Sofia Ahmed et al.

/International Journal of Chemical and Analytical Science 2010, 1(8),174-176

Available online through www.ijcas.info Analysis of Amino Acids By Paper Chromatography

Research Article
ISSN: 0976-1209
Sofia Ahmed1, Muhammad Ali Sheraz 1* , Muhammad Aminuddin 1, Iqbal Ahmad1, Karamat Mahmood2 and Kefi Iqbal3 1 Institute of Pharmaceutical Sciences, Baqai Medical University, Toll Plaza, Super Highway, Gadap Road, Karachi74600, Pakistan 2 Department of Chemistry, The Islamia University, Bahawalpur, Pakistan. 3 Baqai Dental College, Baqai Medical University, Karachi, Pakistan.

Received on: 20-05-2010; Revised on: 16-06-2010; Accepted on:15-07-2010 ABSTRACT

Paper chromatography method for separation and identification of amino acids in a mixture is described. The method is based on the use of ninhydrin as a locating reagent. The procedure has been successfully applied to the study of establishing a relationship between the zone area of the separated spots and the concentration of the amino acid (s) for quantitation of the solute. A suitable solvent system n-butanol-acetic acid-water (65:15:25) has been evolved which is found to give better resolution of the components during development of chromatograms. The amino acids in the concentration range varying from 104 to 10 1 mol / liter has been studied and found to give satisfactory results.

Keywords: Paper chromatography, amino acids, ninhydrin, qualitative and quantitative analysis.
INTRODUCTION Ninhydrin (1,2,3-triketo-hydrindene hydrate) is the mostly widely used colorimetric assay for amino acids[1]. Moore and Stein [2] developed this reaction as a convenient photometric method for the determination of amino acids. The reaction produces a blue color product, which absorbed maximally at 590 nm[3]. Several methods for separation, detection and quantitation of amino acids by paper chromatography in various samples have been reported[423]. The other methods are thin layer chromatography[2427], HPLC[2831] and electrophoresis[3234] which are used for the determination of amino acids. The micelle-induced interaction between ninhydrin and tryptophan has also been reported[35]. Ninhydrin ferric reagent has been reported to react highly and specifically with lysine at pH value of 1.0[36]. Ferric ion inhibits the reaction of ninhydrin with proline, ornithine, glycine, arginine and histidine. Separation of amino sugars and related compounds can be accomplished by two-dimensional thin-layer chromatography on cellulose layers[37]. The determination of N-methylhistidine in human urine has been possible using orthophthalaldehyde reagent[38]. Lysine and ornithine, however, can be determined using acid ninhydrin [39]. Recent development includes the application of capillary electrophoresis, laser induced fluorescence detection of amino acids[4042]. The present study is undertaken to determine the feasibility of combining the simple ninhydrin procedure with the o-phthalaldehyde based chromatographic method thus improving the chromatography technique for a rapid, reproducible and reliable results. EXPERIMENTAL Materials purchased either from Merck or BDH were of analytical grade. Spectrophotometer (Spectronic20, USA) was used for the absorbance measurements. 1. Preparation of Solutions i. Solutions of amino acids One molar solution (1 mole / L) was prepared by dissolving the amino acids in water. This one molar solution was suitably diluted with water to obtain 0.1 M, 0.01 M and 0.001 M solution of amino acids. ii. Ninhydrin solution (1% w/v) One gram of ninhydrin was dissolved in a few ml of acetone and volume made up to 100 ml with acetone. iii. Solvent systems for mobile phase a)n-butanol-acetic acid-water (60:15:25) b)n-butanol-acetic acid-water (50:20:30) c)Phenol-water (3:1.5) d)Phenol-water (4:1) iv. Sample preparation Mixture of amino acids made:
Mixture No. 1 2 3 4 5 6 Amino acids in mixture Glycine + Alanine Serine + Proline + -Phenylalanine Valine + Leucine + Hydroxyproline + Arginine Lysine + Alanine Tyrosine + Glycine + -Phenylalanine Alanine + Serine + Valine

2. General Procedure for Separation of Amino Acids i. Strips of 20 20 cm sizes were cut from the Whatman No. 1 chromatographic sheets. A line was marked with lead pencil at one edge of the paper at approximately 1.5 cm above from the lower end of the paper, marking out positions of spots for application of the sample solution. A small drop of standard or sample solution was applied at specified position by means of a capillary tube. The solution spots were dried by means of warm air draught from a hair-dryer. It was developed using different solvent systems. The developed paper was then removed from the chromatographic chamber, dried and sprayed with the locating reagent. This was then dried with dryer and then heated in an oven at 110C for 5 min. An outline was drawn around the developed colored spots by an ordinary lead pencil.

ii.

