Radial/Circular Paper Chromatography of Amino Acids
Experimental Problem To separate and analyze a mixture of amino acids Educational Purpose By performing this lab, the student will: 1. Learn how to perform paper chromatography- separation technique. 2. Understand the basics of paper chromatography 3. To determine the relationship between the chromatographic properties and the chemical structures of amino acids 4. Use paper chromatography to investigate the chemical structure of an unknown amino acid Tutorials
Paper chromatography
What To Turn In 1. Questionnaire: prelab and postlab 2. Developed Chromatogram
1. INTRODUCTION Amino Acids The thousands of different cellular proteins carry out distinct biological processes. The specific process mediated by a protein is dependent depen dent on the protein’s three dimensional shape. Ultimately, this three dimensional shape is dependent on the chemical structure of the protein. Proteins consist of long polymers called polypeptides, strings amino acids linked together by peptide bonds. All polypeptides p olypeptides are composed of the same set of twenty amino acids. Different proteins vary in the order and number of amino acids in their polypeptide chains. All twenty amino acids share a common structure called the “conserved region” of the amino acid. This conserved region consists of a central carbon called the αα-carbon. This α-carbon α-carbon is linked to a carboxyl group, an amino group and a hydrogen atom. These groups along with the ααcarbon make up the “conserved region”. All twenty amino acids have this structure. The ααcarbon is also attached to a variable structure called the R group. The R group is what differs among the twenty amino acids.
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Chromatography is a technique used for separating the components of a mixture. It can be used for purification of compounds, identification of the components, of a mixture and their separation. The separation of the components of a mixture is brought about by means of two immiscible substances (may be solid/liquid) which adsorbs the components of the mixture and the second the mobile phase (may be liquid/gas) which while passing through the stationary phase transports the components of the mixture on to it (Table 1). The components of the mixture are transported at different rates. The most strongly adsorbed components move slowly while the weakly adsorbed components moves faster with the moving/mobile phase. Thus as the mobile phase passes through the stationary phase i t separates the components of the mixture. Table1: Chromatographic methods Types chromatography
of Stationary phase
Mobile phase
Principle
Column
Solid
Liquid
Adsorption
Thin Layer
Solid
Liquid
Adsorption
Paper
Liquid
Liquid
Partition
Gas Liquid
Liquid
Gas
Partition
The stationary phase in paper chromatography is a liquid i.e. water (adsorbed on cellulose of the Whatmann filter paper) and moving phase is also a liquid and the two phases are immiscible. The basis of separation of the components of the mixture is their partition between two phases. Paper chromatography classified as three types 1. Ascending chromatography: the solvent moves up along the paper. Here the development of paper occurs due the solvent movement or travel in upward direction on the paper. The solvent reservoir is at the bottom of beaker. The paper tip with sample spots just dips into the solvent at bottom such that spots remain well above the solvent.
Fig. 1: Ascending chromatography
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2. Descending chromatography: the solvent moves down along the paper ere the development of paper occurs due to solvent travel downwards on the paper. The solvent reservoir is at the top. The movement of solvent is assisted by gravity besides capillary action.
Fig. 2: Descending chromatography 3. Radial/Circular chromatography: the solvent moves from the centre of the circular paper towards its circumference horizontally Here the solvent travels from center(mid point) towards periphery of Circular chromatography paper. The entire system is kept in a covered petridish for development of chromatogram. The wick at the center of paper dips into mobile phase in a petridish by which the solvent drains on to the paper and moves the sample radially to form the sample spots of different compounds as concentric rings (Fig. 3).
Fig. 3: Radial chromatography
Paper Chromatography Chromatography is an analytical tool for distinguishing different biomolecule based on their chemical properties. One of the oldest and most reliable forms of chromatography is paper chromatography. In this assay, a biomolecule (or mixture of biomolecules) is spotted on a piece of filter paper. The filter paper is composed mostly of cellulose and is very hydrophilic. Next a hydrophobic organic solvent is drawn up the paper by capillary action. As the solvent moves over the location of the biomolecule, the biomolecule begins to move up the paper. The rate at which the biomolecule moves up the paper is related to its relative affinity for the paper (which is hydrophilic) and the solvent (which is hydrophobic). Hydrophobic molecules will move faster because they are more attracted to the hydrophobic solvent than the hydrophilic paper. On the 4
other hand, hydrophilic molecules will move slower because they are attracted more to the paper than the hydrophobic solvent. Uses and applications of paper chromatography
Paper chromatography is especially useful in characterizing amino acids, carbohydrates and to determine organic compounds, biochemicals in urine etc. Sometimes used for evaluation of inorganic compounds like salts and complexes. Calculation of R f values
The different amino acids move at differing rates on the paper because of differences in their R groups. The rate of movement of a biomolecule during paper chromatography is reported as its relative mobility/retention factor (R ). R is simply the distance the biomolecule moved throu gh f
f
the filter paper divided by the distance the solvent moved through the paper. Measure the distance from the start line to the solvent front and to the front of each spot. For each spot, calculate the R f value (R f means relative to front or retention facor ): distance moved by spot from the centre distance moved by solvent front from the centre The identity of an amino acid can be established by calculating its R f value and comparing with the literature (Table 1). Since the Rf value varies with the type of the paper used, thickness of the adsorbent on TLC plate, purity of solvents and excat composition of mobile phase and hence such a comparison is somewhat risky and not dependable. The best way to identify the amino acid is to run the sample of known amino acids with the unknown mixture of amino acids so that Rf value obtained under exactly identical conditions can be compared. Even pyridine isatin reagent can also be used to visualize amino acids. Table 1: Literature R f value of amino acids Amino acid
R f value
alanine
0.38
arginine
0.20
asparagine
0.5
aspartic acid
0.24
cysteine
0.4
glutamine
0.13
glutamic acid
0.30
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glycine
0.26
histidine
0.11
isoleucine
0.72
leucine
0.73
lysine
0.14
methionine
0.55
phenylalanine
0.68
proline not a true amino acid shows up as yellow
0.43
serine
0.27
threonine
0.35
tryptophan
0.66
tyrosine
0.45
valine
0.61
Chemistry: Amino acids have no colour. Therefore all of these procedures need to be carried out "blind", and the results will be seen when a revealing agent (ninhydrin) is sprayed on the resulting chromatogram.
