Rapid Quantitative Determination of Amino Acids by High Temperature Paper Chromatography 1. B. HIMES and 1. D. METCALFE Armour & Co., Chicago, 9, 111.
b
Horizontal paper chromatography is used for quantitative determination of the amino acid composition of a mixture of amino acids or a protein hydrolyzate in a small sealed horizontal development tank. A 2-hour resolution of amino acid hydrochlorides is obtained because of the rapid movement of the solvent at higher temperatures. A solvent system of methyl ethyl ketone, propionic acid, and water (1 5 5 6 ) is used to develop the paper chromatogram at 60" C. The amino acid spots are developed with ninhydrin and absorbance i s read with a densitometer.
A
acid mixtures are generally analyeed quantitatively by ion exchange chromatography or paper chromatography. The ion exchange chromatographic method, developed by Stein and Moore (17, I8), has given accurate and consistent results in this laboratory, but is too slow for the rapid, routine, quantitative analysis of large numbers of amino acid mixtures or protein hydrolyzates. Paper chromatography appeared to be the most promising approach to rapid, routine amino acid analysis. However, investigation of a number of paper chromatographic methods (2, 3, 6, 9, 15, 19) indicated that these methods suffer from the same disadvantage. A number of authors (1, 4-8, 10, I S ) observed that the rate of development of paper chromatograms increased with increasing temperatures. This observation was confirmed in this laboratory. Roberts and coworkers (10, 1 1 , 18, 14) used a technique of horizontal solvent development a t an elevated temperature for paper chromatography of sugars and amino acids. They reported a separation of six amino acids in a mixture of 20 amino acids using a buffered phenol system on buffered paper. I n the present investigation, a modification of the Roberts and Kolor method was used. However, as buffered phenol proved incapable of resolving a number of amino acids, the more generally applicable solvent of Clayton and Strong (5) [a mixture of methyl ethyl ketone (2-butanone), propionic acid, and water] was used. At MINO
1192
ANALYnCAL CHEMISTRY
elevated temperatures a development chamber of small volume was needed to maintain equilibrium conditions, because the constant relationship of liquid and vapor compositions could not be maintained in a large, classicaltype development chamber. The chamber described by Roberts and coworkers (10) was modified in size and detail to meet requirements. Horizontal paper chromatography employing the Clayton and Strong solvent system and the modification of the Roberts development chamber was successfully used to separate 18 amino acids in 2 hours a t 60" C., the solvent front moving about 20 cm. The paper was air-dried in 40 minutes, then dipped in an acetone solution of ninhydrin for color development of the chromatographed amino acids. The intensities of the colored spots were read with a densitometer for quantitative results.
APPARATUS AND PROCEDURE
Chromatographic Chamber. A stainless steel pan, 9 X 13 X 2 inches, with a l/A-inch flange is used as a tank for developing the chromatograms. A removable stainless steel rack (l13/& X 8l/2 X 11/2 inches) is set inside to support a glass trough (11/2 X 8 inches) and six glass rods, parallel to the solvent trough, spaced 11/* inches apart. The pan is covered with plate glass (10 X 14 X 1/4 inches) which has two 3/~-inchholes, each 11/2 inches from an end. Tygon sheeting (1/1* inch thick, 1 inch wide) is glued on the glass cover for a seal between pan flange and glass. (This chamber may be obtained from Labline, Inc., Chicago, Ill.) Filter Paper. Whatman No. 1 chromatographic filter paper sheets (18 X 221/2 inches) are cut into 8 x 111/, inch sheets. Sample Preparation. Approximately 10 mg. of a n amino acid mixture or protein hydrolyzate is dissolved in 0.1 ml. of 1N hydrochloric acid and the solution is diluted to 1.0 ml. with water containing 10% isopropyl alcohol. A solution containing a known mixture of amin? acids, resembling the unknown, IS made in the same manner t o chromatograph on the same paper. The
