COLUMN CHROMATOGRAPHY
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It was disco iscov vered red by Mikhail Tswett in 1906 is one of of the most most useful useful method methods s for the sepa separatio ration n and purifi purificatio cation n of both both solids solids and liquids. The princi principle ple of column column chroma chromatog tograph raphy y is based based on differe differentia ntiall adsorptio adsorption n of substan substance ce by the adsorbent. The The sepa separa rati tion on can can be be liqu liquid id/s /sol olid id ( adsortion ) or liquid/liquid ( artition) in column chromatography. The stationary phase, a solid adsorbent, is usually placed in a vertical glass column and the mobile phase, is added from the top and let flow down through the column by either gravity or eternal pressure
PR!NC!PL"
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!hen !hen a mitur miture e of compon componen ents ts dissol dissolved ved in the the mobile mobile phase phase is intro introdu duced ced into into the column, column, the individu individual al compone components nts move with differe different nt rates rates depend depending ing upon upon their their relative affinities. The compo compound und with with lesser lesser affini affinity ty towards towards stati stationary onary phase phase moves moves faste fasterr and it is eluted eluted out of the column first. The one with greater affinity towards stationary phase moves slower down the column and hence it is eluted latter. Thus the compounds are separated !hen !hen a mitur miture e of compon componen ents ts dissol dissolved ved in the the mobile mobile phase phase is intro introdu duced ced into into the column, column, the individu individual al compone components nts move with differe different nt rates rates depend depending ing upon upon their their relative affinities. The compo compound und with with lesser lesser affini affinity ty towards towards stati stationary onary phase phase moves moves faste fasterr and it is eluted eluted out of the column first. The one with greater affinity towards stationary phase moves slower down the column and hence it is eluted latter. Thus the compounds are separated
A#$OR%"NT$ USUAL ADSORBENTS USED"
silica, alumina, calcium carbonate, calcium phosphate, magnesia, starch, etc., selection of solvent is based on the nature of both the solvent and the adsorbent. SELECTION OF ADSORBENT
#dsorbent in $.$ should meet following criteria " • • • • •
%articles should be spherical in shape & uniform in si'e echanical stability must be high They should not react chemically It should be useful for separating for wide variety of compounds It should be freely available & inepensive
$OL&"NT
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The polarity of the solvent, which is passed through the column, affects the relative rates at which compounds move through the column. %olar solvents can more effectively compete with the polar molecules of a miture for the polar sites on the adsorbent surface and will also better solve the polar constituents. highly polar solvent will move even the highly polar molecules rapidly through the column. If a solvent is too polar, movement becomes too rapid, and little or no separation of the components of a miture will result.
$"L"CT!ON O' $OL&"NT
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The solvents should also have sufficiently low boiling points to permit ready recovery of eluted material. owever, polarity as seen the most important factor in adsorption chromatography. *ifferent mobile phases used" ( in increasing order of polarity)" %etroleum ether, carbon tetrachloride, cycloheane, ether, acetone, ben'ene, toluene, esters, water, etc It can be used in either pure form or as miture of solvents
COLUMN COLUMN CHARACT"R!$T!C$
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The main function of all the columns is to support the stationary phase. The material of the column is mostly good quality neutral glass since it should not be affected by solvents. #n ordinary burette can also be used as column for separation. $olumn dimensions + length & diameter ratio (-",-" or --") arious accessories are attached to the top and bottom of the column for maintenance of the elution process.
PR"PARAT!ON O' COLUMN
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It consists of a glass tube with bottom portion of the column 0 pac1ed with glass wool/cotton wool or may contain asbestos pad #bove which adsorbent is pac1ed #fter pac1ing a paper disc 1ept on the top, so that the adsorbent layer is not disturbed during the introduction of sample or mobile phase The level of solvent must never be allowed to fall below the level of adsorbent to prevent crac1s.
PAC(!NG O' COLUMN$
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There are two types of pac1ing techniques used in $.$ i. *ry pac1ing / dry filling ii. !et pac1ing / wet filling The column should be free from impurity, before using column, it should be washed properly and dry it. 2efore filling column with stationary phase, cotton/glass wool is 1ept It should be uniformly filled )"T PAC(!NG
#RY PAC(!NG Dry packing involves the pouring of fine powdered form of the adsorbent into the column.
The column must be tapped while the filling process is going on so as to maintain the soft compactness of the adsorbent in the body of the column.
The column is filled upto 3/4 th of the actual height of the column. The empty head above the surface of the packed column is filled with the mobile phase.
!NTRO#UCT!ON O' TH" $AMPL"
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The sample which is usually a miture of components is dissolved in minimum quantity of the mobile phase. The entire sample is introduced into the column at once and get adsorbed on the top portion of the column. 3rom this 'one, individual sample can be separated by a process of elution.
#"T"CT!ON O' COMPON"NT$
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If the compounds separated in a column chromatography procedure are colored, the progress of the separation can simply be monitored visually. If the compounds to be isolated from column chromatography are colorless. In this case, small fractions of the eluent are collected sequentially in labelled tubes and the composition of each fraction is analy'ed by T4$.
APPL!CAT!ON
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5eparation of miture of compounds %urification process Isolation of active constituents 6stimation of drugs in formulation separation of diastereomers
A#&ANTAG"$ • • • •
#ny type of miture can be separated #ny quantity of miture can be separated !ider choice of obile %hase #utomation is possible
#!$A#&ATAG"$
Time consuming more amount of obile %hase are required #utomation ma1es the techniques more complicated & epensive
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