, squalane.

04.D.I.b. Capillary column

They are next generation, costlier separating columns containing thin coherently distributed layer of stationary phase over their internal wall surface. They have following characteristics

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Column dimensions: Length: 5-100 meters Internal diameter : 0.1-0.75 mm Shape: cylindrical Material of construction: Fused highly cross glass or Stainless steel, or Quartz Film thickness: 0.2-5µm Temperature withstand : 200-400 degree Celsius Loading capacity : 10-1000 ng

These columns are further be subcategorized as 04.D.I.b.1. Micropacked column:

These columns rely on solid particles packed over the whole diameter of column. 04.D.I.b.2. Open tubular capillary column (OTC):

Compare to micropacked column, open tubular capillary columns have an unrestricted flow path at middle for smooth movement of carrier gas and the stationary phase is coated along the internal diameter of whole column. They are further be classified as 04.D.I.b.2.1. Wall coated Open tubular capillary column (WCOT)/wide bore capillary columns

These columns have an internal diameter of 0.53mm & length of 10-30 meters. The stationary phase, in WCOT, is coated uniformly & directly over the internal wall of capillary. Until the introduction of bonded phase polymer column, internal coating for these types of column is a tedious process due to poor wet-ability of liquid stationary phase. Ideally the thin film formed over the internal surface of column should be non-extractable and thermally resisted. A variety of functional group can be efficiently blended into polysiloxane chain to provide stationary phase of differential polarity and selectivity. In order to remove the extrageneous matter or any organic or inorganic contamitent, or pyrolytic product, column must be flashed with pure solvent prior to use. Advantages of WCOT

a.

Provide rapid analysis compare to packed columns.

b.

Offer a short retention time.

c.

Great inertness and long life.

d.

Better resolution with low bleeding.

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e.

Great reproducibility.

f.

Efficient separation and analysis of large molecular weight and high boiling mixtures.

04.D.I.b.2.2

Support coated open tubular column (SCOT)

In these types of column instead of direct coating of stationary phase over the capillary internal wall, stationary phase is coated over finely divided layer of solid support material which then placed onto internal walls of separating column.

Figure 03: Internal view of capillary column 04.D.I.b.2.3.

Porous layer Open tubular capillary column (PLOT)

Unlike the above mentioned two columns (WCOT & SCOT) where partitioning plays an essential role in separation due to liquid stationary phase, in porous layer Open tubular capillary column (PLOT) adsorption plays an key role in separation due to coating of very small uniformly sized suitable solid particles (alumina, carbosieve) onto the internal wall of column. These columns are analogues to packed columns, but differ from them in size of particles which is very less comparatively. However they suffers a drawback of mechanical instability of coating material compare to liquid stationary phase and needs extra care for their handling. 04.E. Detectors A detector is located at the exit end of column and responsible for analysis of individual component of sample as they leave column. Ideally the volume of detector is kept very small to prevent any remixing of components.

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04.E.1. Ideal requirement of detectors includes a. It must be universal in nature. b. Easy to handle and operate. c.

Low response time and high sensitivity.

d. Single detector compatible for detecting multiple compounds. e.

Thermally stable even at high temperature.

f.

Not affected by chemical constituent of sample or flow rate of carrier gases.

g. Insensitive towards undesired compounds. h. Least affected by baseline noise, drift, and must be linear in response.

04.E.2. Types of gas chromatographic detectors 04.E.2.I.

Thermal conductivity detectors (TCD)/Hot wire detectors/ Katherometer

They are one of the oldest known gas chromatographic detectors but because of their large volumes, contamination problem, and low sensitivity are unsuitable to work with capillary columns. However, their excellent response over low flow rate (1ml/min) and better sensitivity with hydrogen or helium gases makes them ideally suitable with capillary column.

Figure 04: Thermal conductivity detector

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Principle, construction & working

Thermal conductivity detector measures changes associated with thermal conductivity of carrier gas both blank and with sample upon passing them through thermal conductivity cell meant for their detection. The detectors is based upon the fact that, amount of heat lost from filament to detector wall is directly proportional to thermal conductivity of carrier gas hence concentration of analyte which is then recorded and plotted as chromatogram. Helium and hydrogen are more commonly used carrier gas with this type of detectors owing to their high thermal conductivity values. TCDs are consist of thermal conductivity cell containing four identical filaments made up of tungsten or tungsten rhenium alloy or alternatively tungsten sheathed with gold and arranged in a Wheatstone bridge circuit such that one set of filament known as reference filaments are surrounded by carrier gas alone while another set of filament known as sample filaments are blanket by column effluent and is laying just opposite to reference filament filaments. Under normal condition when only blank gas is passes through both reference and sample filament there is no change in filament conductivity thus Wheatstone bridge is in balanced condition. However, once the column effluent containing organic compound or separated constituent comes in contact with filament ultimately leads to imbalance of Wheatstone bridge owing to differential thermal conductivity of carrier gas which is then related to net concentration of analyte presence in effluent. This difference in thermal conductivity of blank and carrier gas with sample is measured and fed into recorder producing a chromatogram. 04.E.2.II. Flame ionization detectors (FID)

These detectors are one of the most sensitive (mass sensitive rather than concentration sensitive) and their sensitivity is usually expressed in terms of mass per unit time, universally accepted universal detector (not universal but close to universal detectors), free from contaminating problem is employed for gas chromatographic system especially for routine analysis employing capillary gas chromatographic columns. They are flow rate independent detectors having better linear response against wide varieties of organic compounds except oxidized carbon containing compounds (alcohols, or carbonyl compounds) which produces less or no ions upon ionization. Modification in parent FID increase their spectrum and sensitivity towards quantification & detection of inorganic compounds like nitrogen or phosphorous by alkali flame detectors (AFD), or employing flame photometric detectors (FPD) for analysis of compounds containing phosphorus or sulphur. As FID and their analogues detectors (AFD, FPD) are operate at as high as 400 degree Celsius they are free from any reasonable contamination from condensation and are easily used with hyperthermic columns. Nitrogen or Argon is choice of carrier gas for FID and usually mixed with hydrogen before passing to detector.

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Figure 05: Flame ionization detectors Principle and Working

These detectors are based on fact that upon pyrolysis of organic compounds in presence of hydro-oxy flame (hydrogen and oxygen flame) results in their ionization (production of ions) thereby producing a current which is then sensitized (by electrometer) & well amplified for further plotting of a chromatogram and determination of individual components of mixture. Simply a FID consist of pair of electrodes, out of which one electrode is itself acting as burner jet having negatively charge while another electrode is mounted top near or extended into tip of flame and acting as a positively charged electrode. Carrier gas containing column eluent is mixed with hydrogen and burned in an atmosphere of air providing sufficient energy to ionized analyte, if present in eluent. The ions so produced are collected at electrode constituting an ion-current which is suitably detected. In order to avoid drifts and moisture FID is enclosed in tight jacket and heated sufficiently before use. Head & tails of FID

FID shows no or low response against oxidized compound as mention above, but their sensitization can be improved by converting them into reduced form. Another advantage of FID is their insensitiveness against moisture and permanent gases like carbon dioxide, carbon monoxide, sulphur dioxide, ammonia etc hence can be used for analysis of moist compounds or trace of organic compounds against above mention carrier gas as a background without affecting its sensitivity. FID response proportional to number of -CH 2- groups when ionized into flame i.e. it response twice to an equimolar concentration of butane than ethane.

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04.E.2.III. Electron capture detector (ECD)

Compare to FID, electron capture detectors measures a net reduction in current when column effluent passes through it. Numerous highly sensitive and selective ECD detectors are available for quantification and determination of various compounds that are problematic with FID (but may not be with counter part) like halogens, carbonyl compounds, anhydrides along with sulphur containing compounds, nitrates, organometallic compounds, nitrites etc. ECD are highly sensitive if it coupled with carrier gas like nitrogen or with methane-nitrogen mixture and better efficiency can be achieved while conjugating it with capillary system using helium or hydrogen gas as par. Principle, construction & working

ECD under normal condition employ ionization of carrier gas mostly nitrogen (argon for high electron affinity compounds) by radioactive substance like tritinium or nickel-63, slow electrons so produced under steady potential are collected by electrodes thus generating a constant baseline current. When the column effluent containing electronegative organic compounds such as halogen or oxygen atoms, react with slow electron, and get them replaced with higher mass negative ions thereby resulting in reduction in current flow which is further sensitized and amplified to give a chromatogram. 04.E.2.IV. Photoionization detectors (PID) Principle, construction & working

PID are some of most recently introduced ionization detectors working on same principle of FID instead of flame, PID make use of high intensity UV radiation for ionizing organic compounds present in column effluent. Beside this there is no need of ancillary gas supply other than carrier for PID which makes the system portable and easy to handle compare to FID. PID is a universal detector for most of organic compounds whose bond energies fall between its irradiation ranges (106-149nm) or compounds having low ionization potential. Requirement of suitable intensity UV lamp, its maintenance, life time, contamination of lamp window are some of its limitation. 05.

CHROMATOGRAPHIC BEHAVIOR OF SOLUTE

05.A. Retention time It is the time of emergence of maximum peak after the injection of sample

Or It is the time period of emergence of peak after injection of sample into chromatographic system.

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Or It is the sum of total time spends by the solute in both mobile phases as well as in stationary phase. It is denoted by tR Mathematically retention time can be expressed as

Where, tR =Retention time t’R =Adjusted retention time tM= Time spend by component in mobile phase. As different component have different retention time hence they come out from column at different interval and get separated out. 05.A.I. Factors affecting retention time The retention time of a solute depends upon following factors a.

Dimension of column (length and diameter)

b.

Nature or mode of column packing

c.

Particle size of packing

d.

Nature of sample to be handled

e.

Solvent system

f.

Solvent flow rate

g.

Temperature during separation

h.

Nature of adsorbent.

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Figure 06: Chromatogram 05.B. Adjusted retention time It is the total time spends by a solute in stationary phase and denoted by t’R. Mathematically, it is expressed as

Where, t’R =Adjusted retention time tR =Retention time tM= Time spend by component in mobile phase. 05.C. Capacity factor (K) It is the ratio of total time spend by solute in stationary phase to ratio of time spend by solute in mobile phase. It is expressed by K and mathematically expressed as

Where, K= Capacity factor t’R =Adjusted retention time tM= Time spend by component in mobile phase.

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05.D. Retention volume Retention volume is related to carrier gas and indicates volume of carrier gas required to elute one half of component from the column as indicated by peak maximum. It is denoted by vR & mathematically expressed as

Where, vR=Retention volume tR = Retention time f c= Mobile phase flow rate Porosity refers to ratio of interstial volume of the packing to volume of total mass. For solid, porous, and capillary packing total porosity is 0.35-0.45, 0.75-0.90, and 1.00 respectively. 05.E. Adjusted retention volume It is the volume of gas that holds up by column, injector, and detector system due to their intercalated volume. It is denoted by v’R and mathematically expressed as

Where, V’R=Adjusted retention volume t’R =Adjusted retention time f c= Mobile phase flow rate 05.F. Net retention volume The average flow rate of carrier gas is differs from the outlet flow rate due to compressibility and pressure gradient of gas exist down to the separating column. It is denoted as vN and mathematically expressed as

Where, vN= Net retention volume V’R=Adjusted retention volume J= Compresibility factor

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05.G. Partition coefficient When solute enters into a chromatographic system it immediately distributed itself between mobile and stationary phase. If flow of mobile phase is stopped at any particular time it is assumed that there is a distribution of solute in mobile and stationary phase. The concentration of solute in each phase is given by

Where, k= Partition coefficient (k=1; solute distributed equally in both phases) Cs= Solute concentration in stationery phase Cm= Solute concentration in mobile phase For symmetrical peak i.e. when the peak maximum appears at the exit of column half of the solute eluted in the retention volume and half remains distributed between volume of stationary phase and volume of mobile phase 05.H. Partition ratio It relates the equilibrium distribution of sample with the column to the thermodynamic property of column and to the temperature. For a given set of operating parameter partition ratio is measure of time spend in stationary phase relative to the time spend in mobile phase or alternatively it is the ratio of mole of solute in the stationary phase to mobile phase. It is denoted as k’ & mathematically expressed as

Where, k’=Partition ratio k=Partition coefficient β=Volumetric phase ratio 05.I. Relative retention The relative retention (α) of two solute when solute 1 elute before solute 2 given by

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Where, symbols have their usual meanings. 06. COLUMN EFFICIENCY Column efficiency of a chromatographic system can be expressed by following parameters. 06.A. Height of effective theoretical plate (HETP) It is a dimensionless quantity which describes efficiency of chromatographic column. It may be defined as length of column required by a solute molecule for single equilibration. It is represented as Heff and mathematically expressed as

Where, Heff =Height of effective theoretical plate L=Length of column N=Number of theoretical plate From above equation it is clear that, better column efficiency could be achieved by reducing overall height of theoretical plates which in turn enhanced number of theoretical plate along with column length. 06.B Number of plate or plate number (N) It may be defined as number to times a solute molecule undergoes equilibration between stationary and mobile phase. As HETP, it is also a dimensionless quantity, denoted by N and mathematically expressed as;

Or,

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Where, N=Number of theoretical plate L=Length of column Heff =Height of effective theoretical plate 06.C. Resolution or degree of separation Resolution may be defined as distance between two bands of peak divided by average band width or alternatively, it is the degree of separation between two adjacent bands distinctly. It is denoted by Rs and expressed as

Where, 2

1

tR - tR = Retention time (w1-w2)=Band width A good resolution (See figure 07) in chromatographic system is characterized by separation of two bands distinctly with sharp peak height and less band width also the band should be free from tailing and fronting. 07. APPLICATION OF GAS CHROMATOGRAPHY Acceptability of gas chromatography increased day by day in various scientific fields for both quantitative (by area normalization & addition of internal standard) and qualitative determination of wide varieties of organic and inorganic compounds. The technique can be efficiently used for determination of volatile content or volatile impurities of various drugs such as halothane, methoxy-flurane, and ethanol. The technique is equally effective in determination of chloroform or ethyl acetate in colchicines, ethanol in doxycycline, dichloromethane in ampicillin sodium, propan-2-ol in warfarin sodium etc. Beside this gas chromatography is also used in detection of various fragrance components and their quality in perfumery and cosmetics.

Gas chromatography is extensively used in analysis of crude petroleum product for determination of gasoline, LPG, nitrogen or/and sulphur, waxes, and unsaturated components. The technique also employed for determination and analysis of color and flavoring agent in food and beverages including determination of residual solvent in various spices, and presence of insecticide or pesticide in foods. Gas chromatograph when conjugated with quadrapole mass spectroscope (for detail on this technique please refer chapter on mass spectrometry) used for isolation of Uranium.

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Figure 07: Comparative diagrammatic representation of good & poor resolution

In biochemical analysis and clinical medicine gas chromatography along with mass spectroscopy used for quantitative estimation of various metabolites of drugs along with serum determination of hormones and blood gases. For example with an aid of propan-2-ol as an internal standard quantitative determination of ethanol in blood sample can be done satisfactorily.

Figure 08: Structure of some derivatizing & metal complexing agents

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08.

182

EXERCISE a. Write a brief note on gas chromatography.

b. Classify gas chromatography and mention their pros & cons. c. Enumerate with detail various factors that affect gas chromatography. d. Illustrate instrumentation of gas chromatography by a well labeled diagram. e. Write a brief note on various carrier gases used in gas chromatography f. Enlist ideal characteristics of a carrier gas to be used as mobile phase in gas chromatography. g. Write a short note on; a.