amino acid solutions are kept frozen until ready for use. Procedure. Using a n ultramicropipet, 1.0- and 2.0-J. aliquots of both the known and the unknown solutions are placed on the paper so that the spots are inch apart, 11/2 inches in from one of the 8-inch edges. As many as three known and three unknown solutions may be run per sheet. The first and last sample spots should be "4 inch from the edges of the paper. A ll/E-inch tab is folded at a right angle to the paper, inch below the line of sample spots. The opposite end of the paper is folded to form a l/T inch tab. The paper is placed in the chromatographic pan, which has been in a 60" C. air oven for 1 hour, and is positioned so that it lies over the glass rods with the l1/8-inch tab extending into the glass trough a t one end of the pan. The plate glass is placed over the pan. By means of a funnel, 30 ml. of solvent (75 parts of methyl ethyl ketone, 25 parts of propionic acid, 30 parts of water) are delivered to the glass trough through one of the holes in the glass cover. Another 25 ml. of solvent is placed in the bottom of the pan through the other hole in the cover. The holes are closed with cork stoppers. The chromatographic chamber is maintained a t 60" C. during the 2-hour development. The paper is removed from the pan a t the end of 2 hours, hung in an exhaust hood a t room temperature for 20 minutes, and then placed in the 60" C. oven for 20 minutes. The dried chromatogram is dipped in an acetone solution containing 0.2% ninhydrin and 1% acetic acid (16, 19). Again, it is hung in the exhaust hood for 20 minutes and then placed in the 60' C. oven for 20 minutes. Estimation of Amino Acids. The intensities of the colored amino acid spots are read on a densitometer with a slit width of 1 mm. (A Photovolt densitometer Model 525 was used in this work.) Measuring the maximum color density of the individual amino acid spots (9,12) was found satisfactory. When this technique is used, the absorbance of the unknown must fall within the absorbance range of the known solution. Therefore, one of the two dilutions of the unknown is compared to the one dilution of the known which it most closely resembles. The percentage of amino acids in each unknown solution is calculated from the total weight of the sample in the original spot of the paper, the weight
the samples in question. The absorbance us. the distances traveled on the chromatogram by the amino acids is illustrated in Figure 1. Estimation of the individual amino acids in the mixture agrees reasonably well with the actual composition. Cystine, methionine, valine, isoleucine, phenylalanine,
0 7
0.6
0 s
0.4
Table I. Rf Values of Individual Amino Acids on Whatman No. 1 Filter Paper a t 60" C.
0.3
0 . 2
(Solvent. Methyl ethyl ketone, propionic acid, and water) Amino Rf Amino Rf Acid Valuea Acid Valuea Cystine 0.21 Alanine 0.54 Lysine 0 . 3 1 Proline 0.58 Histidine 0 . 3 4 Tyrosine 0.66 Arginine 0.38 Valine 0.71 Serine 0.42 hlethionine 0.73 Aspartic acid 0.43 Tryptophan 0.78 Glycine 0 . 4 6 Isoleucine 0.81 Threonine 0.49 Phenylalanine 0.82 Glutamic acid 0.51 Leucine 0.85 a Average of three calculations
" 1
10
0
2 0
3 0
k 0
50
6 0
7.0
9 0
E O
\\ 100
DISTASCE [cm)
Figure 1.
