Rapid Paper Chromatographic Method for Quantitative Determination of Tryptophan HENRY R. ROBERTS, MICHAEL G. KOLOR, and WESLEY BUCEK Research and Development Division, National Dairy Products Carp., Oakdale, Long laland N .Tryptophan can b e separated from the other commm amino acids in 2 hours a t 60' C. using horizontal p a p e r chromatography and a developing solvent composed of equal volumes of collidine and pH 9.0 buffer. Using the maximum density technique, a quantitative procedure for tryptophan has been developed. Statistical evaluation of this method shows that the accuracy and precision is excellent. Application of this procedure to the analysis of the tryptophan content of a-casein following a n alkaline hydrolysis gave a value of 1.54 mg. per 100 mg. of a-casein, which compares favorably with the previously published value of 1.6 mg.
I
the amino acid Composition . . of a protein by paper chromatography, an acid hydrolysis is required for all the amino acids except tryptophan, which needs an alkaline hydrolysis. Following this hydrolysis, horizontal paper chromatography at an elevated temperature has been utilized to separate tryptophan from the other amino acids in 2 hours. The accuracy of this rapid paper chromatographic procedure is reported here, as well as its application in determining the tryptophan content of rr-casein following an alkaline hydrolysis. N DETERMINING
EXPERIMENTAL
Whatman No. 1 filter paper buffered a t pH 9.0 (63,and measuring 12 x 17 ern., is used. The origin is 2.5 cm. from one end. A 1.5-cm. tab, folded a t a right angle to the paper, is inserted into the solvent trough. The chromatographic chamber and the manner in which the filter paper strip is supported in a horizontal position have been described (8). The developing solvent is prepared a t C. in a temperature-controlled 22 room by equilibrating equal volumes of redistilled 2,4,Rcollidine (Koppers Co., Ine., refined grade) and pH 9.0 buffer (50 ml. of O.OG7.W boric acid and potassium chloride plus 21.30 ml. of 0.0G7.M sodium hydroxide). The solvent-rich phase is placed in the trough, and the bottom of the chamber is covered with the buffer-rich phase. The covered chamber is housed in a mechanical convection oven a t a temperature of GOo C.
1626
ANALYTICAL CHEMISTRY
Seven amino acid solutions are spotted along a line 2.5 em. from the tab end of the filter paper strip. Four standard t w t o p h a n solutions (pH 6.5) containing, respectively, 0.025, 0.050, 0.075, and 0.100 y of amino nitrogen are applied consecutively to the paper in 0.5-pl. volumes using a Gilmont ultramicroburet. Three dilutions of the unknown tryptophan solution are a p plied alternately with the knowns. The unknown solution is spotted to fall within the 0.025- to O.1M)-rrange of the standard solution. After spotting, the filter paper strip is placed in the chamber and a solvent development of 2 hours at 60' C. separates tryptophan from the other common amino acids. Following solvent development, the chromatogram is dried for 30 minutes at 60' C. in a mechanical convection oven, then dipped in water-saturated butanol containing 0.4% ninhydrin plus 4% acetic acid. The color is developed by placing the chromatogram in an oven a t GOo C. for 15 minutes (7). The maximum densities of the tryptcphan spots are measured with a Densichron transmission densitometer (Model 3835B, W. M. Welch Scientific Co., Chicago, Ill.) using a 1-nun. diameter aperture and the green filter built into the instrument. A standard curve is prepared by plotting the logarithm of the known concentration against the densities. The concentration of tryptophan in the unknown solution is then calculated by referring to the standard curve. The standard curves are not reproducible from one paper strip to another, and the standards must be run each time on the same chromatogram with the unknown. The rr-casein used in this study was obtained from whole casein employing an alcohol fractionation (6) and Warner's isoelectric precipitation procedure (11). On a dry basis, the acasein sample contained 15.24% nitrogen and 1.12% total phosphorus. The electrophoresis pattern gave one component with a mobility of -6.19 X 10-6sq. em. per see.-volt. The analysis was made in Verona1 buffer a t pH 7.78, with ionic strength 0.1, containing 0.08Nsodium chloride. Three 25C-mg. mmples of the casein preparation were hydrolyzed under reflux for 20 hours in 25-ml. solutions of 14% barium hydroxide. Following clarification and concentration of the hydrolyzate as described by Block (Z), the dried residue was dissolved in 10% isopropanol, adjusted to
V
Two-hour solvent development in collidine-pH
9.0 buffer (1 to 1) A. 8.
