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Thin-layer chromatography of amino acids
MICROCHEMICAL
16, 391-394 (1971)
JOURNAL
Thin-Layer
Chromatography
ALAN Department
of Amino
Acids
F. KRIVIS AND C. C. ONG
of Chemistry,
University
Received
February
of Akron, Akron, Ohio 44304 16, 1971
As part of a program to characterize adhesives which are secreted by certain marine organisms, it became necessary to separate and identify a number of amino acids. Amino acid separations have been performed by ion-exchange chromatography (5), paper chromatography (3), and thin-layer chromatography (TLC) (I, 2). Although automated procedures and equipment for amino acid analyses (e.g., Spackman et al. (6) ) have become paramount in those laboratories performing protein or peptide research, most general analytical laboratories must employ simpler, less expensive, and less restricted equipment for their less frequent amino acid separations. On the basis of convenience, speed, and ease of operation, TLC is more attractive for this purpose than some other forms of chromatograPhY. The TLC procedures which have been proposed in the past suffer from several faults with regard to the characterization of the adhesives mentioned above. For example, a single plate rarely will show complete resolution of all of the amino acids of interest; even two-dimensional development may not produce a satisfactory separation (2), and several plates may be necessary. Furthermore, the highly mobile amino acids, e.g., valine, tyrosine, and phenylalanine are not well resolved. Because of these problems, we became interested in developing a TLC procedure which would separate all of the pertinent amino acids equally well but, at the least, permit satisfactory resolution of the mobile acids. EXPERIMENTAL
METHODS
ChemicaZs.All inorganic chemicals and organic solvents were reagent grade and were used without further purification. The amino acid standards were obtained from Bio Rad Laboratories and were used as 2.5 mM solutions. Cellulose MN 300 was obtained from Macherey Nagel and Co. A slurry of the cellulose was prepared by mixing 140 ml of water, 20 ml 391
392
KRIVIS
AND
ONG
of ethanol, and 30 g of cellulose MN 300 and homogenizing in a blender for several minutes. This slurry then was used to coat the plates. Developing solvents were prepared from isopropanol:methylethylketone: 1M hydrochloric acid in v/v ratios of 60: 15 :25 (Solvent I) and from methanol: water :pyridine in ratios of 20: 5 : 1 (Solvent II). The visualization reagent was based on a Ninhydrin-copper mixture (4). A solution of 50 ml of 0.2% Ninhydrin in absolute ethanol, 10 ml of acetic acid, and 2 ml of 2,4,6-collidine was prepared. A second solution containing 1% copper nitrate trihydrate in absolute ethanol was prepared. Just before use, 50 parts of the Ninhydrin solution were added to 3 parts of the copper solution. Procedure. Glass plates (20 X 20 cm) were coated with a cellulose layer 500 p thick and then air-dried at room temperature. Sampleswere spotted on the plates at a point 1.5 cm equidistant from two adjoining edgesand air dried for a convenient period of time (ca. 0.5 hour). Ascending development o’f the spotted and dried plates were carried out in Solvent I. When the solvent front had moved about 15 cm, the plates were removed from the tank and air-dried for 15 minutes, followed by another 15 minutes in an oven at 60°C. The latter treatment was needed to remove residual hydrochloric acid. The yellow strip across the plate at the solvent front was removed and development in the second dimension was carried out in Solvent II. When the solvent had moved about 16 cm, the plates were removed from the tank, airdried for 15 minutes, and then at 100°C for 15 minutes. After cooling, the plates were sprayed with the Ninhydrin-copper reagent mixture. Heating at 110°C for 15 minutes visualized the chromatographic spots. Many of the amino acids produced unique individual colors with this reagent. RESULTS
AND
DISCUSSION
The chromatographic separation of amino acids on either filter paper or a cellulose TLC support is influenced greatly by several factors. Amino acids are soluble in water and insoluble (or only slightly soluble) in nonaqueous solvents (including polar solvents). Amino acids also are amphoteric compounds so that the pH of the solution medium will control the form existing in that particular medium. These properties are at the base of most of the procedures used for TLC separations of the amino acids. A cellulose TLC support contains a stationary water phase which permits partitioning of the amino acids between it and the developing solvent. The latter, therefore, may be tailored for specific purposes by adjustment of either or both of the polarity-solubility or the pH aspects.