Absorbance of amino acids vs. distances traveled on chromatogram
of each individual amino acid in the corresponding known mixture applied to the paper, and the ratios of the color densities. Thus,
% amino acid where TI',
=
R,, = R, TI',
=
=
=
TI', x uR x 100
R,
in the mixture solution were calculated from each paper (Table 11). In all tables, the figures for this method are averages of calculations of two or more paper chromatograms of
TY,
neight of each amino acid on the paper in standard mixture absorbance reading of amino acid spot of unknown sample absorbance reading of amino acid spot of standard mixture total weight of sample in solution on the paper
If duplicate samples are analyzed, they are chromatographed on different papers and the calculations averaged. RESULTS AND DISCUSSION
To achieve good resolution, the amino acids must be in the form of their hydrochlorides. The separation of the free acids is very poor. To ensure complete conversion of the amino acids to the hydrochlorides, the samples are dissolved in 1S hydrochloric acid. A thorough investigation of the solvent has indicated that varying the ratio of its components or varying the time of solvent development a t 60" C. does not improve observed R, values. The R/ values of chromatographed individual amino acids (Table I) were found to be consistent and reproducible under a given set of operating conditions. A solution of a synthetic mixture of amino acids was prepared as described, and was chromatographed on four different papers, using applications of both 1.0 and 2.0 pl. of the sample on each paper. Identical volumes of a suitable known standard n-ere also applied. The percentages of the amino acids
Table 11.
Amino Acid Cystine
Found,a
%
4.0 6.8 3.9 2.5 3.9 12.9 11.7 3 5 9 5 5.9 7.6 2.2 4.7 2.8 3.9 3.7 4.0
2Eine Arginine Serine AsDartic acid Glicine Threonine Glutamic acid Alanine Proline Tyrosine Valine Methionine Isoleucine Phenylalanine Leucine Average of four calculations.
Analysis of Known Mixture
Av. Dev.
Actual, %
1 0 .1 k0.3 10.3 f0.3 1 0 .1 10.8 kO.9 hO.1 f0.7 10.7 3~0.6 10.1 f0.3 k0.3 k0.3 10.3 f0.4
Diff., Found ilctual
3.7 6.7 3.8 2.4 4.0 13.2 12.4 3.5 9.6 5.8 7.2 2.2 5.1 3.1 4.2 4.0 4.4
%
Error
0.3 0.1 0.1 0.1 -0.1 -0.3 -0.7 0.0 -0.1
8.1 1.5 2.6 4.2 -2.5 -2.3 -5.6 0.0 -1.0 1.7 5,6 0.0 -7.8 -9.7 -7.2 -7.5 -9.1
0.1
0.4 0.0 -0.4 -0.3 -0.3 -0.3 -0.4
0
Table 111.
Analysis of Feather Meal and Blood Feather Meal Blood This methodJa Moore-Stein, This method," Moore-Stein, CI % % 70 /O
Amino Acid Cystine 8.7 4.0 Lysine Histidine 1.5 Arginine 7.2 6.8 Serine Aspartic acid 8.0 Glycine ... Threonine 2.7 Glutamic acid 7.6 Alanine ... Proline 3.9 Tyrosine 1.8 Valine 7.4 Methionine 0.9 Isoleucine 5,1 Phenylalanine 4.4 Leucine 6 6 a Average of two calculations.
8.9 4.7 1.3 6.4 6.0 7.9 3.3 7.3
6.5 10.1 7.6 6.4 4.5 8.1 3.6 3.6 4.2
3.7 2.3 6.8 0.9 6.0 6.2 7.0
4.6 1.6 8.6 1.9 2.4 2.7 10.8
... ...
11 .o
6.0 10.7 7.4 6.6 5.3 7.9 3.1 4.1 4.1 11.1 5.7 1.4 8.2 1.8 3.3 2.1 11.0
VOL. 31, NO. 7,JULY 1959
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and leucine give consistently the greatest errors, but all 17 constituents were within =klO.O% of the actual amino acid composition of the mixture. This compares favorably with other quantitative paper chromatographic methods. Cattle blood and chicken feathers were hydrolyzed with 6N hydrochloric acid in a sealed test tube a t 110' C. for 16 hours. The hydrolysates were analyzed for amino acids by this method and the column chromatographic method of Moore and Stein (Table 111). The methods agree fairly well. I n general, the procedure described provides a very rapid, less tedious, reasonably accurate method of quantitative analysis of protein hpdrolyzates.
LITERATURE CITED
(2)
Baker, B. E., I
