1964
ANALYTICAL CHEMISTRY ACKNOWLEDGMENT
The authors are indebted to D. J. Pompeo for suggestions and encouragement in the use of ionization chamber detectors, to S. T. Abrams, 1%'. B. Conner, Jr., and S. AT. Brassington for their part in the design and construction of the cells, and to J. F. iJ7aller and H. U. Cole for their part in applying the cells in practical gas partition chromatography, LITERATURE CITED
(1) Deisler, P. F., Jr., hlcHenny, K. W., Jr., Wilhelm, R. H., ANAL. CHEM.27, 1366 (1955).
(2) James, A . T., Martin, A. J. P., Bwchem. J . 50, 679 (1951). i3j James, A.T., Martin, 8 . J. P., Brit.Med. Bul., 10, 170 (1954). (4) Jesse, W.P., Sadauskis, J., Phys. Rev. 100, 1755 (1955). (5) Loeb, L. B.,"Kinetic Theory of Gases," p. 555, RlcGraw-Hill, New York, 1934. (6) Otvos, J. W., Stevenson, D. P., J . Am. Chsm. SOC.78,546 (1956). O" Phillips, C.S. G., Discusdons FaTaday SOC.7, 241 (1949). (8) Pompeo, D.J., Otvos, J. W., U.S. Patent 2,641,710(1953). (9) Scott, R. P.W., h'ature 176,793 (1955). (10) Smith, V. N.,Otvos, J. R., ANAL.CHEM.26, 359 (1954).
RECEI V E D for review April 26, 1956. dccepted June 15, 1956. Division of Analytical Chemistry. Symposium on Vapor Phase Chromatography, 129th Meeting, ACS, Dallas, Tex., -4pril 19.50.
Quantitative Determination of Phenylalanine on Paper Chromatograms ARTHUR E. PASIEKA and JOSEPH F. MORGAN Laboratory of Hygiene, Department of National Health and Welfare, Ottawa, Canada
A quantitative method for the determination of phenylalanine in the presence of varying proportions of 19 other amino acids utilizes the characteristic blue color formed by phenylalanine when ninhydrin-developed paper chromatograms are treated with dilute sodium bicarbonate. The ninhydrin colors of the other amino acids can be removed by water washing without eluting the specific phenylalanine color, which is then extracted with 1-butanol and measured in a spectrophotometer at 600 m p . The intensity of the clearly defined phenylalanine spot can also be read directly on the chromatograms with a densitometer. The application of the method to complex biological media is reported.
D
U R I S G studies on the amino acid metabolism of animal tissues cultivated in vitro in synthetic media (8),paper chromatography indicated that an uptake of phenylalanine occurred during the cultivation period. I n these studies, the synthetic medium employed was 31 150 (4, 6), which contains 60 ingredients including 20 amino acids dissolved in a modified Tyrode's solution ( 2 ) . The DL-phenylalanine content of this medium is 50 mg. per liter, representing 4.596 of the total amino acids present. The disappearance of phenylalanine from the medium suggested its importance for tissue culture nutrition and metabolism. Under certain conditions, phenylalanine forms a characteristic gray color when developed by the conventional ninhydrin procedure, and by this method microgram quantities can be detected (1, 3, 9). Under the conditions of neutral and acidic solvent systems used in the present study, however, the phenylalanine-ninhydrin color was found to be nonspecific and indistinguishable from that of various other amino acids. For this reason, an effort was made to develop a selective method for phenylalanine that could be applied in the presence of high concentrations of other amino acids following development by neutral and acidic solvents. Previous studies from this laboratory ( 7 ) have shown that a characteristic blue color, specific for phenylalanine, is produced when the ninhydrin-developed chromatograms are subsequently treated with dilute sodium bicarbonate. Development of this specific color is associated with a shift in the absorption maximum from 560 to 600 mp as well as increased color stability. The results reported in the present communication show that this characteristic blue color can be used as the basis for a quantitative method that permits the specific determination of phenylalanine on paper chromatograms in the presence of 19 other amino acids, even under conditions of poor separation and resolution.
/I
0.25 w
0.20
-
I
0
Figure 1.