‘TLC
OF
AMINO
TABLE
I
R, VALUES FOR 16 AMINO
Compound
393
ACIDS
Solvent:
Arginine Lysine Cystine Histidine Glycine Serine Aspartic acid Threonine Glutamic‘acid Alanine 2; Tyrosine Methionine Phenylalanine Valine ! # Leucine Isoleucine
ACIDS
I
II
0.2 0.15 0.08 0.09 0.33 0.35 0.43 0.40 0.53 0.52 0.66 0.78 0.75 0.76 0.78 0.88
0.14 0.15 0.17 0.21 0.37 0.43 0.44 0.56 0.53 0.59 0.59 0.65 0.73 0.80 0.81 0.83
In the present case, the amino acids were developed first in an acidic solvent containing an alcohol and a ketone. This was followed by development in the second dimension with an organic base in an alcoholwater medium. Both solvent systems were intentionally chosen to keep the number of components down to a minimum and to avoid the need for stringent preparatory conditions. To our knowledge, the combination of these very simple solvent systems (I and II) has not been reported previously for the separation of amino acids on a single thinlayer chromatogram. Table 1 lists the RI values in both dimensions for 16 amino acids. The mobile amino acids of interest were well separated. Although arginine, lysine, cystine, and histidine are not physically as well separated as desired, their unique color reactions with the spray reagent permitted easy location and detection of each. SUMMARY A two-dimensional thin layer chromatographic procedure for separation of amino acids has been developed. The method is based on simple solvent systems and is especially effective for the separation of the highly mobile amino acids. ACKNOWLEDGMENT The authors thank the National Institute of Dental Research for support of this work through Contract No. PH-43-67-1172.
394
KRIVIS
AND
ONG
REFERENCES f. Haworth, C., and Heathcote, J. G., An improved technique for the analysis of amino acids and related compounds on thin layers of cellulose. J. Chromatogr. 41, 380-385 (1969). 2. Jones, K., and Heathcote, J. C., The rapid resolution of naturally occurring amino acids by thin-layer chromatography. J. Chromatogr. 24, 106-I 11 (1966).
3. Levy, A. L., and Chung, D., Two-dimensional chromatography of amino acids on buffered papers. Anal. Chem. 25, 396-399 (1953). 4. Moffat, E. D., and Lytle, R. I., Polychromatic technique for the identification of amino acids on paper chromatograms. Anal. Chem. 31, 926-928 (1959).
5. Moore, S., and Stein, W. H., Chromatographic determination of amino acids by use of automatic recording equipment. Methods Emymol. 6, 819 (1965). 6. Spackman, D. H., Stein, W. H., and Moore, S., Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30, 1190-1206 (1958).
16, 391-394 (1971)
JOURNAL
Thin-Layer
Chromatography
ALAN Department
of Amino
Acids
F. KRIVIS AND C. C. ONG
of Chemistry,
University
Received
February
of Akron, Akron, Ohio 44304 16, 1971
As part of a program to characterize adhesives which are secreted by certain marine organisms, it became necessary to separate and identify a number of amino acids. Amino acid separations have been performed by ion-exchange chromatography (5), paper chromatography (3), and thin-layer chromatography (TLC) (I, 2). Although automated procedures and equipment for amino acid analyses (e.g., Spackman et al. (6) ) have become paramount in those laboratories performing protein or peptide research, most general analytical laboratories must employ simpler, less expensive, and less restricted equipment for their less frequent amino acid separations. On the basis of convenience, speed, and ease of operation, TLC is more attractive for this purpose than some other forms of chromatograPhY. The TLC procedures which have been proposed in the past suffer from several faults with regard to the characterization of the adhesives mentioned above. For example, a single plate rarely will show complete resolution of all of the amino acids of interest; even two-dimensional development may not produce a satisfactory separation (2), and several plates may be necessary. Furthermore, the highly mobile amino acids, e.g., valine, tyrosine, and phenylalanine are not well resolved. Because of these problems, we became interested in developing a TLC procedure which would separate all of the pertinent amino acids equally well but, at the least, permit satisfactory resolution of the mobile acids. EXPERIMENTAL
METHODS
ChemicaZs.All inorganic chemicals and organic solvents were reagent grade and were used without further purification. The amino acid standards were obtained from Bio Rad Laboratories and were used as 2.5 mM solutions. Cellulose MN 300 was obtained from Macherey Nagel and Co. A slurry of the cellulose was prepared by mixing 140 ml of water, 20 ml 391
392
KRIVIS
AND
ONG
