Experiment 2: Separation of Amino Acids by Paper Chromatography Chingcuangco, Ma. Renelyn D. Galan, Charize Mae U. Group 2, Chem 31.1; AB2; Ms. Denise Arganda April 29, 2009 Abstract In any chemical or bioprocessing industry, the need to separate and purify a product from a complex mixture is a necessary and important step in the production line. Chromatography is a very special separation process; it can separate complex mixtures with great precision. Even very similar components, such as proteins that may only vary by a single amino acid, can be separated with chromatography. It is also used to separate and identify all sorts of substances in police work; drugs from narcotics to aspirin can be identified in urine and blood samples. It does not only provide methods of separation, chromatographic techniques can also provide method methods s of analys analysis. is. This experi experimen mentt was done done to demons demonstra trate te the separa separati tion on of amino amino acids acids by paper paper chromatography and use chromatography to identify unknown amino acids. Definition of Terms: Chromatography – a technique used to separate mixtures into components Paper Chromatography – one of the methods of chromatography by using a paper like filter paper or any special paper Mobile Phase – gas or liquid that carries the components Stationary Phase – part of the system that does not move with the sample
I. Introduction Chromatogr Chromatography aphy is a powerful powerful method to sepa separa rate te mixt mixtur ures es of subs substa tanc nces es into into thei their r respective substances. All forms of chromatography have a mobile phase, which can be a liquid or gas, and a stationary phase which can be a solid or liqui liquid d supp suppor orte ted d on a soli solid. d. As the the samp sample le is introduced into the mobile phase, the mobile phase flows through the stationary phase and caries the mixture’s components with it. Different components travel travel at differ different ent rates rates due to vario various us factor factors. s. Chroma Chromatog tograp raphic hic proced procedure ures s can be adsorp adsorptio tion n chromatogr chromatography aphy and partition partition chromatogr chromatography. aphy. In adsorption chromatography, there is an equilibrium betwee between n the mobil mobile e and stati stationar onary y phase phase which which accounts for the separation of the components. In parti partitio tion n chroma chromatog tograp raphy, hy, the compon component ents s are dist distri ribu bute ted d betw betwee een n the the mobi mobile le phas phase e and and the the stationary liquid. II. Methodology 12 mL of butanol, 3 mL glacial acetic acid and 5 mL distilled water were mixed in a bottle without wetting the sides with the solvent and it was cov covered ered.. A 19 cm x 14 cm Whatma atman n No. 1 chromatography paper was laid on a clean sheet of bond paper and a very light pencil line was drawn across its length 1 cm from the bottom. Starting 2 cm from the left edge, light pencil cross marks were drawn on the line 3 cm apart. With a capillary tube, amino acid solution was applied four times at the
center center of the cross marks. marks. The spots spots were dried dried first first before before applyi applying ng the succee succeedin ding g spots. spots. The The spots were labeled with pencil marks to identify the amin amino o acid acids s and and the the unkn unknow own. n. The The paper paper was was rolled into a cylinder and the edges were stapled 1 mm apar part. It was then hen put put insi inside de the bott bottlle containing the developing solvent with the sample edge edge down. The line line containi containing ng the amino amino acid solution spots is not immersed in the solution. The bottle bottle was closed tightly and was not moved while the solven solventt is risin rising. g. The The proces process s was stopped stopped when the solvent front is only about 1 cm from the top edge of the paper. The paper was removed and was was drie dried. d. The The stapl staples es were were remo remove ved d and and the the paper was laid down on a clean bond paper with its front side down. Ninhydrin Ninhydrin solution was brushed brushed lightly and evenly on the paper along the direction direction of solvent flow under the dryer. The edges of the resulting resulting violet spots were traced traced and the center center with the darkest spot was marked. The Rf values of the amino acids were calculated and the unknown was identified. III. Results and Discussions The distance distances s of the solute solute and solven solventt were measured and were taken as follows:
Amin Amino o Acid Acid Standards
Glycine
Experiment 2: Separation of Amino Acids by Paper Chromatography
Distance Travel Traveled ed by the amino acid (cm) 20
Distance Trav Travel eled ed by the the Solve olvent nt (cm) 116 Page 1 of 3
Lysine Leucine Tyrosine Unknown
102 70 49 41
116 116 116 116
Based on the measurements obtained, we can get the Rf values of each amino acid by this formula: Rf = distance traveled by the amino acid distance traveled by the solvent Rf or Retention factor is the ratio between the distance traveled by the solute and the distance traveled by the solvent. This value is between 0 and 1 and has no unit. This is used to identify the unknown by comparing its Rf value to a Rf value of known compounds. Glycine = 0.17/116 = 0.17 Lycine = 102/116 = 0.88 Leucine = 70/116 = 0.60 Tyrosine = 49/116 = 0.42 Unknown = 41/116 = 0.35 Since the Rf value of the unknown amino acid, which is 0.35, is closest to the Rf value of Tyrosine, which is 0.42, we can identify this unknown as Tyrosine. IV. Guide Questions 1. Identify the stationary and mobile phase in paper chromatography. Stationary phase – water Mobile phase – Butanol 2. Explain briefly the differences in Rf values of the amino acid component of your mixture. The different polarity of the amino acids contribute to their differences in Rf values. Compounds with larger Rf have lesser polarity because it interacts less strongly with the polar adsorbent.