Sample pyrolysis

b.

Sample derivatization

c.

Metal complexation

h. What is packed column? Briefly explain its components. i.

Define peak tailing. Enumerate various methodologies to overcome this problem.

j.

Classify capillary column and explain WCOT, SCOT, and PLOT column briefly.

k. What are the various types of detectors used in gas chromatography? Give their idealistic properties. l.

Compare TCD with FID.

m. Write a short note on advantages and disadvantages of flame ionization detectors. n. Explain thermal conductivity detector in terms of principle, working and application. o. Write a short note on a. Retention time b. Adjusted retention time c. Capacity factor d. Net retention volume e. Partition coefficient f. Partition ratio g. Relative retention

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p. Write a short note on HETP and resolution. q. Co-relate HEPT with number of plate. r. Discuss briefly application of gas chromatography. 09.


Gas chromatography can be used for a. Qualitative analysis b. Quantitative analysis c. Both d. None of the above c Quantitative analysis in gas chromatography could be done by a. Area normalization method b. Internal standard addition method c. Both d. None of the above c The technique of gas chromatography can handle a. Volatile substances b. Thermostable substances c. Low molecular weight substance d. All the above d Limitation of gas chromatography includes a. Highly polar compounds b. Compound of non-volatile nature c. Thermolabile and ionic compounds d. All are true d The technique of gas chromatography was first introduced by a. Martin & Synge (1914) b. Martin & Synge (1941) c. Martin & Synge (1994) d. Martin & Synge (1944) b

Chowrasia, Deepak

True experimentation on gas chromatography was initially done by _______in 1954 with lower fatty acid. a. Martin & James b. Marry & James c. Martin & Jones d. Martin & Jacque a Mobile phase used in gas chromatography is a. Highly pressured liquid b. Liquid at critical temperature c. Pure and inert gas d. None of the above c In gas solid chromatography (GSC), stationary phase is a. Solid b. Liquid c. Gas d. None of the above a In gas liquid chromatography (GLC), stationary phase is a. Solid b. Liquid c. Gas d. None of the above b Out of GSC & GLC which technique has limited application a. GSC b. GLC

GAS CHROMATOGRAPHY


c. Both d. None of the above b Limited applicability of GSC is due to its a. Retention of carrier gas over stationary phase b. Reduction of overall surface area c. Tailing problem d. All of the above d In GSC and GLC separation of component is usually takes place via a. Adsorption & partition b. Partition & adsorption c. Adsorption & absorption d. None of the above a Any reduction of stationary phase particle size ultimately a. Reduces surface area b. Increases resolution c. Increases surface area d. Both b & c d Number of theoretical plate ________ with increase in particle size of a. stationary phase a. Increases b. Decreases c. Remain constant d. None of the above b Resolving power of chromatographic column ______ with increase in length a. Increases b. Decreases c. Remain constant d. None of the above a Reduction in column diameter a. Reduces efficiency

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b. Increases efficiency c. No effect on efficiency d. No comment b Carrier gas used in gas chromatography for a. Elute out any residue left after separation b. As a cleaning agent c. As a mobile phase d. None of the above c Most commonly used carrier gas in gas chromatography is a. Hydrogen b. Neon c. Oxygen d. Helium d Pick out the gas mostly used with thermal conductivity detectors a. Hydrogen b. Neon c. Oxygen d. Helium d Purity of carrier gas used for gas chromatography is a. 99.95% to 99.995% b. 99.5% to 99.995% c. 99.99% to 99.999% d. 99.995% to 99.9995% d All are the property/s of carrier gas used in gas chromatography except a. Low density b. Oxygen free c. Explosive d. Highly pure c

GAS CHROMATOGRAPHY


Hydrogen mostly avoided as carrier gas owing to its property of a. Impurity b. Availability c. Incompatible with unsaturated compounds d. Inflammability c,d Sample injection port usually heated to a temperature of ____ degree Celsius above the lowest boiling point of sample a. 40 b. 50 c. 60 d. 70 b Samples that are unable to vaporize are ________ prior to their injection into gas chromatographic system a. Derivatized b. Deified c. Defined d. Developed a Capacity of micro-syringe for introduction of liquid sample is a. 1-100µL b. 0.1-1000µL c. 0.1-100µL d. 10-100µL c Micro-syringe used for introduction of ________ sample a. Solid b. Liquid c. Gas d. All the above b

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Gas tight syringe used for injection of gaseous sample into chromatographic system is having capacity of a. 5-10ml b. 0..5-100ml c. 00.5-10ml d. 0.5-10ml d Can gas chromatography handle solid sample directly a. Yes b. No c. Can’t say d. None of the above b An Insoluble, non volatile, extensive polar, high molecular weight and thermally unstable sample can however be used for separation by gas chromatography by adopting the technique/s a. Pyrolysis b. Derivatization c. Complexation d. All the above d Sample pyrolysis is adopted for compounds that are a. High molecular weight b. Non-volatile in nature c. Both d. None of the above c The technique of pyrolysis is done in the presence of a. Heat alone b. Heat and surplus oxygen alone c. Heat and water d. Heat and trace oxygen atmosphere d

GAS CHROMATOGRAPHY


The technique of Sample pyrolysis is also termed as a. Pyrolysis gas chromatography (PGC) b. Pyrolytic gas chromatography (PGC) c. Pyrolysis gas chromatogram (PGC) d. Pyrolysis gas chromatograph (PGC) a Mostly the technique of pyrolysis gas chromatography (PGC) is done for a. Semi synthetic compounds b. Polymer c. Synthetic chemical compounds d. All the above b Samples are derivatized in order to increase their a. Volatility b. Hydrophobicity c. Thermal stability d. All the above d In derivatization technique, active hydrogen of sample is replaced with

a. b. c. d.

Trimethylsilyl group Tetramethylsilyl group Triethylsilyl group None of the above

a The process of replacing active hydrogen with trimethylsilyl group is termed as a. Silyllation b. Sillylation c. Silylation d. Selylation c

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Pick out silylating reagent/s a. N-trimethyl silylimidazole (TSIM) b. N-O-bis-trimethyl silyl trifluoro acetamide (BSTFA) c. N-O-bis-trimethylsilyl acetamide (BSA) d. All the above d Metal are difficult to be analyzed by gas chromatography due to their a. Low volatility b. Thermal stability c. Both d. None of the above c Analysis of metal can be done by gas chromatography by adopting a process known as a. Metal complexation b. Metal complementation c. Metal ionization d. None of the above a “The phenomenon of complexation is better suited for metal ions that form neutral metal complexes with β-diketone ligands thereby making them easy to handle”. The statement is a. Incorrect b. Correct c. Need modification d. None of the above b Which of the halogenated derivative of acetylacetone is used for metal complexation? a. Fluorinated b. Chlorinated c. Brominated d. None of the above a

GAS CHROMATOGRAPHY


Pick out suitable acetylacetone derivative for metal complexation in gas chromatography a. Trifluoroacetylacetone b. Hexafluoroacetylacetone c. Both d. None of the above c Separating column used initially in gas chromatography is a. Packed column b. Capillary column c. Both d. None of the above a Typical dimension of a packed column is a. 3-50m; 1.6-9.5mm b. 0.33-5mm; 16-95mm c. 0.3-0.5m; 1.6-9.5mm d. 3-5m; 1.6-9.5mm d Packed gas chromatographic column are usually made-up of a. Metal, plastic, & glass b. Metal, plastic, & thermally resisted glass c. Metal, ordinary plastic, & glass d. None of the above b Pick out the plastic used in manufacturing of packed column a. Polytetrafluoroethylene b. Polytetrachloroethylene c. Polytetrabromoethylene d. Polytetraiodoethylene a Polytetrafluoroethylene is chemical name of a. Taflon b. Telfon

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c. Teflon d. Tedlon c Packed column usually consist of a. Backing material b. Packing material c. Stationary phase d. Both a & c d Dicalite, Chromosorb P, Sterchamol, Celit, Chromosorb W are some of the commercial name of a. Diatomaceous earth b. Kieselguhr c. Both d. None of the above c The purpose of above material in packed column is a. Act as stationary phase b. Act as backing support for stationary phase c. As a cleaning agent d. None of the above b Backing material mostly used in packed column is a. Carborundum b. Teflon c. Microglass beads d. Kieselguhr d Internal pore size of Kieselguhr is ___ & ____ when derived from fire brick and filter aid respectively. a. 2.0 µm; 9.0 µm b. 0.2 µm; 9.0 µm c. 2.0 µm; 0.9 µm d. 20 µm; 9.0 µm a

GAS CHROMATOGRAPHY


“For better resolution, it is advisable that internal diameter of column should be 8 times more than that of support material”. The statement is a. May be true or false b. Exactly false c. Exactly true d. None of the above c Pick out less commonly used backing material for packed column in gas chromatography a. Carborundum b. Teflon c. Microglass beads d. Kieselguhr abc The problem mostly encountered while using diatomaceous earth is a. Peak enlarging b. Peak tailing c. Peak fronting d. None of the above b Which functionality/s is commonly associated with peak tailing in diatomaceous earth? a. Selanol group b. Salanol group c. Silinol group d. Silanol group d Which type of compounds mostly affected by peak tailing a. Polar group b. Hydrophilic group c. Non-polar group d. Hydrophobic group ab

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The problem of peak tailing can be reduced by use of a. More polar stationary phase b. More polar mobile phase c. Use of silanizing reagent. d. Both a & d d Pick out examples of silanizing reagents a. Demethyldichlorosilane b. Hexamethyldisilazane. c. Both d. None of the above c The stationary phase used in packed column is a. Solid b. Liquid c. Both d. None of the above c Which stationary phase is advantageous in terms of analytical procedure in gas chromatography? a. Solid stationary phase b. Liquid stationary phase c. Both d. None of the above b The liquid stationary phase in what form used in gas chromatography a. As a thin layer coated over column b. As a thin layer coated over backing material c. Both d. None of the above b

GAS CHROMATOGRAPHY


Pick out highly polar stationary phase used in gas chromatography a. Polyglycols or carbowaxes b. Apiezon-L, squalane c. Glycol, hydroxy acid, glycerol d. None of the above

189

Pick out the costlier gas chromatographic column

a. Capillary column b. Packed column c. Both d. None of the above

a a Squalane is used as stationary phase for separation Site of stationary phase in capillary column of in gas chromatography is a. Amino acid b. Proteins a. Over the bed of backing material c. Carbohydrate b. Over external wall of column d. Hydrocarbon c. Over internal wall of column d Pick out stationary phase that can resist high d. None of the above c temperature Typical dimension of capillary column is a. Glycol b. Paraffin oil a. L-50-100m; I.D-0.1-0.75mm c. Silicone gum rubber b. L-0.5-100m; I.D-0.75mm d. All the above c. L-5-10m; I.D-0.1-0.75mm c d. L-5-100m; I.D-0.1-0.75mm The nature of Silicone gum rubber is d a. Polar Sample loading capacity of capillary b. Non polar column is c. Slight polar d. Slight non polar a. 10-1000 mg b b. 10-1000 ng Dinonyl phthalate is c. 10-1000 µg a. Polar stationary phase b. Moderate polar stationary phase d. 10-100 ng c. Strongest polar stationary phase b d. None of the above All are the sub category of capillary b column except Carbowax 200 can withhold temperature ____ in a. Packed column ` degree Celsius b. Micro-packed column a. 50 b. 150 c. Open tubular column c. 200 d. None of the above d. 250 a b

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GAS CHROMATOGRAPHY


In micropacked column the solid particles packed over a. Surface of column b. Whole diameter of column c. Both d. None of the above b Open tubular column (OTC) differs from micropacked column in a. Unrestricted flow path at middle of column b. Coating of stationary phase over internal wall of column c. Both d. None of the above c Wall coated open tubular (WCOT) column are a. Narrow bored column b. Wide bored column c. Middle bored column d. None of the above b In wall coated open tubular (WCOT) column stationary phase is a. Packed inside column b. Coated inside column c. Coated indirectly inside column d. Coated directly inside column d Wall coated open tubular (WCOT) column have stationary phase coated directly over the _____ wall of column chromatography a. Internal b. External c. Both d. None of the above a

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Advantages of WCOT is/are a. Provide rapid analysis compare to packed columns b. Offer a short retention time. c. Great inertness and long life. d. Better resolution with low bleeding abcd SCOT column can be expand as a. Self coated open tubular column b. Support coated open tubular column c. Superior coated open tubular column d. Sound coated open tubular column b In SCOT columns stationary phase is coated a. Directly over internal wall of column b. Indirectly over internal wall of column c. Both d. None of the above b _______ phenomenon plays pivot role in WCOT & SCOT column a. Partition b. Adsorption c. Absorption d. None of the above a In PLOT column _______ phenomenon plays a dominant role in separation a. Partition b. Adsorption c. Absorption d. None of the above b

GAS CHROMATOGRAPHY


Stationary phase in case of PLOT column is a. Solid or liquid b. Solid c. Liquid d. None of the above b “Gas chromatography has universal detector” the statement is a. True b. False c. Can’t say d. None of the above a Thermal conductivity detectors (TCD) is also termed as a. Hot wire detectors. b. Katherometer c. Both d. None of the above c Thermal conductivity detectors (TCD) are not suitable to work with a. Packed column b. Capillary column c. Both d. None of the above b Carrier gas that can be used with thermal conductivity detectors is/are a. Hydrogen b. Helium c. Both d. None of the above c First choice of gas with thermal conductivity detector is a. Nitrogen b. Hydrogen

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c. Argon d. Helium d Detection circuit of thermal conductivity detector is a a. Wheatstone bridge b. Wheatstep bridge c. Wheatstolone bridge d. Wheatstand bridge a Filament of Wheatstone bridge is made-up of

a. b. c. d.