3. Qualitative Analysis This is done by measuring Rf values, being specific for a compound. This was obtained by measuring the ratio of the distance moved by the solute to the distance moved by the solvent present. 4. Quantitative Analysis i. Zone area method The area of the spot is proportional to the concentration of substance. The zone area of the spot was obtained by transferring it to a piece of graph paper and the squares were counted. ii. Calibration curve Amino acids (pure) in concentrations varying from 101 to 10 4 were used. The zone area of the spots was plotted against the known log concentration of the pure amino acid(s). This gave a linear relationship, obeying Beer-Lambert law. RESULTS AND DISCUSSION The analytical system was optimized for complete separation of amino acids from sample matrices by studying the parameters such as solvent system, solute concentration and the sample size. The present work involved the investigation of the effectiveness of various solvent mixtures for a successful separation of amino acids, singly or in a mixture. The separation was effective when the

*Corresponding author.
Muhammad Ali Sheraz Institute of Pharmaceutical Sciences, Baqai Medical University, Toll Plaza, Super Highway, Gadap Road, Karachi74600, Pakistan Tel.: + 92-21-34410293 Telefax: +92-21-34410439 E-mail:ali_sheraz80@hotmail.com

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Table 1.Rf values of amino acids
Amino acids Alanine Glycerin Valine Proline Glutamic acid Arginine Lysine Leucine Hydroxyproline Tyrosine -Phenylalanine R f values 0.24 0.20 0.40 0.39 0.25 0.13 0.12 0.58 0.21 0.38 0.50

Table 4.Relationship between zone area and concentration of amino acid

Concentration /mol liter Alanine 10 4 10 3 10 2 10 1 Leucine 10 4 10 3 10 2 10 1 Proline 10 4 10 3 10 2 10 1 Valine 10 4 10 3 10 2 10 1 Glycine 10 4 10 3 10 2 10 1 Arginine 10 4 10 3 10 2 10 1 Lysine 10 4 10 3 10 2 10 1 Zone area (squares) 6.0 12.0 18.0 24.0 8.0 16.0 23.0 32.0 8.0 18.0 26.0 32.0 5.25 8.75 12.0 15.0 3.0 5.0 6.0 9.0 9.0 16.0 24.0 32.0 7.0 16.0 24.0 34.0 log concentration 4.0 3.0 2.0 1.0 4.0 3.0 2.0 1.0 4.0 3.0 2.0 1.0 4.0 3.0 2.0 1.0 4.0 3.0 2.0 1.0 4.0 3.0 2.0 1.0 4.0 3.0 2.0 1.0

Mobile phase: n-Butanol: Acetic acid: water (60:15:35) (Solvent mixture 2) Development time: 3 hours Temperature: 25 C Table 2. Rf values of amino acids
Amino acids Alanine Glycerin Valine Proline Glutamic acid Asparagine Arginine Lysine Leucine Hydroxyproline Tyrosine -Phenylalanine R f values 0.62 0.40 0.80 0.89 0.33 0.39 0.64 0.58 0.82 0.67 0.62 0.86

Mobile phase: Phenol: water (60:15:35) (Solvent mixture 1) Development time: 3 hours Temperature: 25 C

Table 3.Separation and identification of amino acids in mixture

Sample Mixture No. 1 Mixture No. 2 Mixture No. 3 Components Glycine + Alanine -Phenylalanine + Proline + Serine Valine+Leucine+ Hydroxyproline+Arginine Lysine + Alanine Tyrosine + Glycine + -Phenylalanine Alanine + Serine + Valine Rf values 0.20 0.24 0.50 0.39 0.19 0.58 0.40 0.21 0.13 0.12 0.24 0.50 0.20 0.38 0.19 0.24 0.40 Components identified on comparison with standards Glycine Alanine -Phenylalanine Proline Serine Leucine Valine Hydroxyproline Arginine Lysine Alanine -Phenylalanine Glycine Tyrosine Serine Alanine Valine

Further work is related to separation and identification of amino acids in a mixture (Table 3). Mixture no. 1 to 6 belonged to various combination of amino acids in mixture. n-butanol-acetic acid-water (60:15:25) solvent system was used which was found to give excellent results in terms of amino acids resolution. The amino acids were identified on comparison with the pure standard amino acid Rf values. The separation and identification of amino acids using ninhydrin as the locating reagent helped in the quantification of the amino acids as well. Table 4 presented the result showing the relationship between zone area (squares) of the separated spots and the concentration of amino acids. A linear relationship between the logarithm of the weight of the substance and the square root of the spot is predicted[43]. Compounds in tomato, apple, orange, lemon and pomegranate juices are well resolved, however, their identification needed further work. The effect of concentration of the solute on the Rf values is negligible (Table not presented). It revealed that the Rf values may remain unaffected by the change in the solute concentration, especially in the case of solvent, n-butanol-acetic acidwater, used. CONCLUSION Paper chromatography has successfully been employed for the separation of amino acids in the mixture. There is a linear relationship between zone area of the spot and the concentration of the amino acids. REFERENCES
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Mobile phase: n-Butanol: Acetic acid: water (60:15:35) solvent system for developing the chromatogram consisted of n-butanol-acetic acid-water (60:15:25). This solvent system was selected for all qualitative separation of amino acids either from a known mixture or real sample. The Rf values in Table 1 provide a better reflection of a successful resolution of amino acids, if present in a mixture. This table, however gives the Rf values of amino acids when singly present. Table 2 indicated similar result with the other solvent system which consisted of phenol: water (4:1). This solvent system was not used successfully for resolution of amino acids in a mixture. It gave a poor resolution.

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Source of support: Nil, Conflict of interest: None Declared

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