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Most amino acids (except proline and hydroxyproline which gives yellow colour) give a purple blue colour with ninhydrin solution. This is because the final product of the reaction between amino acid and ninhydrin has the same structure for all these amino acids.
Prelab (10 marks): 1. What is the significance of number while saying Whatmann paper No. 1, 2,3? (2) 2. Can a normal filter paper could be used for the paper chromatography. If not then why? (2) 7
3. Write down the structure of amino acids Tryptophan, Lysine, Leucine and Alanine. Arrange them in increasing order of their polarity (2+2=4) 4. Write down the structure of proline. Why proline gives yellow colour with ninhydrin. (2)
Requirements: Petri-dish (50 mL), a pair of tweezer, Whatmann filter paper No.1, Mixture of unknown amino acids, 1 known amino acid, Pencils and Rulers, filter paper, BAW (4:1:1, 30 mL), Ninhydrin solution, cotton, Measuring cylinder 50mL, capillary. Precautions: BAW mixture is corrosive and can cause burns Ninhydrin can stain your hands. Be careful while spraying. Chromatography paper must not be touched with the hands (from middle). Always hold from corner. Do not place it on dirty bench (work on a clean surface) Used BAW mixture should be place back in the Labelled (Used BAW Mixture) bottle NOTE CAREFULLY: You must wear gloves while carrying out this experiment!!! Dispose of used cotton in the waste containers, which have been provided in the hoods. Procedure:
1. In a petri-dish (Chromatography chamber) pour 25-30mL of given BAW mixture. Allow it to equilibrate. 2. Obtain a clean piece of filter paper. Th e fi lter paper shoul d never be handled by bare hands since the skin’s oils show up on the developed chr omatogr am
3. Use a pencil, draw a dot approximately in the centre of the paper. Similarly your partner will draw the dot on other Whatmann paper. 4. Label your paper with paper at extreme right hand corner with amino acid mixture taken for spotting and your initials with the date in pencil 4. Spot with a capillary your sample on the pencil dot carefully. Let all samples dry and then spot it gain at the same dot. Dry and repeat the process 3-4 times in total 5. When the sample is dry, pierce a hole at the dot place carefully without tearing the paper using sharp end pencil. Insert a cotton wick from the hole form reverse side. Be careful your hands are dry and you are touching the paper through corners only. (figure 2). 6. Place the Whatmann paper in such a way that dot lies/cotton wick lies in the centre of petri dish (as shown by the instructor). Place the other half of petri dish carefully and allow the chromatogram to develop for 30-45 minutes undisturbed or until the solvent line is within an inch from the rim of the paper. 7. Remove the paper from the petri-dish and immediately mark the solvent front line in pencil. Allow the paper to thoroughly dry. 8. Your lab instructor will spray your paper with Ninhydrin developer just to wet the developed chromatogram, which is used to detect the location of amino acids. Allow it to air dry and then o kept in a preheated oven at 80 C for 5 min to allow the color to develop.
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9. Measure the distance the solvent migrated and the distance each of the amino acids migrated. Record the measurements on the developed chromatogram. Calculate the retention factor (R f ) for each amino acid. 10. Record and report the changes. Name and Roll No.: Partner’s Name and Roll No.: Your initial… Distance travelled by solvent front (cm) 1. 2. 3. 4. Mean=
Sample “…” Distance travelled by amino acid (cm) 1. 2. 3. 4. Mean=
1. 2. 3. 4. Mean=
R f of Amino acid
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Partner initial..... Distance travelled by solvent front (cm) 1. 2. 3. 4. Mean=
Sample “…..” Distance travelled by amino acid (cm) 1. 2. 3. 4. Mean=
R f of Amino acid
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Sample “…..” contains a mixture of ……amino acids, namely…………………and ……………. with R f values…………………and……………respectively. (3) Submit your Developed chromatogram. (3)
Postlab (10 marks):
1. Explain your chromatograph? (2) [ Hint: comment on amino acid hydrophilic/ hydrophobic nature, migration rates] 2. Which technique is better among ascending, descending and radial paper chromatography and why? (2)
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