Tungsten Tungsten rhenium alloy Tungsten sheathed with gold All the above

d The basic principle behind thermal conductivity detector is a. Heat b. Light c. Heat based Current d. None of the above c TCD is abbreviated as a. Thermal connectivity detector b. Thermal conductivity detector c. Thermal conductivity detection d. Thermally conductivity detector b Pick out universal detector in gas chromatography a. Thermal conductivity detector (TCD) b. Flame ionization detector (FID) c. Electron capture detector (ECD) d. None of the above b

GAS CHROMATOGRAPHY


Flame ionization detectors are insensitive against a. Oxidized compounds b. Reduced compounds c. Both d. None of the above a Alkali flame detectors are modification of a. Thermal conductivity detector (TCD) b. Flame ionization detector (FID) c. Electron capture detector (ECD) d. None of the above b Alkali flame detectors are used for quantification of a. Inorganic compounds b. Organic compounds c. Both d. None of the above a A flame photometric detector (FPD) used for analysis of compounds containing a. Phosphorus b. Sulphur c. Both d. None of the above c The choice of makeup carrier gas for FID is/are a. Helium b. Hydrogen c. Nitrogen d. Argon cd “Can a FID be used for column operated at high temperature?” a. Yes b. No c. Can’t say d. None of the above a

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Sensitivity of FID is expressed as a. Mass per unit mole b. Mass per unit volume c. Mass per unit time d. Mass per unit liter c Most commonly used detector in gas chromatography is a. TCD b. FID c. ECD d. None of the above b Pick out reason/s for FID being a universal most commonly used detector a. Highest sensitivity b. Rapid response time c. Stability & wide linear response rate d. All the above d With oxidized compound FID show low sensitivity, but their sensitivity can be enhanced by converting oxidized compounds into a. Reduced compound b. Increasing their unsaturation c. Both d. None of the above a An alkali flame detector (AFD) is ______time more sensitive to compound containing nitrogen and approximately _____ time more sensitive towards phosphorous containing compound. a. 5; 50 b. 50; 500 c. 500; 5000 d. 5000; 50000 b

GAS CHROMATOGRAPHY


Pick out the correct arrangement of alkane in response of signal strength by FID a. Methane>ethane>propane>butane b. Ethane> propane > methane > butane c. Butane > propane> ethane > methane d. None of the above c Ionization in electron capture detector is done by a. Flame b. Electron bombardment c. Radioactive substances d. None of the above c Radioactive substances that are commonly used in ECD is/are a. Tritinium b. Nickel-63 c. Both d. None of the above c In Photoionization detectors (PID) ionization is caused by a. UV light b. IR rays c. Gama rays d. Beta rays a Ancillary gas is required by all except a. FID b. TCD c. ECD d. PID d A scientist want to analyse a pesticide by gas chromatography which type of detector he choose for better result a. FID b. TCD

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c. PID d. ECD a The sum of total time spends by the solute in both mobile phases as well as in stationary phase is termed as a. Retain time b. Rate time c. Retention time d. Retention peak c Factor that affect retention time is/are a. Dimension of column (length and diameter) b. Nature or mode of column packing c. Particle size of packing d. Nature of sample to be handled abcd Time spend by solute alone in stationary phase is termed as a. Adjusted retention time b. Retention time c. Repetition time d. None of the above a In chromatographic system the term K represents a. Calling factor b. Coronary factor c. Collision factor d. Capacity factor d Column efficiency can be expressed in terms of a. HETP b. Number of plate c. Resolution d. All the above d

GAS CHROMATOGRAPHY


Expand HETP a. Height of effective theoretical place b. Height of effective theoretical plateau c. Height of effective theoretical plate d. Height of effective theoretical plane c “HETP is a dimensionless quantity”. The statement is a. True b. False c. Can’t say d. None of the above a Technically, degree of separation is also known as a. Retention b. Resolution

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c. Redissolution d. None of the above b ________ used as an internal standard for quantitative estimation of ethanol in blood sample a. propan-1-ol b. propan-2-ol c. propan-3-ol d. propan-4-ol b Area normalization in gas chromatography is used for _______ analysis a. Quantitative b. Qualitative c. Both d. None of the above a

GAS CHROMATOGRAPHY

Chapter - 11 ION EXCHANGE CHROMATOGRAPHY - Deepak Chowrasia

Chowrasia, Deepak

ION EXCHANGE CHROMATOGRAPHY

ION EXCHANGE CHROMATOGRAPHY (Chapter Overview) 01. INTRODUCTION .................................................................................................................. 199 02. ION EXCHANGERS ............................................................................................................ 199

02.A. Ideal characteristic of ion exchanger ........................................................199 03. TYPES OF ION EXCHANGER ......................................................................................... 200 03.A. Organic ion exchangers ............................................................................................. 200 03.A.I. Anionic organic ion exchanger. .....................................................................200 03.A.II. Cationic organic ion exchangers. ................................................................201 03.A.III. Amphoteric organic ion exchanger. ...........................................................201 03.B. Inorganic ion exchangers .......................................................................................... 201 03.B.I. Inorganic anionic ion exchangers ...............................................................201 03.B.II. Inorganic cationic ion exchanger ................................................................201 03.C. Synthetic inorganic ion exchanger ..........................................................................201 04. MECHANISTIC PROFILE OF ION EXCHANGERS ................................................202 05. DISTRIBUTION COEFFICIENT (D) .............................................................................. 203 06. FACTORS AFFECTING ION EXCHANGE CHROMATOGRAPHY ...................203 06.A. 06.B 06.C. 06.D. 06.E. 06.F. 06.G. 06.H.

Surface area of stationary phase ............................................................................. 203 Number of exchange sites ......................................................................................... 203 pH ................................................................................................................................... 203 Temperature ................................................................................................................ 203 Degree of Swelling ......................................................................................................204 Flow rate of eluent ......................................................................................................204 Cross linking of resin .................................................................................................204 Concentration .............................................................................................................. 204 06.I. Complex formation ......................................................................................... 204 06.J. Column geometry ........................................................................................... 204

07. INSTRUMENTATION .........................................................................................................205 07.A. Column preparation and procedure ...................................................................... 205 07.B. Detection of column effluent ....................................................................................206 08. APPLICATIONS ...................................................................................................................206 09. EXERCISE .............................................................................................................................. 207 10. MULTIPLE CHOICE QUESTIONS ................................................................................207

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ION EXCHANGE CHROMATOGRAPHY 01. INTRODUCTION Thompson and Way around 1850 successfully exchange calcium ion present in soil against ammonium ion as a solution of ammonium sulfate, publishing their result, stimulated an extensive exploration of compounds having an inherent capacity of exchanging ions. However, some of them are failed due to adaptation of defaulted methodology, instability of exchange process against wide range of pH, and other circumstances. Earlier in nineteen, fruitful synthesis of sulfonic acid & polyamide resin (1935) and styrene & acrylic resin (1944) ultimately breakthrough the possibility of utilizing ion exchange phenomenon for various analytical and industrial purposes including analysis and purification of drinking water, separation of biological macromolecules like proteins and nucleic acid, and analysis of semi conductors etc. 02. ION EXCHANGERS Ion exchangers are the key component that plays an essential role of exchanging ions with an analyte (test solution) and can be describes as “ A 3-dimensional resin or polymeric macromolecular, charged, insoluble solid substances either organic or inorganic in nature, may be synthetic or natural in origin, employed in ion exchange chromatography ( IEC ), and works on the principle of isoelectric exchange of ions during chromatographic processes. Speaking strictly under equilibrium condition ion exchangers are electrically neutral in nature (may or may not be) i.e. they contain equal number of positive and negative ions. For example an anionic ion exchanger is a positive charge macromolecular system with negative charged counter ions (mobile ions) likewise a cationic ion exchanger which is widely used is a negatively charged macromolecular structured substance having positively charged counter ions. Counter ions, also known as mobile ions are the heart of ion exchangers and are responsible for overall efficiency of ion exchange process by exchanging themselves against the ions of analyte or sample. Basically, the counter ions are exactly opposite in charge with that of fixed ions of resin and get exchanged during the separation process with isoelectric ions of analyte. Sodium, chloride, hydrogen, hydroxyl, sulfate are some of the most commonly employed mobile ions present in ion exchange resin ( IER).

02.A.

Ideal characteristic of ion exchanger a. Essentially it must be highly porous and open tree like structure. b. Insoluble in both polar as well as non polar solvents. c. Under normal condition of separation, it must be electrically neutral i.e. exchange opposite change ion in same ratio. d. Must be stable in terms of mechanical, chemical and physical properties.

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e. Works over wide range of pH. f. Should be free from any hazardous interaction against both analyte and analyst g. Must be highly cross linked macro-porous in structure to provide extensive large surface area for efficient separation. h. Should be denser than water & insoluble in same. i.

It must be cheap, easily available, and versatile in separation process.

Figure 01: Diagrammatic representation of ion exchange mechanism 03.

TYPES OF ION EXCHANGER

03.A. Organic ion exchangers Organic ion exchangers consist of an irregular three dimensional hydrocarbon chain (formed due to polymerization of suitable compounds via addition or condensation phenomenon) to which different functional groups are attached accordingly to their electrical properties. Advantages of organic ion exchangers include their practical insolubility in most of the commonly used organic and inorganic solvents, rigorous stability against physical, chemical, & mechanical conditions, and inherent capacity of ion exchange. Organic ion exchanger are further be classified as’ 03.A.I. Anionic organic ion exchanger contains primary, secondary, tertiary, or quaternary amino groups.

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03.A.II. Cationic organic ion exchanger s have sulfonic, phenolic, carboxylic, or phosphoric groups. 03.A.III. Amphoteric organic ion exchanger has both anionic and cationic functional groups. 03.B. Inorganic ion exchangers

03.B.I. Inorganic anionic ion exchangers Zeolite (analcite), clays (montmorillonite), Glauconite 03.B.II. Inorganic cationic ion exchanger Apatite & hydroxyapatite 03.C. Synthetic inorganic ion exchanger Synthetic inorganic ion exchangers are somewhat superior over organic ion exchanger in terms of ion exchange capacity, stability against temperature, better tolerance against radiation, and good selectivity for inorganic ions. For example Molybdate, Tungstate, Phosphate, Vanadate and Arsenate forms of tetravalent metals are superior cationic exchanger in terms of above mention qualities as compare to their hydrous oxides of tri and tetravalent metal forms which are unstable in the presence of acid or bases. Ion exchanger Strongly acidic cation exchange resin

Working pH 1-14

Uses Fractionation of cations, lanthanide, vitamins, peptides, amino acids

5-14

Fractionation of cations, biochemical separation, transition metal ions, antibiotics, organic bases

Strongly basic Quaternary anion exchange ammonium polystyrene resin

0-12

Fractionation of anions, vitamin B complex, halogens, fatty acid, alkaloids

Weakly basic anion exchange resin

0-9

Fractionation of anionic complex of metals, anions of different valences, amino acids and vitamins

Weakly cation resin

Example Sulphonated polystyrene

acidic Carboxylic exchange polymethacrylate resin

Phenol formaldehyde and polyamine polystyrene resin

Table 01: Some ion exchangers & their applications

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04. MECHANISTIC PROFILE OF ION EXCHANGERS It is a well known fact that ion exchanger irrespective of their origin and physiochemical properties, is a highly cross linked macromolecular structured compounds containing fixed ions (functional group-cationic/anionic in nature) which is bind with a backing material mostly a hydrocarbon chain and contains mobile or counter ion whose charge is exactly opposite to that of fixed ions which when comes in contact with analyte solution (sample solution) get themselves exchanged with isoelectric ions of analyte solution thus promoting the process of separation. Since, analyte solution have several ions of same charge, but with differential affinity against mobile ions of exchanger thus separation is possible.

To elaborate this phenomenon, let us consider an anionic ion exchanger resin containing a positive charge function group ( C+) attached with polymeric backing hydrocarbon chain (Res) and have negatively charge counter ions which are here for the purpose of ease designated as (A-). Therefore, complete structure of an ion exchanger can be summarized as below; +

-

(Res-C )A

When a solution of analyte containing anion ( a-) is passes through resin, anion ( a-) from solution get exchanged with counter anion ( A-) of resin. +

-

(Res-C )A

+

(Anionic Ion exchanger)

-

a

→→→

(Test solution anion)

+

-

(Res-C )a

-

+

(Separated test sol. Anion) -

-

A

(Exchanged resin counter anion) -

If the solution of analyte contains multiple anions ( b , c , d etc) all will get separated out depending upon their differential affinity towards ion exchanger resin. Thus an overall ion exchange mechanism for anionic and cationic ion exchanger can be summarized as below Anionic ion exchange mechanism +

-

R A

+

-

a

→→→

+ -

+

A

- +

+

C

Ra

-

Cationic ion exchange mechanism -

+

RC

+

c

+

→→→

Rc

+

05. DISTRIBUTION COEFFICIENT (D) At steady state, distribution coefficient of ions in ion exchange chromatography may be defined as the ratio of concentration of ions present in stationary phase to that of mobile phase. It is denoted by D.

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Where, D= Distribution coefficient Cs= Concentration of ions in stationary phase Cm=Concentration of ions in mobile phase. It must be noted that for an effective and efficient separation of ions, value of distribution coefficient (D) must be contrasting in nature. As per situation, when it is required to remove unwanted ions from system or analyte solution the value of D must be higher i.e. greater than 1000. On other hand, a lower value of D (<1) ultimately collect ions of interest. However, ions which are having same or near about value of distribution coefficient are difficult to be get separated. 06. FACTORS AFFECTING ION EXCHANGE CHROMATOGRAPHY There are numerous factors that affect the separation efficiency of ion exchange chromatography (ICE) like concentration, number & types of ions present in sample solution, properties of individual ions, structure and exchange capacity of ion exchanger, surface area, degree of cross linking, swelling properties, sorption phenomenon of stationary phase, pH, overall temperature of system, solvent employed for elution, diffusion effects, and electrochemical phenomenon. 06.A. Surface area of stationary phase Smaller the size of resin, larger the surface area per unit volume; a good separation is thus obtained. 06.B Number of exchange sites Greater the number of exchange sites present on resin better is the ion separation. 06.C. pH Cationic exchanger work best at basic pH, while exchange capacity of anionic exchanger reduces with increase in pH. 06.D. Temperature As per other separation process, an increase in temperature up to certain degree leads to show positive effect on ion separation, but beyond a temperature increment ultimately leads to degradation of analyte (if thermolabile) along with reduction in resolution efficiency.

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06.E. Degree of Swelling Swelling is directly proportional to ion exchange capacity of exchanger since swelling provides larger surface area by generating macroporous structure inside the resin. Polar solvents increase the degree of swelling while non polar reduces. 06.F. Flow rate of eluent An optimum flow rate nor fast neither slows improves separation efficiency of ion exchanger. 06.G. Cross linking of resin An efficient cross linking provides mechanical stability to resin but at the same time affect its swelling property also. Thus only optimum cross linking will ultimately promotes exchange of ions. 06.H. Concentration Concentration of ions affects ion exchange chromatography in following manners (I).

At low concentration and normal temperature the extent of exchange increase with increasing charge of exchanging ions

(II)

At low concentration and normal temperature the extent of ion exchange increase with reduction in size of hydrated cations and anions of single charge species.

(III) At low concentration and constant valency, the extent of ion exchange increase with increase in atomic number. ( IV )

At low concentration, the extent of exchange of cation or anion in solution against a ion of different sign increase with ion of higher charges

From above mention points it is clear that low concentration favors exchange process. 06.I. Complex formation Formation of complex during exchange process rigorously influences separation efficiency. 06.J. Column geometry Basically, longer columns are of choice for cation separation while shorter one are preferably selected for anions along with cations separation at spend of less time. Length of column is extended only to certain maximum limit called as critical length above which any further

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increment causes no effect on separation. Mostly a ratio of 100:1 or 10:1 between length and diameter is maintained for good separation. 07.

INSTRUMENTATION

07.A. Column preparation and procedure Basic phenomenon of separation in ion exchange chromatography is somewhat analogous to that of classical column chromatographic technique, instead, ion exchange chromatography makes use of suitably charged ion exchanger resin for fractionation rather than plane or bonded silica in case of column chromatography. ( Note: No adsorption process involved in exchange of ions).