IO 20 30 40 50 PHENYLALANINE CONCENTRATION, 7
Standard curve for specific phenylalanine determination
0 Graded concentrations of DL-phenylalanine dissolved in A1 150 with variable ratio of phenylalanine t o other amino acids present X Graded concentrations of DL-phenylalanine with constant ratio of phenylalanine to other amino acids PROCEDURE
Chromatography. Samples for analj-sis (5.0 ml.) are concentrated to dryness in vacuo over concentrated sulfuric acid and reconstituted in 0.2 ml. of deionized water. With the biological media studied, 10-pl. aliquots of the concentrates were found suitable for analysis. One-dimensional descending paper chromatograms are employed with Schleicher and Schuell S o . 597 or Whatman No. 1 paper. The solvent systems recommended for use are either 1-butanol-acetic acid-water or l-butanol-ethanolwater, prepared as described previously (6-8). The chromatograms are developed a t room temperature by the descending technique for 18 hours, dried a t 110' C. for 2 to 3 minutes, and redeveloped in the same solvent for an additional 18 hours in the same dimension. Separation and resolution aere considerably increased by this second development period. The chromatograms are then dried at 110' C. for 2 t o 3 minutes and sprayed with 0.4YC ninhydrin dissolved in either watersaturated 1-butanol or 95YG ethanol. The chroniatograms are dried a t room temperature for 5 to 10 minutes, reheated to 110' C. for 3 minutes, and dipped into l.OYC sodium bicarbonate. The phenylalanine region on the chromatograms immediately appears as a deep blue spot, which is clearly visible even in the presence of the purple color of other amino acids. This blue color does not fade on prolonged standing, although the color intensity of the other amino acids on the chromatograms decreases steadily (7). Measurement of Concentration. SPECTROPHOTOLIETER. The blue phenylalanine area is then cut into several strips, which are placed in a test tube with 5.0 ml. of deionized water. The tube from each spot is shaken mechanically for 1 minute, the liquid decanted, and the washing procedure repeated. The tubes are
V O L U M E 28, NO. 1 2 , D E C E M B E R 1 9 5 6 then drained, 5.0 ml. of butanol is added to each,-and shaking continued for 10 minutes. This extract is then decanted and the color intensity read in a s ectrophotometer a t 600 mfi (a Beckmann Model D U was use&, employing a 1-butanol blank. The phenylalanine concentration is calculated from a standard curve prepared by the same procedure. DENSITOXETER. As an alternative method, the ninhydrinand bicarbonate-treated chromatograms may be washed with water until the contaminating colors have been removed. The period of washing will depend on the concentration of contaminating ninhydrin-positive materials, but is usually complete within 5 minutes. The intensity of the clearly defined blue phenylalanine spot may then be measured with a densitometer (Welch Densichron), and the phenylalanine concentration calculated from a standard curve iepared in the same way. Because the henylainine spots are somewhat irregular, both area and peak d3ensity must be measured in the densitometer procedure. For this reason, the butanol extraction method is recommended as the procedure of choice. In the development of this method, specific types of paper and developing solvents were used, but it appears probable that other papers and solvent systems R ould prove equally satisfactory. Standard Curves. Graded levels of DL-phenylalanine were disbolved in medium 11 150, samples concentrated to dryness, and reconstituted in deionized water a8 described previously. At the mme time, samples of M 150 containing 50 mg. per liter of obphenylalanine were concentrated to dryness and reconstituted in varying quantities of deionized water. Portions of 10 pl. of these concentrated samples were then spotted on paper chromatograms so that the concentration of phenylalanine per spot ranged from 10 to 50 y. In this manner two series of phenylalanine spots were prepared. In one series, the ratio of phenylalanine to other amino acids remained constant, while in the second series the ratio changed progressively. The specific phenylalanine color was then developed, follox-ed by butanol extraction and measurement of absorbance. The results of these determinations (Figure 1) show a linear response over the concentration range of 10 to 50 y. Higher concentrations of phenylalanine could not be measured in this system because of solubility limitations. Because the response obtained is linear and identical whether the ratio of phenylalanine to other amino acids remains constant or changes, interference by other amino acids is precluded. In preparing these curves, eight replicate chromatograms were used in each series and separate measurements were made a t each phenylalanine concentration. Calculation of the standard error in terms of the absorbance measurements (0.047 & 0.004, 0.168 & 0.011, and 0.257 =!= 0.016 for 10, 30, and 50 e, of phenylalanine, respectively) shows that the precision of the method is good. RESULTS AND DISCUSSION
Data from a typical recovery experiment, summarized in Table I, show that excellent recoveries can be obtained by this method.