Tryptophan Solution containing 20 common amino acids
pH 6.5, and made up to a final volume of 10ml. RESULTS AND DISCUSSION
The chromatogram obtained nhcn a solution containing 20 common amino acids is chromatographed a t 60' C. for 2 hours in collidine-pH 9.0 buffer (1 to 1) is shown in Figure 1. Tryptophan, having the greatest R, value, is clearly separated from the other amino acids. The descending paper chromatographic procedure using the same buffered paper and solvent systrm requires a development of 24 hours (6'). Because an alkaline hydrolysis of 1:irotein is required for the paper chromatographic determination of tryptophan Z),this new chromatographic procedure ias added significance as it provides a apid identification and assay of tryptoihan in the alkaline orotein hvdrolvsate. " " n addition, the sensitivity is increased. ?he concentrations of the tryptophan tandards applied to the paper are oneenth the concentrations required for he quantitative descending paper hromatographic procedure (7). The results of the determination of the ryptophan content of a-casein following
an alkaline hydrolysis are presented in Table I. The average value of nine quantitative chromatograms was 1.54 mg. of tryptophan per 100 mg. of CYcasein, &-hich compares well with the value of 1.6 mg. reported previously (4). It is appreciably lower, however, than the value of 2.2% reported by Gordon and conorkers (3’) employing the method of Spies and Chambers (10)on unliydrolyzed a-casein. The accuracy and precision of the method were established by determining the tryptophan content of a solution containing 18 common amino acids (40 mg. of each amino acid per 100 ml. total volume, pH 6.5). The average value of 39 2 nig. obtained from nine yiiantitatiT-e chromatograms (Table 11) differed from the theoretical value by 2 0%. The standard deviation, 1.14 rng. per 100 ml., indicates that the assay can be repeated with good precision. TThile the accuracy of the procedure is about the same as that previously reported for descending paper chromatography and the maximum density method (9). the precision of the horizontal method. 1.14 mg. per 100 nil., is an improvement over the previous value of 2.34 mg per 100 ml. ACKNOWLEDGMENT
The authors n-ish to thank Elizabeth Stanton and George Kissel for the sample of a-caspin and Margaret &I.
Table 1. Tryptophan Content of QCasein as Determined b y Horizontal Paper Chromatography
Table ll. Accuracy and Precision of Horizontal Paper Chromatographic Determination of Tryptophan
Calcd. Concn.,
Calcd. Concn.,
Mg./100 Mg.
Chromatogram
a-Casein
1 2 3
4
5
Chromatogram
1.54 1.36 1.54 1.48 1.60
1 2 3 4
1 67
6 7 8 9
5
_ .
9
Average Standard deviation (9) Chemical determination ( 4 ) Chemical determination on unhydrolyzed a-casein ( 3 )
Fitzpatrick analysis.
for
the
1.54 1.54 1.60 1 54 0 087 16 2 2
electrophoretic
LITERATURE CITED
(1) Block, R. J., ANAL. CHEX 22, 1327 (I950). (2) Bliqck, R. J., Durrum, E. L., Zweig, G., Manual of Paper Chromatography and Paper Electrophoresis,” 2nd ed., Academic Press, Xew York, 1958. (3) Gordon, W. G., Semmett, W. F., Bender, hf.. J. Am. Chem. SOC. 75, 1678 (1953).’ (4) Gordon, W. G., Semmett, IT. F., Cable, R. S., Morris, M., I b i d . , 71, 3293 (1949).
Average Standard deviation (9) Deviation from theoretical value, 40 mg./100 ml., yo
Mg./lOO
Wl.
39.4 37.9 37.9 39 4 39.4 40.8 39.4 37.9 40.8 39.2 1.14 2.0
(5) Hipp, Y. J., Groves, AI. L., Custer J. H., McMeekin, T. L., J . D a i r y Sci 35, 272 (1952). (6) McFarren, E. F., AKAI,.CHEJI. 23, 168 (1981). ( 7 ) McFarren, E. F., Mills, J. A,, Ibid., 24, 650 (1952)). (8) Roberts, H. R., Zbid., 29, 1443 (1957). (9) Roberts, H. R.: Kolor, bl. G., Zbid., 29, 1800 (1957). (10) Spies, J. R., Chambers, L). C., Zbid., 21, 1249 (1949). (11) Warner, R. C., J . i i m . Chem. SOC. 66, 1725 (1944).
RhCE1T.m for review February 27, 1958. Accepted &la>-31, 1958.
Determination of Lithium and Sodium Chlorides by Potentiometric Titration Following 2-Ethyl-1-hexanol Separation GLENN R. WATERBURY, EDWARD H. VAN KOOTEN, and BRUNO MOROSIN’ University o f California, 10s Alamos Scientific Laboratory, Los Alamos, N. M.
b Sodium and lithium chlorides are titrated potentiometrically with silver nitrate following two extractions of the lithium chloride with 2-ethyl-1 -hexanol. Using glass and silver-silver chloride electrodes, the detection of the end point i s enhanced in the organic medium. For 17 determinations, an average of 99.99y0 was obtained for lithium, with a standard deviation of 0.1670, and an average of 99.9% for sodium, with a standard deviation of o.4y0. A study of the effect of 20 other metals on the analysis showed that lithium i s also separated b y the extraction from aluminum, barium, and tungsten; so-
dium may b e determined in the ence of lithium and plutonium, mium, cobalt, iron, magnesium, dymium, nickel, tin, uranium, or
T
prescadneozinc.
purpose of this investigation was twofold: to develop a precise procedure for the analysis of lithiumrich mixtures of sodium and lithium using the 2-ethyl-1-hexanol separation, and to determine the applicability of the method for the separation and determination of lithium or sodium in other metals, especially plutonium or uranium. I n the original method described by Caley and Axilrod ( I ) , a solution of the alkali chlorides is deHE
hydrated in contact with 2-ethyl-lhexanol which selectively dissolves the lithium chloride. The insoluble sodium or potassium chloride is removed by filtration, and the separated alkali metals are determined as the chlorides or sulfates by time-consuming gravimetriC methods. White and Goldberg ( 5 ) applied the Volhard method (3) for the titration of chloride to the determination of lithium chloride in alcoholic solution following the dehydration. This method requires a back1 Present address, Chemistry Department, University of Washington, Seattle, Wash.
VOL.
30, NO. 10, OCTOBER 1958
1627