Figure 02: Typical instrument for ion exchange chromatography

An inert mechanically stable glass or stainless steel column of optimum dimension is packed with slurry made in same solvent used as eluent. The slurry is passed into the column in several small but equal parts with sufficient gap to allow each part to get settled down between successive additions. It must be noted that dry packing of column is strictly prohibited, slurry of resin must always be prepared outside the column, and must be provided with sufficient time of contact with solvent for their proper swelling. Once packing is done solution containing analyte is added from top of the column and allow to passes through bed of ion exchanger under positive effect of gravity resulting in exchange of isoelectric ion between ion exchanger and analyte solution. As the process repeated several times all the ions from analyte are get replaced against counter ion of exchanger and any further addition of analyte solution ultimately does not causes any further ion separation. At

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this stage, exchanger is said to be exhausted and un-separated analyte ions are detected in the final eluate which is known as breakthrough point of these ion. Exhausted ion exchanger can be regenerated successfully by passing a solution containing ion (same as that of resin counter ions) which are get replaced with analyte ion adhere to ion exchanger. This process of converting exhausted ion exchanger into their original form is known as exchanger regeneration. 07.B. Detection of column effluent Effluent can be analyzed by utilizing suitable technique as per the ingredient present in the effluent. A colorimetric or spectroscopic method is employed when effluent contains a color forming or UV/visible absorbing species. Polarographic and conductometric methods are used for determination of diffusion current at constant potential and electrical conductivity of elutes coming from column. If the eluent contains some radioactive or isotopic substance they can be suitably detected with an aid of radiochemical methods like ionizing chambers or Geiger Muller counter etc. 08. APPLICATIONS In biological field, ion exchange technique plays a key role in separation of various ions of biological interest including serum electrolytes, fractionation of blood, electrodialysis, separation of amino acids, peptides, nucleic acid etc. Therapeutic agents incorporated with charged resins are used for formulation of various dosage form especially sustained and delayed action formulations. Suspension or ointments containing charged resins are used in treatment of various diseases related with dermatological disorders. Cationic exchangers are used for treatment of hypertension & edema by removing excess sodium ions from patient blood also they act as a diagnostic aid in diseases like hyperacidity while on other hand anionic exchanger are used for treatment of peptic ulcers. Further more, ion exchange process is successfully used for fractionation and analysis of vitamins, hormones, antibiotic, alkaloids, serum proteins, local anesthetic, carbohydrates, fatty acids, radioisotopes and other chemicals of pharmaceutical interest. Some of its application is briefly describes below

a. Determination of sodium and potassium content of mixture. b. Deionization of water. c. Treatment of food and beverages. d. Separating of transition metals. e. Separation of metals alloys and high alloy steels. f. Identification of ions g. Conversion of salts to acid or bases h. Separation of amphoteric metals from non-amphoteric metals.

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EXERCISE a. Give a brief account on ion exchange chromatography. b. Classify ion exchanger and enumerate their ideal characteristic. c. Write a short note on organic and inorganic ion exchangers. d. Establish mechanistic profile of ion exchangers. e. What is distribution coefficient? Give its importance in terms of ion exchange phenomenon. f. Enumerate various factors that affect efficiency of ion exchanger. g. Discuss instrumentation of ion exchange chromatography. h. Explain various applications of ion exchange chromatography.

10. MULTIPLE CHOICE QUESTIONS The ion exchange chromatography works on the principle of a. adsorption b. absorption c. selective ion exchange d. None of the above c The sulfonic acid & polyamide resins were synthesized in a. 1920 b. 1935 c. 1945 d. 1955 b Ion exchange chromatography act as an useful tool in a. Separation of biomolecules b. Purification of water c. Analysis of semi-conductors d. All the above d Ion exchangers are a. Polymeric macromolecules b. May be synthetic or natural in origin c. Organic or inorganic in nature d. All the above d

Chowrasia, Deepak

The charge of counter ions is always a. Opposite to that of fixed ion b. Same as that of fixed ion c. Both d. None of the above a Counter ions are also termed as a. Motile ions b. Mobile ions c. Motion ions d. None of the above b An anionic ion exchanger is a a. Positive charge macromolecular system with negative charged counter ions b. Negative charge macromolecular system with negative charged counter ons c. Both d. None of the above a A cationic ion exchanger is a a. Positive charge macromolecular system with negative charged counter ions



b. Negative charge macromolecular system with positive charged counter ions c. Both d. None of the above b An ion exchanger may be _______ in nature. a. Organic b. Inorganic c. Both d. None of the above c Pick out inorganic anionic ion exchanger a. Zeolite (analcite) b. Clays (montmorillonite) c. Glauconite d. All the above d All are inorganic anionic ion exchanger except a. Zeolite (analcite) b. Clays (montmorillonite) c. Glauconite d. Apatite & hydroxyapatite d Out of anionic & cationic ion exchanger which is mostly employed a. Cationic ion exchanger b. Anionic ion exchanger a. Both b. None of the above a

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A cationic exchanger works best at _____ pH a. Acidic b. Basic c. Neutral d. None of the above b An anionic exchanger works best at _____ pH a. Acidic b. Basic c. Neutral d. None of the above a Arrange following ions against descending order of their ion exchange a. Th4+>Ca2+> Na+> Al3+ b. Th4+> Al3+> Ca2+> Na+ c. Both d. None of the above b The phenomenon of ion exchange generally promoted at _______ concentration a. High b. Low c. Both d. None of the above b The ion exchanger finds their application as a. Therapeutic agents b. Purifying agent c. Diagnostic agent d. All the above d


Chapter - 12 SIZE EXCLUSION CHROMATOGRAPHY - Deepak Chowrasia

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SIZE EXCLUSION CHROMATOGRAPHY (Chapter Overview) 01. INTRODUCTION .................................................................................................................. 213 02. PRINCIPLE ............................................................................................................................ 213 03. ILLUSTRATION OF TECHNIQUE ................................................................................213 04. STATIONARY PHASE ........................................................................................................214 04.A. Ideal characteristic of Stationary phase used in SEC ........................................214 05. MECHANISM OF SEPARATION ....................................................................................215 06. INSTRUMENTATION AND METHODOLOGY ......................................................... 215 07. APPLICATIONS ...................................................................................................................216 08. COLUMN CHROMATOGRAPHY ..................................................................................216 09. MULTIPLE CHOICE QUESTIONS ................................................................................216

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SIZE EXCLUSION CHROMATOGRAPHY 01. INTRODUCTION Size exclusion chromatography (SEC) or exclusion chromatography is a technique for separating mixture into its individual components according to their size or molecular weight or more specifically their solution volume. On the basis of types of solvent employed during separation process, size exclusion chromatography is classified into two types gel permeation chromatography (GPC) utilizes organic non-polar solvent for separation and gel filtration chromatography (GFC) which rely on polar aqueous solvents. It should be noted that separation phenomenon in both the case of SEC is same either separation strictly on the basis of size of solute molecule without involving any chemical mechanism. The credit for development of SEC was goes to Henry Lathe & Colin R Ruthven (1955) who separated mixture of an analyte according to their respective size using starch gel as a matrix while working at Queen Charlotte’s Hospital London, later on introduction of more efficient gel matrix (Dextran gel-in 1959) by Jerker Porath and Per Flodin elaborates its analytical importance. 02. PRINCIPLE Size exclusion chromatography is based on the principle of separating molecule on the basis of their geometry and size. When solute molecules of different sizes are allowed to pass through a macromolecular differential porosity gel-like stationary phase get retained over their exclusively on the basis of matrix pore size. Solute particles whose size is smaller or somewhat equal to the pore size of stationary phase penetrate interior of gel and hence get retained over there, while on other hand larger size solute particles bypasses smaller matrix pores, thus gets elute out and separated off. This technique of fractionation provides an effective means for separating larger molecular weight bioactive components without disturbing its biological properties with an expense of minimal eluate volume.

It should be noted that gel permeation chromatography is differ from electrophoresis and ion exchange chromatography, in former case smaller particles are migrate first in the presence of electric field while in later case chemical interaction involved in separation. 03. ILLUSTRATION OF TECHNIQUE Let us consider five different compounds A, B, C, D, E of molecular weight 2,90,000; 1,70,000; 1,30,000; 75,000; & 55,000 respectively are separated on a stationery phase whose minimum & maximum fractionation limit is 50,000 and 2,80,000 strictly. Compound A elute out first as it is out of the maximum fractionation capacity of stationary phase and cannot be retain by it while compound E which is having molecular weight of 55,000 is totally gripped into the pores of stationary phase and elute out last. Compound B, C, & D are eluted out of the separating system according to their decreasing molecular weight respectively.

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04. STATIONARY PHASE Stationary phase used in size exclusion chromatography includes inert, high molecular weight, semi rigid, polymeric gel such as Xerogels of polyacrylamide, Sephadex (Dextran), Agarose etc that are highly crossed linked to give a porous three dimensional structure necessary for effective separation procedure. However, these gels are not suitable to work out with HPLC system owing to their inability to bear high pressure solvent flow during operation although they are extensively & satisfactorily be used in classical column chromatographic technique for separation. Porosity is an essential property determining overall separation process can be withheld in limit by controlling the degree of cross-linking during manufacturing of stationary phase.

Mostly & commonly gels used in SEC are hydrophilic in nature and swells when come in contact with aqueous solvents mostly water. A highly cross linked gel have smaller pore size thus needs lesser quantity of solvent to get filled into its pores in compare to lower degree cross linked matrix requiring large quantity of solvent due to their larger pore diameter. The solvent so present in the interstitial space of gel is available during the time of separation of analyte molecules. An analyte molecule which is larger in size in contrast to largest pore diameter of matrix will barred from penetrating into it pores hence elute out first while all other analytes which are having size either comparable or smaller than the largest matrix pore (stationary phase) get retained and thus elute out in order of their size. Sephadex Grade

Excluded Molecular weight

Swelling time in hours

Water regaining capacity (g/g)

1

G-10

700

3

1.0

2

G-15

1500

3

1.5

3

G-25

5000

12

2.5

4

G-50

10000

12

5.0

5

G-75

50000

24

7.5

6

G-100

100000

48

10.00

S.No

Table 01: Stationary phases & their characteristics 04.A. Ideal characteristic of Stationary phase used in SEC a. It must be inert and do not react with any components of analyte

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b. It should be optimally crossed linked to get differential pore size & three dimensional structures. c. It must swell adequately with suitable solvents without undergoing self dissolution. d. It must be cheap, easily available, and versatile in separation process. e. Must be stable in terms of mechanical, chemical and physical properties. f. Should be free from any hazardous interaction against both analyte and analyst g. If possible single stationary phase can be used for multiple separation processes. 05. MECHANISM OF SEPARATION When a pre-swollen gel packed in suitable dimension column, net volume of packed column can be mathematically expressed as

Where V T = Total volume of column packed with gel V G= Volume occupied by solid matrix of gel and constitute about 20% of total packed volume of gel. V L= Volume of solvent present in interstitial space of gel i.e. pores which allows free movement of solvent and analyte particles makeup at least 40% of total packed volume. V 0= Void or free volume of gel particles; constitute about 20%. 06. INSTRUMENTATION AND METHODOLOGY Basically classical glass column (as used in column chromatography) of suitable dimension (height:diameter=10:1 or 20:1) is sufficient for performing size exclusion chromatography. Column preparation can be done initially by introducing cotton plug or glass wool down at the bottom of column thereby preventing loss of gel matrix during separation procedure. Mostly dry packing is avoided since it causes cracking of glass column due to swelling of gel during separation phenomenon. Therefore for better result and to overcome before said problem, column is previously filled with solvent (to avoid air bubble in matrix) use as eluent and then small portion of gel (previously prepared in solvent or suitable electrolyte solution in a ratio of 1:2-gel:solvent) passes into the column from its top with the help of funnel of suitable size till a uniform bed is formed inside the column. Channeling inside the column can be avoided

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by even packing of gel matrix and an optimum unhindered flow rate can be maintained by removing smaller size particle from gel bed by decantation. Once the filling is done upper surface of column bed is covered either by filter paper or plastic net to avoid any disturbance and finally the column is connected with eluant reservoir and run freely overnight prior to use. Sample to be separated should be applied in small volume at the top of column carefully with an aid of suitable mechanical device or a plunger. As a rule of thumb, only 1-2% of sample is sufficient for most of the analytical work however a sample size of 20-30% of total column volume can also be used as per requirement. Column effluent is collected in a fixed amount into suitable fraction collectors and can either be analyzed simultaneously during separation or after completion of process by use of suitable methodology like spectrophotometry or any other recommended procedure. 07. APPLICATIONS Size exclusion chromatography is a universally accepted technique for analysis and separation of wide varieties of organic and inorganic compounds. One of the main and extremely important applications of SEC is characterization of polymer. However some of its other application includes separation of polymers both natural and synthetic in origin, separation of sugars, polypeptides, proteins, asphalts, polyethylenes, polystyrenes, silicon polymer, and asphalts. 08.

COLUMN CHROMATOGRAPHY a. What is size exclusion chromatography? Outline its principle briefly.

b. Illustrate size exclusion chromatography with a suitable example. c. Write a short note on stationary phase used in size exclusion chromatography. d. Enumerate various properties of ideal stationary phase used in size exclusion chromatography. e. Enlist some of the applications of size exclusion chromatography. 09. MULTIPLE CHOICE QUESTIONS In terms of chromatographic procedures, SEC represents a. Separation on the basic of electrical charges b. Size exclusion chromatography c. Size excitation chromatography d. Size excision chromatography b

Chowrasia, Deepak

Size exclusion chromatography separates components according to their a. Size b. Molecular weight c. Solution volume d. All the above d



Expand GPC & GFC a. Gel permeation chromatography & Gel filtration chromatography b. Gel permanent chromatography & Gel filtration chromatography c. Gel permeation chromatography & Gel failure chromatography d. None of the above a GPC & GFC are types of a. Size exclusion chromatography b. Size exclusion chromatogram c. Size excitation chromatography d. All the above a Solvent used by gel permeation & gel filtration chromatography is a. Polar & non polar b. Non polar & polar c. Non polar & non polar d. Polar & polar b The credit for development of SEC was goes to a. Henry Lathe & Colin R Ruthven (1952) b. Henry Lathe & Colin R Ruthven (1953) c. Henry Lathe & Colin R Ruthven (1954) d. Henry Lathe & Colin R Ruthven (1955) d Dextran gel matrix was introduced in 1959 by a. Jerker Porath and Per Flodin b. Jerker Parth and Per Flodin c. Jerger Porath and Per Floding d. None of the above a

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The basic principle of size exclusion chromatography is a. Migration under electric field b. Separation due to chemical interaction c. Separation due to physical interaction d. All are possible c In electrophoresis fractionation of mixture components are done via a. Migration under electric field b. Separation due to chemical interaction c. Separation due to physical interaction d. All are possible a Ion exchange chromatography involved ________ mechanism in separation a. Physical b. Chemical c. Biological d. All are possible b Let us consider three different compounds A, B, C of molecular weight 50,000 1,00,000; & 1,50,000 are separated on a stationery phase whose minimum & maximum fractionation limit is 40,000 and 1,40,000 strictly. Pick out the correct order of component elution from first to last a. A, B, C b. A, C, B c. C, B, A d. B, C, A c Sephadex is another name of a. Xerogels b. Dextran c. Agarose d. None of the above b



Gels used in SEC are mostly __________ in nature and ______ when come in contact with aqueous solvents. a. Hydrophilic; shrink b. Hydrophobic; swells c. Hydrophilic; swells d. All are correct c A matrix system contains smaller pore size when it is a. Least cross linked b. Does not cross linked c. Highly cross linked d. None of the above c

218

A highly cross-linked matrix system have ________ pore size. a. Larger b. Smaller c. Medium d. can’t be predicted b One of the common facets of size exclusion chromatography is a. Characterization of protein b. Identification of chemical byproduct c. Both d. None of the above a

Chowrasia, Deepak


Chapter - 13 CONDUCTOMETRY - Dr. Nisha Sharma, Deepak Chowrasia

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CONDUCTOMETRY (Chapter Overview) 01. INTRODUCTION .................................................................................................................. 223 02. PRINCIPLE ............................................................................................................................ 223 03. ADVANTAGES ......................................................................................................................224 04. DIFFERENCE BETWEEN ELECTROLYTIC AND METALLIC CONDUCTANCE ...........................................................................................................................224 05. ELECTROLYTE CONDUCTANCE ..................................................................................224 05.A. Ohm’s law ....................................................................................................................224 05.B. Specific resistance or resistivity .............................................................................. 225 05.C. Conductance ................................................................................................................ 225 05.D. Specific conductance or conductivity .....................................................................225 06. EQUIVALENCE & MOLECULAR CONDUCTANCE OF AN ELECTROLYTE .............................................................................................................. 226 07. FACTOR AFFECTING ELECTROLYTIC CONDUCTANCE ................................226 07.A. Concentration .............................................................................................................. 226 07.B Number of ions and their velocity ..........................................................................227 07.C. Temperature ................................................................................................................ 227 07.D. Viscosity of medium ...................................................................................................227 07.E. Solvent dielectric constant ........................................................................................ 227 07.F. High voltage ................................................................................................................. 228 08. CONDUCTANCE MEASUREMENT .............................................................................. 228 08.A. Conductivity cell .........................................................................................................228 08.A.I. Platinization of electrodes ............................................................................228 08.B Conductometer ............................................................................................................ 229 09. TYPES OF CONDUCTOMETRIC TITRATION ......................................................... 230 09.A. 09.B. 09.C. 09.D. 09.E.

