Paper chromatography, of amino acids

E. Heimer,/. Chem. Educ. 49, 547 (1972). Paper chromatography of amino acids. 

P Grossman andj Colburn, Editors, Capillary Electrophoresis Theory and Practice (1992), Academic Press (San Diego). An up-to-date and complete review of CE E Heimer,/. Chem. Educ. 49, 547 (1972) Paper chromatography of amino acids F Lai and T Sheehan, BioTechniques 14, 642-649 (1993) A comparison of reagents for amino acid derivatization... 

Paper chromatography of amino acids, pp. 181-200 in Colloid Chemistry Theoretical and Applied, Vol. 8, J. Alexander, ed.,736 pp.,Reinhold, New York, 1950. With H. A. Sober. 

W14. Wiggins, L. F., and Williams, J. H., Use of i-butanol-formio acid-water mixture in the paper chromatography of amino-acids and sugars. Nature (London) 170, 279-280 (1952). 

Paper chromatography of amino acids is best described as partition chromatography between the stationary aqueous (most polar) phase in the cellulose fibers and the mobile (least polar) phase formed by the solvent system used. The actual situation is somewhat more complicated. The stationary phase cannot be described as pure water but rather as a concentrated aqueous carbohydrate solution. Elements of adsorption chromatography are involved as shown by the relatively small/ f values for aromatic amino acids and by the possibility of separating enantiomers (mirror images) of amino acids depending on the chirality of the cellulose in the paper. 

Figure 10.17 Two-dimensional high voltage electrophoresis and chromatography of amino acids. Paper high voltage electrophoresis (4000 V) in an acetic acid-formic acid buffer at pH 2.0 in the first dimension followed by descending chromatography in the second dimension in an n-butanol-acetic acid-water solvent (12 3 5). The spots were visualized with a ninhydrin-collidine reagent.

T Hayashi, H Tsuchiya, H Naruse. Reversed-phase ion-pair chromatography of amino acids. Application to the determination of amino acids in plasma samples and dried blood on filter papers. J Chromatogr Biomed Appl 274 318-324, 1983. 

Day 2 Thin Layer Chromatography of the DNP-Amino Acid and Paper Chromatography of the Acid-Hydrolyzed Dipeptide... 

W9. Warren, S., Gilligan, D. R., and Moor, J. K., Paper partition chromatography of amino acids of gastric juice of patients with gastric lesion. /. Natl. Cancer Inst. 12, 677-689 (1952). 

Smith, I., Eider, L. J., and Lemer, R. P., Chromatography of amino acids, indoles and imidazoles on thin layers of avicel and cellulose and on paper. J. Chromatogr. 26, 449-455 (1967). 

T4. Tocci, P. M., The biochemical diagnosis of metabolic disorders by urinalysis and paper chromatography. In Amino Acid Metabolism and Genetic Variation (W. L. Nyhan, ed.), pp. 461-490. McGraw-Hill, New York, 1967. 

Other workers separate by paper chromatography the amino acids obtained by catalytic hydrogenolysis of the 2,4-dinitrophenylhydrazones of the a-keto acids (H19, K19, T24). 

Levy, A. L., and Chung, D. (1953). Two-dimensional chromatography of amino acids on buffered papers. Anal. Chem. 25 396-399. 

Paper chromatography separates amino acids on the basis of polarity. A few drops of a solution of an amino acid mixture are applied to the bottom of a strip of filter paper. The edge of the paper is then placed in a solvent. The solvent moves up the paper by capillary... 

The first of the two experiments given below illustrates the separation of amino-acids, now an almost classic example of the use of paper chromatography the second illustrates the separation of anthranilic acid and iV-methylanthranilic acid. Both experiments show the micro scale of the separation, and also the fact that a mixture of compounds which are chemically closely similar can be readily separated, and also can be identified by the use of controls. 

Amino acids have high melting or decomposition points and are best examined for purity by paper or thin layer chromatography. The spots are developed with ninhydrin. Customary methods for the purification of small quantities of amino acids obtained from natural sources (i.e. l-5g) are ion-exchange chromatography (see Chapter 1). For general treatment of amino acids see Greenstein and Winitz [The Amino Acids, Vols 1-3, J.Wiley Sons, New York 1961] and individual amino acids in Chapters 4 and 6. 

The column chromatography technique using Dowex 50 ion-exchange resin, introduced in 1951 (M2) and improved in 1954 (M3) by Moore and Stein, first made possible the precise quantitative analysis of amino acids liberated in the course of acid hydrolysis of urine. Similar results were also obtained by Muting in 1954 (M4), who used paper chromatography methods. In this procedure amino acids were quantitatively determined after staining on the paper and elution of the resulting spots. 

High-voltage electrophoresis and subsequent paper chromatography of the fractions obtained made possible the isolation from the analyzed mixture of twenty-two components giving colored spots with ninhydrin and isatin. Among these, fourteen were identified as peptides and their amino acid composition established (Table 5). In the case of eight peptides, also N- and C-terminal amino acids were determined (Table 6). 

TLC has similar applications to paper chromatography. The stationary phase is a coating, such as silica gel, on a glass or plastic plate. Depending on the TLC plate used, components may be separated based on differences in molecular weight, charge, or polarity (see Chapter 11). TLC with a 70% isopropyl alcohol mobile phase and a silica gel plate is an effective substitute for paper chromatography separation of amino acids. Nucleotides may be separated on a special silica gel plate and a 20% ethanol (in water) mobile phase. 

Experiment 61 Identification of Amino Acids in Food by Paper Chromatography... 

Cellulose is itself polar in nature and can cause some adsorption, which may result in the tailing of zones. However, this adsorptive effect may contribute to the separation process in some instances and the use of a polar mobile phase can enhance this effect further, e.g. the separation of amino acids using an aqueous solution of ammonia as the mobile phase. The combination of partition and adsorption generally influences separations on cellulose thin-layer plates, which have superseded paper chromatography in most instances and offer increased speed and resolution. 

Reagents used for the visualisation of amino acids on the dried chromatogram may be applied either by spraying or dipping. Those commonly used produce intensely coloured bands with approximately 20 nmol of each amino acid for paper chromatography and 5 nmol for thin-layer separations, although smaller amounts can be detected. 

Demonstrating the existence of multiple transaminases in the late 1930s and early 1940s was very difficult. Chromatographic separation of amino acids was not available until the mid-1940s and paper chromatography was not in common use until 1948-1950 when Hird and Rowsell finally showed the existence of the wide range of transaminases and the universality of the transamination process. 

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