Table I. Determination of Known Amounts of DL-Phenylalanine Added to Medium M 130
Dtphenylalanine, y per M1. -4dded Recovered 30 30 30 30 40 40 40 40 40 40 50 50 50 50
30 29 30 31 38 41 40
4 3 4 5 3 3 8
40 2
39 3 39 3 47.6 52.4 48.8 49.1
1965 Table 11. Selective Elution of Contaminating Amino Acids and Phenylalanine from Ninhydrin- and Bicarbonate-Treated Paper Chromatograms Removal ofa ContamPhenylinants Eluting Solvent alanine Chloroform ++++ 1-Butanol Acetone Ethanol, 95% Carbon tetrachloride Diethylamine Water, deionized Sodium bicarbonate, 1.0% Hydrochloric acid, 0.1W 1-Butanol (after water extraction) a = complete removal; = very slight removal. b Dilute hydrochloric acid caused characteristic phenylalanine color t o revert t o typical nonspecific ninhydrin color.
++++ +
+
++++ ++++
+++ +
++++
+
cause the phenylalanine color was bound to the paper more strongly than that of the other amino acids present (7), attempts were made to remove the contaminants by solvent extraction. The phenylalanine areas, together with their Contaminants, were cut from the chromatograms, placed in test tubes, and 5.0 ml. portions of appropriate solvents added. The tubes were shaken mechanically for 5 minutes, the liquid was decanted, and absorption curves were prepared. The results of these experiments are summarized in Table 11. With the exception of chloroform, the organic solvents tested failed to elute the ninhydrinpositive materials. Water or 1.0% sodium bicarbonate removed most of the ninhydrin and amino acid colors but did not elute the phenylalanine color. After washing with water or bicarbonate, however, treatment with 1-butanol was found to elute the specific phenylalanine spot effectively; this tn-0-step elution procedure as selected as a differential method. Extraction of contaminating amino acid and ninhydrin colors from a paper chromatogram of medium M 150 (containing 50 mg. per liter of Dbphenylalanine), after development with ninhydrin and treatment with sodium bicarbonate, is illustrated in Figure 2. A 1-minute extraction with water has removed the majority of the ninhydrin-positive materials, as shown by the
0
.
5
/'. 0.4
7
\
Recovery, % 101.3 97.7 101.3 105.0 95.8 103.1 100.2 100.1 98.3 98.3 95.2 104.8 97.6 98.2
I
'* A
WAVE LENGTH,
Removal of Contaminating Amino Acids. In exploratory experiments, chromatograms of medium M 150 treated with ninhydrin and bicarbonate as described previously (7) gave the characteristic blue phenylalanine spot, but heavily contaminated with valine and occasionally with methionine and leucine, Be-
mp
.
-
Figure 2. Removal of contaminating ninhydrin colors by extaction with water A. B. C.
First 1-minute water extract Second 1-minute water extract Third 1-minute water extract
ANALYTICAL CHEMISTRY
1966 high reading of the characteristic absorption maximum a t 560 m p in the eluted material. At the same time, the lack of a peak a t 600 mp indicates thst the characteristic phenylalanine color has not been eluted. Small amounts of color were removed by a second water extraction but only insignificant amounts appeared during the third extraction. Following two I-minute water washes of the chromatogram, the specific phenylalanine color was eluted by I-butanol, as shown in Figure 3, Curve A . This graph shows clearly the characteristic absorption peak a t 600 nw of the specific phenylalanine color. The regularity of this curve and the absence of a peak a t 560 nw show that only negligible amounts of extraneous color have been carried into the butanol extract. For comparative purposes, a graph of the third water extract, following two 1-minute watei extractions of a duplicate chromatogram, is included (Figure 3, curve B). This cnme shows the typical ninhydrin absorption maximum a t 560 m p . The regularity of this curve and the absence of a peak a t 600 mM indicate that negligible amounts of the specific phenylalanine color have been extracted by the water wash.