Strong acid with strong base ....................................................................................230 Strong acid with Weak base .....................................................................................230 Weak acid with strong base .....................................................................................230 Weak acid with weak base ........................................................................................ 231 Very weak acid with strong base ............................................................................231

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10. APPLICATIONS ...................................................................................................................231 11. EXERCISE .............................................................................................................................. 231 12. MULTIPLE CHOICE QUESTIONS ................................................................................232

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CONDUCTOMETRY 01. INTRODUCTION Conductors may be defined as a class of substance allowing an uninterrupted flow of electrical charge or electricity. Depending upon the nature of molecules involved in electrical conductance, conductors are divided into two distinct classes electrolytic conductors where flow of current take place due to migration of charged ions towards opposite electrode accomplished with chemical changes and metallic conductors in which electricity produced due to free movement of electrons from higher negative potential to lower one without any chemical changes.

Conductometry is a type of electro-analytical method, used for determining conductance of an electrolytic solution with the help of device known as conductometer. Principle advantages of conductometric titration include its applicability even to highly diluted or strongly colored solutions where other titrimetric methods become non-functional in determining end-point satisfactorily. Beside this, the method is equally contributed for detection of those chemical reactions (let it may be incomplete one) where relevancy of potentiometric or other electroanalytical methods becomes questionable. 02. PRINCIPLE In a very dilute solution conductance is brought about by independence migration of ionic species i.e. anions & cations. Since both cations and anions migrates in an electrolytic solution with differential degree of mobility towards opposite charged electrodes thereby constituting electrical current and thus conductance, which instead of remaining constant varies with certain factors discussed briefly under separate section of this chapter. Conductometric titrations ultimately measure this variation in conductance (instead of absolute value) when two electrolytic solutions of different degree of ionization or strength are mixed upon.

Addition of one electrolytic solution to another electrolyte solution will affect the conductance of final solution accordingly whether or not an ionic reaction has occurred. In case, when no ionic reaction has been takes place, then overall conductance of resultant electrolytic solution increased due to contribution of all ions present in electrolytic solution. This phenomenon could be illustrated by mixing simple solution of one salt to another salt, as seen in addition of sodium nitrate solution with solution of sodium chloride. However, in case, when ionic reaction takes place upon mixing of two electrolytic solutions, then overall conductance of final electrolytic solution may increased or decreased which ultimately depends upon replacement or substitution of ions of greater mobility in former case while ion of lower mobility in later case. This type of situation could be well observed during mixing of solution of strong base (sodium hydroxide) with that of strong acid (hydrochloric acid) where

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+

there is replacement of higher mobility hydrogen ion (H ) with that of lower mobility sodium + ion (Na ) thereby resulting an reduction of overall conductance of final solution. Basically, only a small volume of highly concentrated titrant (to avoid appreciable volume change) is added in a sequential manner during conductometric titration procedure and the reading so obtained is sketched in a graph systematically between conductance & volume of titrant added. The point at which two lines are intended to intersect is regarded as an equivalence point. Essentially for the purpose of accuracy, sharper the acute angle of intersecting lines better is the accuracy of conductometric titrations. 03.

ADVANTAGES I. Indicator independence end-point determination.

II.

Turbid, highly colored, and dilute solution can be titrated easily.

III.

Easy to perform, since rather than an absolute value only change in conductance is recorded.

IV.

No need to determined cell constant and specific conductance.

V.

Even incomplete reactions can also be titrated efficiently.

VI.

Graphical end-point determination ultimately provides chance for error minimization.

04. DIFFERENCE BETWEEN ELECTROLYTIC AND METALLIC CONDUCTANCE From above discussion it has been cleared that irrespective of nature of molecules involved, both type of conductors (metallic and electrolytic) allow flow of electrical current, but it should be pinpoint that electrolytic conductance differ from metallic conductance in sense that with increase in temperature there is proportional enhancement in electrolytic conductance due to increased electrolytic ionization along with mobility of charged ions. The same statement, however, is false in terms of metallic conductance where reducing temperature up to 3-4K ultimately result in an overall loss of resistance thereby promoting free migration of electrons thus constituting electric current. On emphasizing further, it also be noted that migration of charged species in case of metallic conductance is from negative to positive pole while charged species migrates towards opposite electrodes in case of electrolytic conductance. 05.

ELECTROLYTE CONDUCTANCE

05.A. Ohm’s law As that of metallic conductors, Ohms law is strictly followed by electrolytic solutions also. Accordingly as per the law

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“Strength of current (I) passing through an electrolytic solution is directly proportional to applied electromotive force-EMF (E) and inversely proportional to resistance (R) offered by conductor”.

Thus I = E/R……………………………………………….

(1)

Or, When defining in terms of unit then equation (1) becomes Ampere = Volt/Ohm 05.B. Specific resistance or resistivity From equation (1) it has been cleared that resistance (R) plays a critical role in hindrance regarding mobility ease of charged species and instead remaining constant it varies in direct proportion as per length (l-meter) of conductor and in an inverse proportion to conductors 2 cross section area (a-meter ) therefore, R α l/a……………………………………….

(2)

R = ρ.l/a…………………………………………

(3)

Or,

Where rho (ρ (ρ) is termed as specific resistance or resistivity having unit of ohm m, and remains constant for given conductor. 05.C. Conductance Conductance is nothing but extent of an electrolytic solution or metallic conductor to overcome resistance (R) barrier and transmits migration of charged ionic species thereby conducting electricity. Technically, reciprocal (inverse) of resistance is termed as conductance which is measured in terms of Siemen (S) sometime alternatively also expressed -1 as ohms or mhos. 05.D. Specific conductance or conductivity Specific conductance or conductivity is standard unit of conductance which accounts property of conducting medium dealing with. Symbolically, it is denoted by k (kappa) and technically can be defined as reciprocal of resistance offered by 1 cm cube of liquid at specific temperatures. The term specific conductance become somewhat misnomer while dealing with solution of electrolyte since extent of electrolytic conductance depends upon number of charged ions exist in electrolyte and usually tends to become zero as the solution is get diluted. While on other hand, molecular conductivity of an electrolytic solution tends to

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reaches their highest value with dilution, therefore equivalent conductance and molecular conductance in true sense are technically defining terms expressing electrolytic conductance to their nearby sense. 06. EQUIVALENCE & MOLECULAR CONDUCTANCE CONDUCTANCE OF AN ELECTROLYTE Equivalence conductance may be defined as the net conductance produced by volume of solution containing 1g equivalent of electrolyte when placed between two parallel electrodes apart from each other by a distance of 1-meter. Symbolically equivalence conductance expressed as Λ. Equivalence conductance is product of specific conductance and volume in cubic.cm (v) i.e. Λ = k v………………………………………………

(4)

Λ = k/C…………………………………………………

(5)

Or

Where C is the concentration of solute in gram equivalent per liter present in electrolyte solution, Therefore, k /C…………………………...…………… Λ = 1000. k/C…………………………...…………… (6) However, if concentration of solute in an electrolytic solution is expressed in terms of mole per liter (M) (equation 6), quantity corresponding to equivalence conductance is then expressed in terms of molecular conductance also known as molar conductance, denoted by µ, and represented as µ = k v ………………………………………… (7) Where terms have their usual meaning. 07.

FACTOR AFFECTING ELECTROLYTIC CONDUCTANCE CONDUCTANCE

07.A. Concentration Specific conductance of an electrolytic solution is directly proportional to concentration, that means, with increase in concentration specific conductance also enhanced sharply, but this only true in case of strong electrolyte. In case of weak electrolyte, however, conductance also increased, but in a gradual manner. This differential concentration dependent conductance enhancement in strong and weak electrolyte is just because of their inherent ionizing capacity which is greater for strong electrolyte while having lower value for weaker electrolyte and thus their conductance. However in both the cases either sharp or dull conductance enhancement is brought out by increased population of ions per unit volume of electrolytic solution.

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Equivalent/litre

HCL

KCL

CH 3COOH

1.0

332

111

NIL

0.1

391

129

5.2

0.01

412

141

16.3

0.001

421

146

49.2

0.05

399

133

7.4

0.005

415

143

2.9

0.0005

422

147

67.7

Table 01 07.B. Number of ions and their velocity In case of strong electrolyte which is completely ionized, number of ions remains same at all values of dilution and any change in equivalent conductance with respect to dilution is only because of variation in ions velocity. Further more it also be observed that in case of concentrated electrolytic solution interionic attraction between oppositely charged species + become quite dominant thus holding charged ions in form of pairs i.e. (A B ) thereby reducing their velocities and hence the conductance. However, this phenomenon could be reversed by diluting the electrolytic solution so as to overcome interionic interionic attraction and hence separating oppositely charged species apart thereby enabling their free migration thus enhancing equivalence conductance. 07.C. Temperature The conductance of all most all electrolytes electrolytes varies in a direct relation with that of temperature. However the above statement is hold true for stronger electrolytes but having some boundary. However in a broad sense electrolytic conductance conductance increased with increase in temperature due to reduction in viscosity of conducting medium which ultimately facilitate mobility of charged ion, thus conductance. 07.D. Viscosity of medium As per Walden’s rule, equivalent conductance of an electrolytic solution varies inversely with viscosity of medium i.e. greater the viscosity of medium, lesser is the electrolytic conductance. 07.E. Solvent dielectric constant It is preferable to use solvent with higher value of dielectric constant rather than a solvent having lower dielectric constant since a solvent with higher dielectric constant will easily overcome electrostatic force of attraction between oppositely charged ions, freeing them apart, thus ensuring their free mobility and hence their conductance.

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07.F. High voltage As per Wien’s effect, equivalence conductance increases when electrolytic solutions are exposed to higher voltage. This is due to the fact that at higher voltage ions are move with an enhanced velocity overcoming electrostatic force of attraction thereby improving conductance. The same is true when electrolytic solution is exposed to higher frequency alternating current (AC). This effect of increased in equivalence conductance with respect to increase in frequency of alternating current is known as Debye-Falkenhagen effect . 08. CONDUCTANCE MEASUREMENT MEASUREMEN T Since conductance is defined as reciprocal of resistance thus a conductometer secured with Wheatstone bridge and coupled with conductivity cell can be efficiently used for conductance measurement measurement of an electrolytic solution (see figure 01).

Figure 01: A typical circuit diagram for conductance measurement 08.A. Conductivity cell They are used for measurement of conductance of an electrolyte and sometime also termed as conductivity vessels. The cells are commercially available in various shapes and sizes and generally made up of pyrex glass fitted with electrodes of platinum or gold. In order to overrule problem associated with current imperfection or polarization and to reduce electrode detriment, the electrodes so used are commonly coated with uniform fine layer of platinum black which is brought about by exposing them to a 3% solution of chloroplatinic acid containing little amount of lead acetate by a process well known as Platinization.

08.A.I. Platinization of electrodes Since some scratches may be developed on the blacked platinum electrodes during washing, cleaning, or by improper handling thus making them unsuitable for measurement. However

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these imperfect electrodes can be recoated by undertaking following methodology. “Thoroughly clean conductivity vessel and electrodes by immersing them in warm solution of K2Cr2O7 (potassium dichromate) in concentrated sulphuric acid. Wash further with distilled water until the electrode get totally free from acid, and replatinized them by dipping in solution containing 2-3% of chloroplatinic acid and 0.02-0.03g of lead acetate by passing electric current though it”. The distance between electrodes depends upon conductivity of solution; electrodes are widely distance apart in highly conducing solution while mounted nearby in lower conducting solutions. Once replatinization is completed, wash electrodes with distilled water carefully and immersed them in same till further usage. 08.B Conductometer Conductometer typically blankets a Wheatstone bridge whose arms are attached with sensitive electronic assembly for precise and accurate conductance measurement. The arrangement of Wheatstone bridge was shown in figure 01, is consist of resistance box (A), source of electric current (B), conductivity cell (C), current sensitizing device (D), uniform slide wire (XY), across which moves a contact point (Z).

For conductance measurement, conductivity cell is filled with electrolytic solution whose conductance has to be determined. The cell is further well equipped with electrodes whose terminals are coupled with Wheatstone bridge circuit of conductometer. Practically, the contact point Z slide along the length of resistance wire XY until no current is detected i.e. bridge is in balanced condition, thus the resistance in arms of Wheatstone bridge is expressed as Cr/R = XY/ZY

Where, Cr = Resistance of electrolytic solution present in cell Rs = Standard resistance Ideally magnitude of standard resistance Rs should be such that bridge is balanced at the mid point of XY. Since the value of Rs is known and the ratio of XY/ZY can be measured thus resistance of cell can be calculated efficiently. Since conductance is reciprocal of resistance it value could be measured successfully. Choice of current sensitizing device (D) either Galvanometer or Earphone depends upon nature of electric current used for measurement i.e. former is used with normal AC current while later is employed when current is produced by valve oscillator generating frequency of 1000 cps (within the reach of normal hearing). It must be noted that under any circumstances direct current (DC) should not be employed due to its ability of causing polarization which ultimately give false reading regarding the resistance of electrolytic solution.

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09. TYPES OF CONDUCTOMETRIC TITRATION Conductometry can be used for various types of neutralization titration which are briefly describes as follow; 09.A. Strong acid with strong base Titration of strong acidic solution (HCl) by a strong basic solution (NaOH) results in replacement of higher mobility (conducting) hydrogen ions with sodium ions of lower mobility, leading to an initial fall in conductance which eventually continues until an equivalence point is achieved. At this point conductance is solely due to presence of sodium and chloride ions. However, any further addition of base to resulting solution leads to rise in conductance which is solely brought about by excess of hydroxyl ions. Thus conductance has a minimum value in this type of titration at equivalence point. A V-shaped graph (see figure 02) is sketched while plotting the data so obtained between conductance with respect of volume of volume of alkali (NaOH) added.