of ethanol, and 30 g of cellulose MN 300 and homogenizing in a blender for several minutes. This slurry then was used to coat the plates. Developing solvents were prepared from isopropanol:methylethylketone: 1M hydrochloric acid in v/v ratios of 60: 15 :25 (Solvent I) and from methanol: water :pyridine in ratios of 20: 5 : 1 (Solvent II). The visualization reagent was based on a Ninhydrin-copper mixture (4). A solution of 50 ml of 0.2% Ninhydrin in absolute ethanol, 10 ml of acetic acid, and 2 ml of 2,4,6-collidine was prepared. A second solution containing 1% copper nitrate trihydrate in absolute ethanol was prepared. Just before use, 50 parts of the Ninhydrin solution were added to 3 parts of the copper solution. Procedure. Glass plates (20 X 20 cm) were coated with a cellulose layer 500 p thick and then air-dried at room temperature. Sampleswere spotted on the plates at a point 1.5 cm equidistant from two adjoining edgesand air dried for a convenient period of time (ca. 0.5 hour). Ascending development o’f the spotted and dried plates were carried out in Solvent I. When the solvent front had moved about 15 cm, the plates were removed from the tank and air-dried for 15 minutes, followed by another 15 minutes in an oven at 60°C. The latter treatment was needed to remove residual hydrochloric acid. The yellow strip across the plate at the solvent front was removed and development in the second dimension was carried out in Solvent II. When the solvent had moved about 16 cm, the plates were removed from the tank, airdried for 15 minutes, and then at 100°C for 15 minutes. After cooling, the plates were sprayed with the Ninhydrin-copper reagent mixture. Heating at 110°C for 15 minutes visualized the chromatographic spots. Many of the amino acids produced unique individual colors with this reagent. RESULTS
AND
DISCUSSION
The chromatographic separation of amino acids on either filter paper or a cellulose TLC support is influenced greatly by several factors. Amino acids are soluble in water and insoluble (or only slightly soluble) in nonaqueous solvents (including polar solvents). Amino acids also are amphoteric compounds so that the pH of the solution medium will control the form existing in that particular medium. These properties are at the base of most of the procedures used for TLC separations of the amino acids. A cellulose TLC support contains a stationary water phase which permits partitioning of the amino acids between it and the developing solvent. The latter, therefore, may be tailored for specific purposes by adjustment of either or both of the polarity-solubility or the pH aspects.
‘TLC
OF
AMINO
TABLE
I
R, VALUES FOR 16 AMINO
Compound
393
ACIDS
Solvent:
Arginine Lysine Cystine Histidine Glycine Serine Aspartic acid Threonine Glutamic‘acid Alanine 2; Tyrosine Methionine Phenylalanine Valine ! # Leucine Isoleucine
ACIDS
I
II
0.2 0.15 0.08 0.09 0.33 0.35 0.43 0.40 0.53 0.52 0.66 0.78 0.75 0.76 0.78 0.88
0.14 0.15 0.17 0.21 0.37 0.43 0.44 0.56 0.53 0.59 0.59 0.65 0.73 0.80 0.81 0.83
In the present case, the amino acids were developed first in an acidic solvent containing an alcohol and a ketone. This was followed by development in the second dimension with an organic base in an alcoholwater medium. Both solvent systems were intentionally chosen to keep the number of components down to a minimum and to avoid the need for stringent preparatory conditions. To our knowledge, the combination of these very simple solvent systems (I and II) has not been reported previously for the separation of amino acids on a single thinlayer chromatogram. Table 1 lists the RI values in both dimensions for 16 amino acids. The mobile amino acids of interest were well separated. Although arginine, lysine, cystine, and histidine are not physically as well separated as desired, their unique color reactions with the spray reagent permitted easy location and detection of each. SUMMARY A two-dimensional thin layer chromatographic procedure for separation of amino acids has been developed. The method is based on simple solvent systems and is especially effective for the separation of the highly mobile amino acids. ACKNOWLEDGMENT The authors thank the National Institute of Dental Research for support of this work through Contract No. PH-43-67-1172.
394
KRIVIS
AND
ONG
REFERENCES f. Haworth, C., and Heathcote, J. G., An improved technique for the analysis of amino acids and related compounds on thin layers of cellulose. J. Chromatogr. 41, 380-385 (1969). 2. Jones, K., and Heathcote, J. C., The rapid resolution of naturally occurring amino acids by thin-layer chromatography. J. Chromatogr. 24, 106-I 11 (1966).
3. Levy, A. L., and Chung, D., Two-dimensional chromatography of amino acids on buffered papers. Anal. Chem. 25, 396-399 (1953). 4. Moffat, E. D., and Lytle, R. I., Polychromatic technique for the identification of amino acids on paper chromatograms. Anal. Chem. 31, 926-928 (1959).
5. Moore, S., and Stein, W. H., Chromatographic determination of amino acids by use of automatic recording equipment. Methods Emymol. 6, 819 (1965). 6. Spackman, D. H., Stein, W. H., and Moore, S., Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30, 1190-1206 (1958).