3. What are the factors that could affect the Rf value of a solute. Factors that could affect the Rf values are the solvent system polarity, adsorbent/paper
porosity, direction of the paper’s fibers, and molecular weight of solute (mobile phase). 4. Give reasons for the following procedures. a. The diameter of the amino acid spots should be about 1mm only. The diameter of the amino acid spots should be about 1 mm only because if the spot is big enough, the solute might travel more in a horizontal manner than vertically and might come in contact with those amino acids on its side. b. The solvent mixture should be allowed to saturate the chromatography chamber. The chromatography chamber must be kept saturated so that the solvent will not evaporate while it rises up the filter paper. c. The chromatography paper should not be touched with bare hands. Sweat from the skin will contaminate the chemicals present and skin’s oils will also appear on the chromatography paper which will give inaccuracy with the results. 5. A mixture of amino acids was separated into its component by two-dimensional chromatography using solvents S1 and S2. The data obtained are given below: Distance traveled by solvent fronts: S1 (Butanol, acetic acid, water mixture) =11.5cm S2 (Phenol, water mixture) = 12 cm Distance traveled by the amino acid standard in S1 and S2: Amino Acid S1 (cm) S2 (cm) A 6.1 5.8 B 8.9 2.1 C 6.0 1.0 D 9.0 4.5 Distance traveled by the Amino Acid standard in S1 and S2: Amino Acid S1 (cm) S2 (cm) Ala 3.7 6.5 Phe 9.14 4.9 Lys 6.15 1.3 Leu 2.0 9.6 Glu 2.3 7.5 His 9.0 2.2 Trp 5.9 6.0
Draw clearly the two-dimensional chromatogram and indicate the directions of solvent flow. Identify amino acids A, B, C and D.
Experiment 2: Separation of Amino Acids by Paper Chromatography
Page 2 of 3
A compound of low polarity will have larger R f value because it interacts less strongly with the polar adsorbent which is the filter paper. Different substances (amino acids) have different Rf values. Knowing the Rf values of the 4 basic amino acids, you can compare and know what’s the identity of the unknown amino acid.
VI. References Carrier, Rebecca et al (1994). Intro to Chromatography .http://www.rpi.edu/dept/chemeng/Biotech-Environ/CHROMO/chromintro.html
6. Discuss briefly the basic principles of the following chromatographic techniques. a. Thin-Layer Chromatography It involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. It has faster runs, better separations and the choice between different adsorbents compared to paper. b. Column Chromatography It consists of a column of particular material such as silica or alumina that has a solvent passed through it at atmospheric or low pressure.
I hereby certify that I have given substantial contribution to this report.
Chingcuangco, Ma. Renelyn D.
Galan, Charize Mae U.
c. Gas Chromatography It involves a solid stationary phase and a mobile gas, most often Helium. The stationary phase adheres to the inside of a capillary column or a packed column. d. High Performance Chromatography It is a separational technique in which the mobile phase is a liquid. It utilizes very small packing particles, which is the stationary phase, and a relatively high pressure. e. Reversed Phase Chromatography It is a procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase. V. Conclusion Difference in affinities of components of a mixture to the stationary and mobile phase determines the rate at which they travel on the filter paper. The larger the Rf value of a compound, the larger the distance it travels on the chromatogram. Experiment 2: Separation of Amino Acids by Paper Chromatography
Page 3 of 3