Figure 02: Graph depicting various types of neutralization titrations 09.B. Strong acid with Weak base The graph obtained in this type of titration could be depicted from figure 02 (Green color) which results from titrating strong acid (HCl) with weak base (ammonia solution-diluted). During the course of titration due to disappearance of high mobility ions results in slow but continuous decrease in conductance of solution. However, once the end point has been reached any further titration does not cause any rigorous change in conductance of resultant solution thus the graph becomes almost horizontal in shape. 09.C. Weak acid with strong base Since in this type of titration a weak acid (boric acid) is titrated against strong base (NaOH) thus overall shape of final graph largely depends upon concentration and dissociation constant

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of acid employed during course of titration. The final shape of graph can be well elucidated from figure 02 (Red color). 09.D. Weak acid with weak base A careful inspection of graph (see figure 02-brown color) obtained by a titration between acetic acid and ammonia solution (aqueous) indicates that initially the conductance of the resultant solution increase up to the equivalence point. However, addition of base after equivalence point has no effect on conductivity of resultant solution due to salt formation which causes ionization suppression. 09.E. Very weak acid with strong base In this case, initially the overall conductance is negligible but increases with course of titration. However the value of conductance increases rigorously once the end point has been reached. The graph in this type of titration is depicted from figure 02-Red color and could be obtained from titration between boric acid and sodium hydroxide. 10. APPLICATIONS Conductometric titrations could be used to obtain both direct as well as indirect conductance measurement of both electrolytes as well as solution containing charged species. Some of its applications are briefly discussed here;

1.

Determination of solubility and solubility product of sparingly soluble salts such as lead sulphate, barium sulphate, and silver chloride.

2.

Study kinetic profile of a chemical reaction which in-turn depends upon overall conductivity of chemical reaction during the course of its proceeding.

3.

By utilizing Ostwald equation, conductometry could be used for determination of organic acid basicity.

4.

Determining degree of ionization and ionization constant of weak electrolyte such as acetic acid with the help of equation ◦

α = Λ / Λ

5.

Determination of concentration, degree of hydrolysis, and hydrolysis constant are some other prominent applications of conductometry.

11. EXERCISE Briefly explain theory of conductometric titrations.

a. Enumerate various advantages of conductometry. b. Compare electrolytic and metallic conductance. c. State Ohm’s law and relate same with electrolytic conductance.

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d. Define following terms with their proper symbol and unit 1. Conductance 2. Resistance 3. Specific resistance 4. Specific conductance 5. Equivalent conductance 6. Molar conductance e. Derive an expression for equivalence conductance. f. State and explain various factors that affects conductance of an electrolyte. g. Briefly explain instrumentation of conductometric titration with the help of well labeled diagram. h. Write a note on conductometer. i.

What is replatinization of electrodes? Give suitable methodology for same.

j.

Enumerate various types of conductometric titrations.

k. Sketch graph showing following conductometric neutralization titration; A. Strong acid with strong base B. Strong acid with Weak base C. Weak acid with strong base D. Weak acid with weak base E. Very weak acid with strong base l.

Write a short note on applications of conductometric titration.

12. MULTIPLE CHOICE QUESTIONS Conductors having characteristic of a. Conducting electricity b. Conducting heat c. Both d. None of the above c In electrolytic conductors ______ are responsible for flow of current a. Free movement of electrons b. Free movement of charged ions

Chowrasia, Deepak

c. Both d. None of the above b In metallic conductors ______ are responsible for flow of current a. Free movement of electrons b. Free movement of charged ions c. Both d. None of the above a

CONDUCTOMETRY


A conductometer used for measurement of a. Conductance b. Potential c. Both d. None of the above a Conductometric titrations are useful in determining end point of a. Very diluted solution b. Highly colored solution c. Both d. None of the above c The value of conductance remains constant in an electrolytic solution a. Yes b. No c. May be d. None of the above b/c Conductometric titrations based on the phenomenon of a. Mobility of charged ions b. Differential mobility of charged ions c. Both d. None of the above b Conductometric titrations measure variation in ________. a. Conductance b. Convection c. Convenient d. Conjunction a The overall conductance of final solution _______ when there is no ionic reaction has been takes place upon mixing of two different electrolytic solutions. a. Increase b. Decrease

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c. Not predicted d. None of the above a The overall conductance of final solution _______ when an ionic reaction has been takes place upon mixing of two different electrolytic solutions. a. Increase b. Decrease c. May increases or decrease as per situation d. None of the above c In conductometric titrations, end point is determined by sketching a graph between a. Potential and volume of titrant b. Conductance and volume of titrand c. Conductance and volume of titrant d. None of the above c The accuracy of conductometric titrations increase when interesting line make an _____ angle. a. Acute b. Obtuse c. Right d. None of the above a Migration of charged species in case of metallic conductance is from a. Negative to positive pole b. Positive to negative pole c. Can’t be determined d. None of the above a Pick out relation that defines Ohm law a. I = 1/R b. E = I/R c. I = R/E d. I = E/R d

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The term specific resistance is also termed as a. Resistance b. Resistivity c. Both d. None of the above b The resistivity has a unit of a. Ohm b. Weber c. Ohm.meter d. None of the above c Resistance of a conductor varies directly with _________ of conductor a. Length b. Cross section area c. Both d. None of the above a Conductance may be defined as a. Hindrance for flow of electric current b. Potential of an conductor to overcome resistance c. Both d. None of the above b Technically conductance can be defined as a. Reciprocal of resistance b. Conjunction of resistance c. Both d. None of the above a The unit of conductance is a. Semen (S) b. Simen (S) c. Seamen (S) d. Siemen (S) d

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Ohms a. b. c. d.

234

-1

or mhos are the alternative unit of Resistance Convection Conductance None of the above c Conductivity can also be termed as a. Specific conductance b. Specific convention c. Specific convenient d. None of the above a Symbolically equivalence conductance can be express as a. A b. Λ c. µ d. α b Equivalence conductance is product of a. Specific resistance and volume in cubic.cm (v) b. Specific conductance and volume in cubic.meter (v) c. Specific convection and volume in cubic.mm (v) d. Specific conductance and volume in cubic.cm (v) d Whether the terms specific conductance can be well equally applied to electrolytic solution a. Yes why not b. No c. May be d. None of the above c/b

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Upon diluting an electrolytic solution, the value of equivalence conductance a. Increase b. Decrease c. Become nil d. None of the above b Molar conductance can be denoted by a. A b. Λ c. µ d. α c With increase in concentration, specific conductance of an electrolytic solution a. Increase b. Decrease c. Becomes zero d. None of the above a An increase in temperature results in _______ in conductance a. Increase b. Decrease c. Becomes zero d. None of the above a An increase in viscosity of medium results in _______ in conductance a. Increase b. Decrease c. Becomes zero d. None of the above b “As per Walden’s rule, equivalent conductance of an electrolytic solution varies inversely with viscosity of medium”. The statement is a. True b. False c. Can’t say

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d. None of the above a “It is preferable to use solvent of higher value of dielectric constant for better conductance rather than a solvent having lower dielectric constant”. The statement is a. True b. False c. Can’t say d. None of the above a “As per Wien’s effect, equivalence conductance increases when electrolytic solutions are exposed to higher voltage”. The statement is a. True b. False c. Can’t say d. None of the above a The effect of increased in equivalence conductance with respect to increase in frequency of alternating current is known as a. Hebye-Falkenhagen effect b. Ohm-Falkenhagen effect c. Debye-Folkenagan effect d. Debye-Falkenhagen effect d Platinization of electrode can be done by solution containing a. Chloroplatinic acid solution (1-2%) & lead acetate (High amount) b. Chloroplatinic acid solution (2-4%) & lead acetate (small amount) c. Chloroplatinic acid solution (2-3%) & lead acetate (small amount) d. Chloroplatinic acid solution (0.1%) & lead acetate (high amount) c

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Platinization of electrode is done in order to a. Reduce current imperfection or polarization b. Prevent electrode detriment c. Both d. None of the above c

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Application of conductometric titration includes a. Determination of ionization constant b. Concentration c. Solubility of sparingly soluble salt d. All the above d

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Chapter - 14 COULOMETRY - Deepak Chowrasia

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COULOMETRY (Chapter Overview) 01. INTRODUCTION .................................................................................................................. 241 02. PRINCIPLE ............................................................................................................................ 241 03. ADVANTAGES ......................................................................................................................242 04. TYPES OF COULOMETRY .............................................................................................. 242 04.A. Controlled potential coulometry ............................................................................. 242 04.B. Constant current coulometry...................................................................................242 04.B.I. Primary constant current coulometry .........................................................243 04.B.II. Secondary constant current coulometry ...................................................... 243 05. APPLICATIONS ...................................................................................................................243 06. EXERCISE .............................................................................................................................. 244 07. MULTIPLE CHOICE QUESTIONS ................................................................................244

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COULOMETRY

COULOMETRY 01. INTRODUCTION The technique of coulometry was first described by C.A. Coulomb, deals with measuring net quantity of electricity in coulomb passes through a solution during electrolytic formation of material provided that electrolytic reaction proceed with 100% efficiency with respect to electrodes employed. Basically, the phenomenon of coulometry adhere strictly with Faraday first of electrolysis which states that- “ Extent of chemical reaction occurring at an electrode is directly proportional to quantity of electricity passing through electrode”. In coulometry, the amount of current brought about chemical transformation is recorded with the help of suitable device known as coulometer. Generally, a coulometer is nothing but a second electrolytic cell placed in series with the reaction cell. 02. PRINCIPLE In form of quantitative electro-analytical technique, method of coulometry used for determining the concentration of an analyte present in solution by measuring amount of electricity required to bring about its chemical transformation. The basic requirement of coulometric methodology includes a cent per cent efficiency i.e. 100% of electrode reaction used for determination process . In a simpler word, amount of electrolysis occurring in a solution must be directly proportional to the extent of current passes through it. When an analyte to be determined directly undergoes reaction (oxidation or reduction) at one of the electrode maintained at a constant potential with respect to the solution is termed as primary coulometric analysis. Basically, in this technique with proceeding of electrolytic chemical reaction there is a reduction of current in proportionate with the disappearance of substance from solution. Likewise, secondary coulometric analysis, which is carried out at constant current, the substance to be analyzed is react quantitatively with another substance yield as a product of electrolysis. However irrespective of their nature i.e. primary or secondary coulometry essential requirement of this type of electro-analytical technique is only a single chemical reaction take place at electrode with a current efficiency of 100%.

Thus as per Faraday’s law, amount of substance deposited W on passing Q coulombs of electricity thought an electrolytic solution is, W = w . Q/96500. n

Where, W= Weight of element deposited w= Atomic weight of element Q= Extent of electricity in coulomb n= valance of element


03.

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ADVANTAGES a. Analyze even those substances whose oxidation state is difficult to determine by other chemical route.

b. Compare to electrogravimetry, no need to weight out the product. c. Accuracy increased with reduction in concentration; thus suitable for analyzing very dilute solutions. d. Better results can be obtained as compare to classical method of electro-analysis. e. Method could be used for analysis of even those compounds which are difficult to analyze by conventional titrimetic methodologies because of their irrelevant physical state, unstability, hazardous or toxic properties, and sometime due to difficulties in titrant preparation. f. Coulometic analysis can be satisfactorily imposed with uranium, titanium, bromine, silver, chlorine, and molten state salt media, which limits their analysis by routine titrimetric process. g. A good technique for routine and remote analysis of chemicals, with no need of preparation, standardization, and storage of titrant. 04. TYPES OF COULOMETRY Coulometry is divided into following two types

04.A. Controlled potential coulometry 04.B. Constant current coulometry 04.A. Controlled potential coulometry It also termed as potentiostatic coulometry and also sometime describes as bulk electrolysis, in which substance to be analyzed chemically by reacting with cent per cent efficiency (100%) at electrode maintained at constant potential for its determination. The reaction is said to be completed when quantity of current reduced to a value of zero and amount of substance reacted can be compelled either from coulometer or by current time integrating unit. The technique of potentiostatic coulometry is sensitive, selective, precise and accurate, but however, due to its time consuming methodologies, limited applicability, and requirement of expansive instrumentation controlled or constant current coulometric techniques are preferred. 04.B. Constant current coulometry It also termed as amperostatic coulometry and also sometime describe as coulometric titrations (both are different). The technique of amperostatic coulometry has advantages over

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potentiostatic coulometry in terms of rapid analysis, wide applicability, less time consumption, and required simple inexpensive instrumentation. Generally, this technique rely on electrolysis of solution containing substance to be analyzed and completeness of overall reaction is judge either by visual method-employing suitable chemical indicators or alternatively by use instrumental techniques such as spectrophotmetric observations, potentiometric, and amperometric determination. Constant current coulometry is further be subdivided into two categories 04.B.I. Primary constant current coulometry 04.B.II. Secondary constant current coulometry 04.B.I. Primary constant current coulometry In this technique, the substance to be determined undergoes directs reaction at full efficiency (100%) at its respective electrode. Like oxidation of ferrous ion into ferric ion at anode. 04.B.II. Secondary constant current coulometry It is also termed as coulometric titrations. The coulometric titrations are unique from other titrimetric analysis, instead of adding titrant from the burette into the reaction mixture, titrant either generated internally (estimation of thiosulphate with iodine generate internally from potassium iodide at anode) or externally (titrimetric analysis of azo dyes in presence of titanous ions produced externally at room temperature and then transferred into reaction vessel) at electrode by passing constant current through an electrolyte solution having 100% current efficiency. The technique of secondary constant current coulometry relies on stoichiometric reaction of one of the titrant produced quantitatively at electrode with the ions to be estimated. For example oxidation of ferrous ion into ferric ion in the presence of ceric ion produced from cerous by a phenomenon of oxidation at anode. Halogens (I>Br>Cl) are one of the most satisfactorily used intermediates in secondary constant current coulometric analysis owing to their ease of generation (100%) and versatility as titrimetric reagent. For example iodine is used in Karl Fischer titration for moisture determination; likewise it is also used as an electrolytic intermediate in titration of hydroquineone and antimony, and utilization of bromine for determination of ascorbic acid and oxine. 05.

APPLICATIONS a. Karl Fisher titration uses coulometric estimation of moisture or water content of substance like therapeutic agents, chemicals, paper, food and their additives etc. ( for detail please refer chapter on Karl Fisher titration of this book).

b. Acid base titration can be performed coulometrically with a greater degree of accuracy by employing a platinum cathode immersed in test solution, yield electro-generated hydroxide ions upon passage of electricity.

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c. Coulometric insisted on bromometric titrations which involves estimation of substance such as aniline, mustard gas, arsenic, phenol, uranium, thiocynate, ammonia etc by reacting them with electro-generated bromide ions. d. Coulometric titration employed for determination of cations with the help of ethylenediamine tetra acetate ion yield at mercury cathode. e. Determination of film thickens by measuring amount of electricity needed. f. When conjugated with other conventional analytical method like gravimetry, coulogravimetry used for simultaneous estimation of elements like determination of zinc and cadmium. 06.

EXERCISE a. Explain principle of coulometry.

b. Enumerate various advantages of coulometry. c. Classify coulometry and give a brief account on secondary constant current coulometry. d. Write a brief note on controlled potential coulometry. e. Enumerate various applications of coulometry. 07. MULTIPLE CHOICE QUESTIONS The technique of Coulometry was first described by a. C.A. Coulomb b. A.A. Coulomb c. Both d. None of the above a “ Extent of chemical reaction occurring at an electrode is directly proportional to quantity of electricity passing through electrode”. The statement is a. Faraday I law b. Faraday II law c. Faraday III law d. Faraday IV law a

Chowrasia, Deepak

For an ideal coulometric process, the electrode reaction must be a. b. c. d.

