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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 methods of analysis. This experiment was done to demonstrate the separation of amino acids by 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 Chromatography is a powerful method to separate mixtures of substances into their 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 liquid supported on a solid. As the sample 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 at different rates due to various factors. Chromatographic procedures can be adsorption chromatography and partition chromatography. In adsorption chromatography, there is an equilibrium between the mobile and stationary phase which accounts for the separation of the components. In partition chromatography, the components are distributed between the mobile phase and 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 covered. A 19 cm x 14 cm Whatman 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 of the cross marks. The spots were dried first before applying the succeeding spots. The spots were labeled with pencil marks to identify the amino acids and the unknown. The paper was rolled into a cylinder and the edges were stapled 1 mm apart. It was then put inside the bottle containing the developing solvent with the sample edge down. The line containing the amino acid solution spots is not immersed in the solution. The bottle was closed tightly and was not moved while the solvent is rising. The process was stopped when the solvent front is only about 1 cm from the top edge of the paper. The paper was removed and was dried. The staples were removed and the paper was laid down on a clean bond paper with its front side down. Ninhydrin solution was brushed lightly and evenly on the paper along the direction of solvent flow under the dryer. The edges of the resulting violet spots were traced and the 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 distances of the solute and solvent were measured and were taken as follows: Amino Acid Standards Glycine Distance Traveled by the amino acid (cm) 20 Distance Traveled by the Solvent (cm) 116 Page 1 of 3
Experiment 2: Separation of Amino Acids by Paper Chromatography
17/116 = 0. Factors that could affect the Rf values are the solvent system polarity. The chromatography chamber must be kept saturated so that the solvent will not evaporate while it rises up the filter paper. which is 0. direction of the paper’s fibers. Identify the stationary and mobile phase in paper chromatography. IV. Glycine = 0.3 Leu 2. What are the factors that could affect the Rf value of a solute.1 5.88 Leucine = 70/116 = 0. 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 Distance traveled by the Amino Acid standard in S1 and S2: Amino Acid S1 (cm) S2 (cm) Ala 3. 5.2 Trp 5. A mixture of amino acids was separated into its component by two-dimensional chromatography using solvents S1 and S2.35.5 Phe 9. water mixture) = 12 cm Distance traveled by the amino acid standard in S1 and S2: Amino Acid S1 (cm) S2 (cm) A 6.0 1. a. Explain briefly the differences in Rf values of the amino acid component of your mixture. The solvent mixture should be allowed to saturate the chromatography chamber.0 Draw clearly the two-dimensional chromatogram and indicate the directions of solvent flow. Compounds with larger Rf have lesser polarity because it interacts less strongly with the polar adsorbent.15 1. The diameter of the amino acid spots should be about 1 mm only because if the spot is big enough. The data obtained are given below: Distance traveled by solvent fronts: S1 (Butanol.0 4.9 Lys 6. Page 2 of 3 Based on the measurements obtained.42. c.35 Since the Rf value of the unknown amino acid.5cm S2 (Phenol.0 2. The different polarity of the amino acids contribute to their differences in Rf values. and molecular weight of solute (mobile phase).5 His 9. The diameter of the amino acid spots should be about 1mm only. This is used to identify the unknown by comparing its Rf value to a Rf value of known compounds.17 Lycine = 102/116 = 0. 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. Guide Questions 1. 4.9 6.7 6.0 9. adsorbent/paper Experiment 2: Separation of Amino Acids by Paper Chromatography .42 Unknown = 41/116 = 0.6 Glu 2. b.1 C 6. Identify amino acids A. 3. is closest to the Rf value of Tyrosine.3 7.60 Tyrosine = 49/116 = 0.0 D 9. The chromatography paper should not be touched with bare hands. acetic acid. we can identify this unknown as Tyrosine. which is 0. Stationary phase – water Mobile phase – Butanol 2.9 2. Give reasons for the following procedures. C and D. the solute might travel more in a horizontal manner than vertically and might come in contact with those amino acids on its side. water mixture) =11. This value is between 0 and 1 and has no unit.Lysine Leucine Tyrosine Unknown 102 70 49 41 116 116 116 116 porosity. B.8 B 8.14 4.
better separations and the choice between different adsorbents compared to paper. a. alumina. which is the stationary phase.http://www. Experiment 2: Separation of Amino Acids by Paper Chromatography Page 3 of 3 . Knowing the Rf values of the 4 basic amino acids.rpi. most often Helium. It utilizes very small packing particles. you can compare and know what’s the identity of the unknown amino acid. References Carrier. V. Galan. Charize Mae U. VI. 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. 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. or cellulose on a flat. The larger the Rf value of a compound.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. Thin-Layer Chromatography It involves a stationary phase of a thin layer of adsorbent like silica gel. c. Intro to Chromatography. the larger the distance it travels on the chromatogram. and a relatively high pressure. Gas Chromatography It involves a solid stationary phase and a mobile gas. d. Rebecca et al (1994).html 6. Ma. Reversed Phase Chromatography It is a procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase. I hereby certify that I have given substantial contribution to this report. Renelyn D. It has faster runs.edu/dept/chemeng/Biotech-Environ/CHROMO/chromintro. b. Discuss briefly the basic principles of the following chromatographic techniques. High Performance Chromatography It is a separational technique in which the mobile phase is a liquid. Chingcuangco. The stationary phase adheres to the inside of a capillary column or a packed column. Different substances (amino acids) have different Rf values. e. inert substrate.