18 1%
-
I7 LE I6 P h r N l L a L n N l N c 15 VALINE
14 METHIONINE 3 TRYPTOPHAN
P NROSINE I
PROLINE
D ALANINE
9
THREONINE
8
GLUTAMIC ACID
APPLICATION TO B1OUH;ICAL MEDIA
The new method was tested on synthetic medium M 150 and on a modified synthetic medium (M 496) in which the total amino acid content was approximately double that of M 150. Concentrated water solutions (30%) of yeast extract (Difco) and emymatic casein hydrolyzate (Difco) were also tested for the presence of phenyldanine. The results of these experiments are shown in Figure 4. The left half ( A , B, C, and D)shows development af these four solutions by the conventional ninhydrin procedure and illustrates €he difficulty of detecting phenylalanine ID the presence of other ninhydrin-positive substances. The right half (A,,BP,Ca, and D,) shows a similar chromatogram which has been developed with ninhydrin, treated with bicarbonate, and washed with water. The characteristic blue Spot8 of phenylalanine (position 16) are clearly defined and washed free of contamination with other amino acids. Yeast extract and casein hydrolyzate (C, and D J show a n appreciable content of phenylalanine, although on development by the conventional ninhydrin procedure alone (C and D ) the presence of this amino acid was impossible to detect. Better resolution of the amino acids in the yeast extract and casein hydrolyaate on these chromatograms could have been obtained by the use of less concentrated samples but the conditions were deliberately made as rigorous as possible. The major advantage of this new method is that it permits the specific determination of phenylalanine in the presence of large amounte of other amino acids, even under conditions of poor
0.151
/
'\
1
VEiOPED AS FT HALF, BUT BSEQUENTIY iATED WITH IDIUM BICAR) N A T E AND &SHED W I T H 4TER
hemylalanine . .. A AI.
Iylealum M 151
B,'B*. Medium M 491 c. cs. Yeart extract D . DI. Enrymntio C&QL
separation and resolution on the chromatograms. I n the application of this procedure to biological media, it has been possible to determine phenylalanine readily in synthetic tissue CUIture medium M 150, in which nL-phenylalanine constitutes 50 mg. in 1100 mg. per liter of total amino acids, a concentration of only 4.5%. An unusual feature of this method is that i t can be subsequently applied to paper chrom&tograms that have been developed with ninhydrin for the measurement of other amino acids or peptides. Because the specific color reaction is undergone by free phenylalanine but not by derivatives or peptidesof phenylalanine (7), the method may possiblybe applied, in conjunction with the conventional ninhydrin procedure, for quantitative measurements of the degree of protein hydrolysis. LITERATURE CITED
(1) Dent, C. E., Biochern. J . 43, 169-80 (1948). (2) Hanks, J. H., Wallace, R. E., Proc. Sac. Ezntl. Biol. Med. 71, 196200 (1949). (3) Hardy, T. L.. Holland, D. 0.. Nnyler, J. H. G..ANALCREW.27, 971-4 (1955). (4) Morgan, J. F., CarnDbell, M. E., Morton, H. J., J . Natl. Cancer Inst. 16,557-67 (1955). (5) Morgan. J. F., Morton, H. J.. Parker, R. C., P ~ c SOC. . Ezptl. B i d . Mad. 73, 1-8 (1950). (6) Pasieks, A. E., Morgan, J. F., Bioehirn. et BaOphys. Acta 18,236-
Figure 3.
Elution of speoifie phenylalanine oolor with 1-butanol
10-minute extraction with I-butanol following two 1-minute water ertraotiona B . Third 1-minute water ertraction following two initial I-minute wsterextraction$aforA
A.
40 (1955). (7) Ibid.. 19,366-71 (8) Pasieka, A. E., h Inst. 16, 995-I( (9) Pratt. J. J.. Jr., P R ~ O E W Efor D review Fc