10% 30% 50% 100% d

In primary coulometric analysis a. No reaction takes place b. The analyte undergoes direct reaction at electrode c. Both d. None of the above b

COULOMETRY


The secondary coulometric analysis occurs at a. Constant potential b. Constant current c. Constant voltage d. None of the above b The primary coulometric analysis occurs at a. Constant potential b. Constant current c. Constant voltage d. None of the above a Constant current coulometry is also known as a. Amperostatic coulometry b. Potentiostatic coulometry c. Both d. None of the above a

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The Potentiostatic coulometry occurs at a. Constant potential b. Constant current c. Constant voltage d. None of the above a The coulometric titrations are a. Primary constant coulometry b. Secondary constant coulometry c. Both d. None of the above

current current

b End point detection in Karl Fischer titration is done by a. Coulometry b. Potentiometry c. Conductometry None of the above a

COULOMETRY

Chapter - 15 POTENTIOMETRY - Deepak Chowrasia

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POTENTIOMETRY (Chapter Overview) 01. INTRODUCTION .................................................................................................................. 251 02. PRINCIPLE ............................................................................................................................ 251 03. ADVANTAGES ......................................................................................................................251 04. Electrochemical cell & basics of potentiometric titration.............................................252 05. Electrodes of potentiometric titrations .............................................................................253 05.A. Classification of potentiometric Electrode ...........................................................253 05.A.I. Reference electrode ........................................................................................ 253 05.A.I.a. Hydrogen electrode ....................................................................... 253 05.A.I.a.a Advantages of reference hydrogen electrode ....... 254 05.A.I.a.b. Disadvantages of hydrogen electrode ................... 255 05.A.I.b. Calomel electrodes ........................................................................ 255 05.A.I.b.a. Advantages of calomel electrodes .........................256 05.A.I.b.b. Disadvantage of calomel electrode ....................... 256 05.A.I.c. Silver-silver chloride electrodes...................................................256 05.A.I.c.a. Advantage of silver-silver chloride electrode ...... 257 05.A.I.c.b. Disadvantages of silver-silver chloride electrode .......................................................................... 257 05.A.I.d. Mercury (I) sulphate electrodes ..................................................257 05.A.II. Indicator electrode ......................................................................................... 257 05.A.II.a Hydrogen electrodes .....................................................................258 05.A.II.b. Glass electrodes ............................................................................258 05.A.II.b.a. Advantages of Glass electrodes .............................259 05.A.II.b.b. Disadvantage of glass electrodes ...........................259 05.A.II.c. Quinhydrone electrode .................................................................260 05.A.II.c.a. Advantages of quinhydrone electrode ....................260 05.A.II.c.b. Disadvantages of quinhydrone electrode .............260 05.A.II.d. Antimony electrode .......................................................................260 05.A.II.d.a. Advantages of antimony electrode ......................... 260 05.A.II.d.b. Disadvantages of antimony electrode ................... 261 05.A.II.e. Ion selective electrode: .................................................................261

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06. APPARATUS ..........................................................................................................................261 06.A. Reaction vessel. ........................................................................................................... 261 06.B. Simple potentiometer. ................................................................................................ 261 07. METHODOLOGY FOR POTENTIOMETRIC TITRATION .................................. 262 08. END POINT DETECTION .................................................................................................262 09. APPLICATIONS ...................................................................................................................263 09.A. Neutralization titrations ............................................................................................ 263 09.B. Redox titrations........................................................................................................... 263 09.C. Precipitation titration ................................................................................................ 264 10. EXERCISE .............................................................................................................................. 264 11. MULTIPLE CHOICE QUESTIONS ................................................................................265

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POTENTIOMETRY 01. INTRODUCTION Direct potentiometry is a simplest and widely accepted technique used for determining potential of large batches of solution with the help of ion selective electrodes (ISE) but suffer drawback of variation in liquid junction potential of reference electrode sometime up to several millivolt (mV) during transferring electrode from one to another solution thereby making the result erroneous. Potentiometric titrations, however, free from this effect and act as a valuable tool for determining solution potential where there is great variation in concentration thus electrode potential. Potentiometric titration involves measuring potential of solution (mV) or electromotive force (EMF) with the help of pair of electrode (indicator and reference electrode). Since potential of solution depends upon temperature, concentration, nature of ionic species and thus pH. Therefore, potential of solution can be efficiently and accurately measured by use of either pH meter or alternatively by a potentiometer. 02. PRINCIPLE Potentiometry rely upon potential measurement of a solution with the help of electrodes by connecting them to suitable device known as potentiometer. On other hand potentiometric titrations involves measurement of change in potential or pH of a solution at constant current on addition of titrant with the help of highly sensitized electrodes i.e. indicator electrode (indicate potential or pH) and reference electrode (value remains constant throughout the process). In more simple words, potentiometry is a type of voltametry at zero current. During titrimetric process addition of titrant to a test solution results in change in concentration of ions present in test solution and thus potential or pH accordingly. The end point of such titration is detected by an immediate change in potential on a graph sketched manually or digitally between volume of titrant added and change in potential or pH of test solution. On the basis of type of chemical reaction takes place during titrimetric process, a suitable indicator electrode is employed like silver electrodes are used in precipitation reaction occurring between halide & silver nitrate, a pH sensitive glass electrode for neutralization reaction, and platinum wire or foil for redox reactions. 03.

ADVANTAGES I. Versatile method suitable for determination of potential as well as pH of solution.

II.

Highly colored solutions can be easily titrated.

III.

Potential of very diluted or concentrated solution can be determined.

IV.

No need to determined actual value of reference electrode provided its potential remains constant throughout the titrimetric process.

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V.

An inexpensive technique does not require any specialized highly sophisticated instruments. Sometime a simple pH meter is sufficient to determine pH thus potential of solution.

VI.

Methodology can be easily and efficiently preformed in normal analytical laboratory for research and educational purposes without any major hindrance.

VII.

No need for prior strength determination of acid or base in neutralization reaction for selection of suitable indicator for end point determination.

VIII.

Easily measured potential of multi-component mixtures.

IX.

Automation in potentiometric titration enhanced their analytical value and save precious time.

04. Electrochemical cell & basics of potentiometric titration An electrochemical cell is a device which utilizes bidirectional flow of chemical energy into electrical energy and electrical energy into chemical energy depending upon their construction and utilization as per the requirement. Note-single cell only perform single directional flow of energy i.e. electrical to chemical and vice versa. Phenomenon of electrolysis, electrolytic cells, electrolytic purification of metals, platinization of electrodes, charging of lead storage battery involves conversion of external electrical energy into chemical energy while on other hand devices such as cells used in radio & digital watches, dry and wet batteries convert chemical energy into electrical energy. Irrespective of their shape, size, & nature of utilizations, an electrochemical cell usually consists of two electrodes which are made up of different metals and are dipped into dry or wet electrolytic solution thus each constituting half cell or half electrode reaction. These electrodes are responsible for phenomenon of oxidation (anode) and reduction (cathode) thereby setting up a potential difference across interface of electrolytic solution and their surface (metal electrodes) which is known as electrode potential (EP). The electrode potential depends upon activity i.e. concentration of ions present in electrolytic solution. Thus when activity of ions in an electrolytic solution is considered as unity then the electrode potential at such unit activity of ions is termed as standard electrode potential (SEP). The difference in potential between electrodes result in a flow of current from electrode maintained at higher oxidation potential to an electrode maintained at lower oxidation potential and this flow of current between two electrodes of different potential is known as electromotive force (EMF) of cell which is abbreviated as emf and expressed in terms of volts. This electromotive force, however, depends upon the electrode potential which ultimately further depends upon activity of ion present in the solution and constitutes basic of potentiometric titrations. The electromotive force of cell could be efficiently and accurately determined by use of simple potentiometer provided that under experimental condition no remarkable current is drawn which may results to polarization of electrodes thereby reducing accuracy of the final result. That why

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potentiometer is preferred over other potential measuring devices like voltmeter in determination of electromotive force of cell and potential or EMF in potentiometric titrations owing to its genius capability of consuming very little (negligible) amount of current without affecting any appreciable change in electrode potential and thus final result. 05. Electrodes of potentiometric titrations William Whewell coined the term electrode, which is nothing but a conductor allowing passage for flow of electric current. In potentiometric titrations, a pair of electrode is used for sensitizing any change in potential or electromotive force or pH of test solution upon addition of reagent or titrant. 05.A. Classification of potentiometric Electrode Electrodes used in potentiometric titration are broadly categorizes into following two t ypes 05.A.I. Reference electrode 05.A.II Indicator electrode

05.A.I. Reference electrode They are also known as standard electrodes having their own well defined and stable potential. Universally, references electrodes are assigned an arbitrary potential of zero (standard hydrogen electrode-SHE or normal hydrogen electrode-NHE) at all value of temperature. A reference electrode constitutes half cell reaction and helps in determination of other half cell potential of all other electrodes employed during experimentation. Hydrogen electrodes are the oldest, well established, and globally accepted reference electrodes sometime also termed as primary reference electrode (PRE) while electrodes other than hydrogen electrodes are known as secondary reference electrodes (SRE). For example silversilver chloride electrodes and saturated calomel electrodes (most commonly used) etc are generally considered as secondary reference electrodes. Generally secondary reference electrodes are superior over primary reference electrodes owing to their lesser maintenance cost, ease of preparation, and easiness in handling. 05.A.I.a. Hydrogen electrode (Reference hydrogen electrode (RHE), Standard hydrogen electrode (SHE) or Normal hydrogen electrode(NHE)

Hydrogen electrode is a primary and universally accepted references electrode which is assigned an arbitrary electrodes potential of zero at all range of temperature as mentioned above, its potential remains constant throughout the process, and electrodes potential of all other reference electrodes are compared with it. Symbolically reference hydrogen electrode is denoted as Pt, H2 (1atm), H+ (a=1) and its overall electrode reaction is expressed as +

-

H (a=1)+e ↔1/2 H2 (P=1 atm)

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The potential of standard hydrogen electrode is expressed in terms of Nernst equation which is as follows ◦

1/2

+

E= E -RT/F ln P H2 /aH

A prototype of reference hydrogen electrode is depicted in figure 01, is consist of small rectangular platinum foil coated electrolytically with platinum black and placed in a solution (acidic solution in case of reference electrode and sample solution in case of indicator electrode) where activity of hydrogen ions is remains as unity. The whole assembly is enveloped with suitable inert glass jacket. The electrode is placed in the solution in such a manner that it is partly immersed as well as expose to atmospheric hydrogen gas. Simultaneously, pure hydrogen gas bubbled into the solution at a pressure of 1 atmosphere. Continuation of the process ultimately leads to establishment of equilibrium between hydrogen ions present in solution and hydrogen molecules in the atmosphere the electrode itself behaving as if it were a metallic electrode and is reversible with that of hydrogen ions present in solution. The net potential of hydrogen electrode is then controlled by activity of + hydrogen (H ) ions present in the solution thus when activity of hydrogen a H+ is taken as unity and pressure of hydrogen gas inside the hydrogen electrodes system is at one atmosphere (1atm) then overall standard potential of references hydrogen electrodes is assigned a value of zero at all range of temperature against which potential of other electrodes are measured. Since the value of references hydrogen electrode is zero thus the measured potential of electrodes is their standard electrodes potential (SEP) which may be positive or negative with respect to reference hydrogen electrode.

Figure 01: Hydrogen electrode 05.A.I.a.a Advantages of reference hydrogen electrode

I.

An ideal electrode against which other electrode potential is measured.

II.

Ideal for measurement of pH over a wide range.

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III.

Free from salt error.

IV.

Depending upon the nature of solution used for its immersion it can be behave as reference and indicator electrode if dipped in standard acid solution in former case and sample solution in later case.

05.A.I.a.b. Disadvantages of hydrogen electrode

I.

Easily affected by purity and pressure of hydrogen gas.

II.

Not suitable to work up with oxidizing and reducing agents.

III.

Poisoning of platinum surface affects potential.

IV.

Difficult to construct, prepare, and maintained.

V.

Problem in handling and somewhat fragile in nature.

05.A.I.b.

Calomel electrodes

It is most commonly and widely used reference electrodes due to ease of its preparation, compactness in size, high temperature coefficient, and maintenance of constant potential during whole process of titration. In its simplest form, calomel electrodes consist of glass jacket containing mercury at its bottom over which a paste of mercury-mercurous chloride is placed which in turn further covered with solution of potassium chloride. A platinum wire of suitable length is passes at the centre of glass jacket, remains in contact with sensitizing solution filled in glass tube at one side while its other end used for making electrical contact with external circuit most preferably with a potentiometer. On the basis of concentration of potassium chloride solution which ranges from 0.1M, 1.0M or saturated (mostly used), the calomel electrodes thus named as decimolar calomel electrodes (DCE), molar calomel electrodes (MCE) or normal calomel electrodes (NCE), and saturated calomel electrodes (SCE), having a respective potential (including liquid junction potential) at 25ºC of 0.3358V, 0.2824V, and 0.2444V. The electrode reaction for calomel electrode while it acting as cathode can be represented as, -

-

½Hg2Cl2(s)+e ↔Hg+Cl (acl-)

Likewise its potential can be denoted as ◦

E= E cl-  Hg2Cl2  pt-RT/F ln acl-

It has been interesting to note down that potential of a calomel electrode is inversely proportional to activity of chloride ions. As per the requirment and suitability of electroanalytical process, useful modifications are effectively be done in calomel electrodes. In order to avoid interference of potassium ions under some situations they are replaced with sodium

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ions effectively by substituting the solution of potassium chloride (KCl) with sodium chloride (NaCl) solution. Likewise, presence of chloride ions can be avoided by employing electrodes made up of mercury (I) sulphate for satisfactorily result. Saturated calomel electrodes are preferred over other type reference electrodes due to its reliability of sensitization, ease of maintenance and preparation. However at higher temperature (80ºC) all calomel electrodes are subjected to problem of accelerated disproportionation of mercury (I) ion to mercury (II) ion.

Figure 02: A Calomel Electrode 05.A.I.b.a. Advantages of calomel electrodes

I.

Easy to prepare and maintained.

II.

Compact in size.

III.

Inert towards solvent employed in electroanalytical process.

IV.

Good sensitivity against broad range of compounds & pH.

05.A.I.b.b. Disadvantage of calomel electrode

Not suitable for working at higher temperature. 05.A.I.c. Silver-silver chloride electrodes These electrodes are similar in importance as that of calomel electrodes, but are sometime difficult to construct. As per construction, silver-silver chloride electrodes are somewhat analogous to that of calomel electrode but differ from them in substituting mercury of calomel

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electrodes by silver or platinum electrode and calomel by silver chloride. Silver-silver chloride electrode commercially is available in two forms wire form and cartridge form. Wire form of Silver-silver chloride electrode consists of silver or silver plated platinum wire over which a thin layer of silver chloride is plated electrolytically which is then immersed in a known concentration of potassium chloride solution saturated with 0.1M silver nitrate solution. For analytical purposes, saturated solution of potassium chloride is most widely accepted however potassium chloride at a concentration of 0.1M and 1.0M is also employed as per requirement of process. The standard electrodes potential of Silver-silver chloride electrode with respect to standard hydrogen electrodes is 0.2224V at 298K. The cartridge form of silver-silver chloride electrode consists of metal which is in contact with the paste of moisturized salt sealed in inner glass tube. The representation of silver-silver chloride electrode & its reaction can be done as follows -

Ag  AgCl  Cl- (acl ) AgCl+e-↔Ag+Cl- (acl-) 05.A.I.c.a. Advantage of silver-silver chloride electrode

I.

Stable potential over wide range of time and temperature.

II.

Use of non toxic components.

III.

Inexpensive.

05.A.I.c.b. Disadvantages of silver-silver chloride electrode

I. Silver-silver chloride electrode should not be used to determine potential of solution containing reducing agent, proteins, halogens, and sulfide ions. 05.A.I.d.

Mercury (I) sulphate electrodes

It is also used as reference electrode where presences of chloride ions are not suitable due to their interference in final result. Construction of Mercury (I) sulphate electrodes is same as that of calomel electrodes except they contains mercury in solution of sulphuric acid (0.05M) saturated with mercurous sulphate. The standard electrodes potential of Mercury (I) sulphate electrodes with respect to standard hydrogen electrodes is 0.6801V at 25 ºC. 05.A.II. Indicator electrode As their name suggested, these electrodes point out or indicate even a minute change in potential or pH of test solution upon addition of titrant. On comparing with reference electrode, potential of indicator electrode varies as per the concentration of analyte present in solution.

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05.A.II.a

258

Hydrogen electrodes

Please refer section on reference electrodes of this chapter 05.A.II.b. Glass electrodes

Glass electrode was discovered by F. Fritz & K. Klemensiewicz who observed that when two solutions of unknown pH where separated by low melting and high conductivity glass results in exchange of protons between the solutions and glass membrane ultimately results in development of potential which depends upon differential value of pH of both solution. Glass electrodes are universally accepted and widely used as pH sensitive indicator electrode. These electrodes are based on the simple phenomenon of potential development on dipping them into a solution. The extent of potential so developed is directly proportional to the concentration of hydrogen ions present in the solution. Glass electrodes possess self potential which depends on thickness of glass bulb, its surface area, geometry of membrane, and overall composition of glass. On other hand, factors like alkaline or sodium ion error, acid error, dehydration of electrode, error due to un-buffered solution, variation in liquid junction potential, temperature, and high resistance of glass membrane also affects the final potential measurement with glass electrode.

Figure 03: The glass electrode

The assembly of glass electrode As Shown in Figure 03 is consist of highly sensitive thin glass membrane bulb containing 0.1M HCl and made up of sodium or calcium or alternatively lithium silicate with lanthanum and barium ion added. The so added ions act as lattice tighter and prevents alkaline hydrolysis of silicate thereby hindered the mobility of alkali ions predominately sodium. This pH responsive glass bulb is then fused onto a piece of

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comparatively thick & highly resistance glass tube. External contact of internally filled 0.1M HCl solution is made with the help of silver wire coated with uniform thin layer of silver chloride acting as an internal reference electrodes. S.No

Glass Types

Composition

1 2

Hard glass Soda lime glass

3

Lithium based glass

Cao-6% SiO2-72% Na2O-22% SiO2-62% LiO2-29% Cs2O-2% Bao-4% Ca2O3-72%

Remark

No use Good result between pH 1-9, PH>9-alkaline error

Excellent working at higher pH; free from alkaline error

Table 01: Composition of glass electrode 05.A.II.b.a. Advantages of Glass electrodes

I.

Rapid in response.

II.

Sensitive to wide range of pH.

III.

Not affected by oxidizing and reducing agents.

IV.

Efficient potential measurement of highly colored solution, solution containing dissolved gases, and solutions containing mild to moderate salt except sodium salt.

V.

Easy to rejuvenation by dipping the bulb in a solution of 0.1M HCl or alternatively by immersing it into solution of acid or alkali in order to reduce effect of residual sodium ion.

VI.

Easy to operate.

05.A.II.b.b. Disadvantage of glass electrodes

I.

Highly fragile in nature.

II.

Minute or minor electrode imperfections, scratches, and surface deposition of colloids or dehydrating agents affect its sensitivity.

III.

Not suitable for working at higher pH.

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IV.

Unsuitable to work up with simple potentiometer due to higher value of internal resistance.

V.

Should not be dried and must be washed thoroughly with distilled water after each measurement.

05.A.II.c. Quinhydrone electrode

Quinhydrone electrode is another commonly utilized indicator electrode for pH sensitization. Compositionally, it consists of quinone and hydroquinone in 1:1 ratio. Quinhydrone electrode is sparingly soluble in acidic solution and yield quinone and hydroquinone on decomposition. For pH determination, it is initially saturated with quinhydrone and then an inert electrode made up of bright platinum or gold wire is immersed into it. Finally, it is placed in a test solution along with saturated calomel electrode acting as reference electrode in order to form a complete cell. 05.A.II.c.a. Advantages of quinhydrone electrode

I.

Easy to prepare just by adding a pinch of quinhydrone (1mg/100ml) in test solution whose pH has to be determined.

II.

Rapid in equilibrium attainment and response.

III.

A good substitute for hydrogen electrode where they are contraindicated.

IV.

Free from error cause due to salt, non reducing gases.

V.

Effectively supersede hydrogen electrodes for solution containing lead, zinc, nickel, calcium, and tin.

05.A.II.c.b. Disadvantages of quinhydrone electrode

I.

Prone towards contamination of test solution.

II.

Unsuitable for solution having pH of more than 8.

III.

Readily get oxidized in air at higher pH.

05.A.II.d. Antimony electrode

It is a reversible oxygen electrode employed for pH determination. It consists of antinomy stick coated over with fine uniform layer of antimony trioxide and placed in the test solution along with saturated calomel electrode for pH measurement. The activity of solid antimony, antimony trioxide and water is ideally taken as unity 05.A.II.d.a. Advantages of antimony electrode

I.

Ideal electrode for pH determination between 2-8.

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II.

Non-fragile in nature.

III.

Free from effect caused by colloids and mild oxidizing agents.

IV.

Due to its sturdy nature, easy and useful for continuous pH determination.

V.

Effective for solutions which are highly turbid or viscous in nature.

05.A.II.d.b. Disadvantages of antimony electrode

I.

Problematic for pH measurement below 3 and above 9.

II.

Strong oxidizing agents and complexing agent interfere with electrode.

III.

In presence of metals like silver, copper, and other metals below antimony electrochemical series creates interferes in measurement.

IV.

Suitable for approximate result but not for precise work.

V.

Suffers salt error.

VI.

Required calibration for every application.

05.A.II.e. Ion selective electrode:

Lead work in the field of ion selective electrode development has been credited to Pungor who ultimately indicates possibility of determining anionic concentration with an aid of silcone rubber and plastic membrane. As their name indicates, these electrodes work on the principle of selective concentration dependent signal generation against specific ion in the presence of other ions without itself getting affected. Construction of a typical ion selective electrode is analogues to that of glass electrode whose selectivity could be modified as per requirement by altering the composition of material of construction. 06. APPARATUS Apparatus used in potentiometric titration consist of following two main assemblies 06.A. Reaction vessel 06.B. Simple potentiometer Reaction vessel used for carrying test solution and consist of suitable size glass vessel or any other vessel made up of inert material having provision for passage of nitrogen tubes (inlet & outlet), electrodes (indicator & reference electrode), and burette from lid. The external circuit of electrode is suitably connected with potentiometer having a typical assembly of a high resistance slide wire of uniform cross section area, a cell of high emf and a readout device which may either be a galvanometer or magic eye.

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Figure 04: A typical titrating assembly 07. METHODOLOGY FOR POTENTIOMETRIC TITRATION The reaction vessel is equipped with all the necessary assemblies, solution to be titrated is placed, and proper leveling had been done with suitable solvent such that tip of both the electrode get dipped into it. A high resistance type potentiometric unit assembled with sensitive galvanometer (mostly) or magic eye is connected to the external circuit of electrodes in order to detect change in potential during the course of titration.

Initially potential is applied and its corresponding value is recorded from galvanometer. Now a definite pre-measured volume of titrant in successive step is added into the test solution and resultant value of emf or pH is recorded. The end point of the titration is determined either by visualizing temporary deflection of needle in case of galvanometer or alternatively by blinking of magic eye. 08. END POINT DETECTION End point detection in potentiometric titration is done by sketching graph by following three methodologies; Methodology I.

Ordinate EMF (E) or pH

Abscissa Volume of titrant (V)

End point detection Volume of titrant gives maximum slop of curve.

II.

EMF (E)/V or, pH/V

Volume of titrant (V)

Drawing a perpendicular line from peak of graph linearly towards volume of titrant axis

III.

 E/ V

2

2

Volume of titrant (V) Maximum slope curve

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263

APPLICATIONS

09.A. Neutralization titrations Potentiometric titration could be efficiently used for determining electromotive force (emf) of solution (HCl vs NaOH) by dipping electrode reversible to the hydrogen ions and coupling them with reference electrode in order to complete cell. Upon addition of sodium hydroxide solution to HCl solution result in change in the pH thus potential which would be in accordance with equation E = E º+ 0.0591 pH

On plotting a graph (see figure 05) between potential (E) or pH against volume of titrant (NaOH) added shows, pH of the solution first rises in a gradual manner and then rapidly at end point. However, any further addition of alkali (NaOH) after attainment of end point only results in a minor increment in solution pH. 09.B. Redox titrations They are also performed potentiometrically in the same manner as that of neutralization reaction provided that reaction should not undergo any cooling or heating effect during overall titration process. Also the reversible hydrogen ion electrode here must be replaced with inert platinum wire electrode which is dipped in test solution and coupled with suitable reference electrode system to make the reading viable. The potential of indicator electrode used in redox titration is given by E = E º + 0.0591/n log (Oxidation)/(Reduction)

Graphically end point can be determined by sudden inflection in curve.

Figure 05: Potentiometric titration of acid with base

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09.C. Precipitation titration Potentiometric assisted precipitation reaction includes measuring potential of solution by precipitating ions from solution with the addition of titrant. For example, titrations of silver nitrate solution in presence of sodium chloride by immersing silver wire in former solution. On addition of sodium chloride solution into silver nitrate results in formation of silver chloride thus silver-silver chloride electrode coupled with reference electrode is suitable for potential measurement measurement in this type of titrations. 10.

EXERCISE a. Briefly explain principle involved in potentiometric potentiometric titration.

b. Enumerate various advantages associated with potentiometric potentiometric titrations. c. Explain following terms I.

Electrode potential

II.

Electromotive force

III.

Standard electrode potential

d. What are electrodes? Give their necessity in potentiometric titrations. e. Compare indicator and reference electrode with suitable examples. f. Write a short note on following I. II.

Standard hydrogen electrode Calomel electrode

III.

Glass electrode

IV.

Silver-silver Silver-silver chloride electrode

V.

Antimony electrode

g. Give advantages and disadvantages of standard hydrogen h ydrogen electrode electrode h. Discuss construction of calomel electrode with the help of well labelled diagram. i.

Write a short note on end point detection in potentiometric titration. titration.

j.

Briefly explain instrumentation and methodology involved in potentiometric titration.

k. Give suitable applications of potentiometric potentiometric titrations.

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Potentiometry deals with measurement of a. Potential b. Voltage c. Conductance d. All the above a Device used for measuring potential in potentiometry is known as a. Voltammeter b. Ammeter c. Potentiometer d. None of the above c Electrode/s used in potentiometric potentiometric titration are a. 1 b. 2 c. 3 d. 4 b The electrode whose value remains constant throughout the titrimetric process is termed as a. Indicator electrode b. Reference electrode c. Both d. None of the above b The purpose of a reference electrode is a. Complete circuit b. For reference purpose c. Both d. None of the above c

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The potential of indicator electrode varies with titrimetric process a. Yes b. No c. May be d. None of the above a Can potentiometric titration be used for pH determination a. Yes b. No c. May be d. None of the above a Expand EMF a. Electromotive force b. Electromotion force c. Electromotive fact d. None of the above a Electrode potential is a. Potential difference across electrode & electrolytic solution b. Potential difference across electrode & metal container c. Potential difference across interface of electrode & electrolytic solution d. None of the above c The electrode potential is a. Dependent quantity b. Independent quantity c. Both d. None of the above a

POTENTIOMETRY


Standard electrode potential is a. Electrode potential at infinite ion activity b. Electrode potential at unit ion activity c. Both d. None of the above b An electromotive force is a. Flow of current from electrode maintained at higher oxidation potential to an electrode maintained at lower oxidation potential b. flow of current from electrode maintained at lower oxidation potential to an electrode maintained at higher oxidation potential c. Both d. None of the above a EMF of cell is expressed in terms of a. Resistance b. Ohm c. Volt d. None of the above c Potentiometric Potentiometric titration involves potentiometer for potential measurement because a. It consume high amount of current b. It consumes negligible amount of current c. It is never used in potentiometric titration d. None of the above b

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Superiority of potentiometer over voltmeter in potential measurement is in a. Affecting electrode potential b. Not affecting electrode potential c. Utilization of none to least current d. Both b & c d The term electrode was coined by a. William Whehell b. William Waswell c. William Whowell d. William Whewell d Reference electrode electrode is also al so known as a. Refer electrode b. Standard electrode c. Both d. None of the above b References electrodes are assigned an arbitrary potential of a. 1.0 b. 0.1 c. 0 d. 0.01 c Pick out primary reference electrode a. Calomel electrode b. Mercury electrode c. Silver-silver Silver-silver chloride electrode d. Hydrogen electrode d Pick out Secondary reference electrode a. Calomel electrode b. Silver-silver Silver-silver chloride electrode c. Hydrogen electrode d. Both a & c d POTENTIOMETRY


Advantages of reference hydrogen electrode is/are a. Easily affected by purity and pressure of hydrogen gas. b. Not suitable to work up with oxidizing and reducing agents. c. Poisoning of platinum surface affects potential. d. Ideal for measurement of pH over a wide range d Disadvantage/s of reference hydrogen electrode is/are a. Easily affected by purity and pressure of hydrogen gas. b. Not suitable to work up with oxidizing and reducing agents. c. Poisoning of platinum surface affects potential. d. All the above d Pick out feasible indicator electrode for neutralization titration a. Glass electrode b. Calomel electrode c. Silver electrode d. Platinum wire a Pick out feasible indicat or electrode for precipitation titration a. Glass electrode b. Calomel electrode c. Silver electrode d. Platinum wire c Pick out feasible indicator electrode for redox titration a. Glass electrode

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b. Calomel electrode c. Silver electrode d. Platinum wire d Generally, a calomel electrode is named on the basis of ______ concentration a. NaCl b. KCl c. HgCl d. BaCl b On the basis of KCl concentration, a calomel electrode is of ______ type/s a. 1 b. 2 c. 3 d. 4 c Concentration of KCl in decimolar calomel electrode is a. 0.1M b. 1.0M c. 0.01M d. None of the above a Concentration of KCl in normal calomel electrode is a. 0.1M b. 1.0M c. 0.01M d. None of the above b Concentration of KCl in molar calomel electrode is a. 0.1M b. 1.0M c. 0.01M d. None of the above b

POTENTIOMETRY

Deepak Chowrasia, Nisha Sharma-Analytical Chemistry. a Qualitative & Quantitative Approach (2015)

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