complex the desired constituent and thereby introduce a second migrating species. ACKNOWLEDGMENT
The authors wish to thank the Purdue Research Foundation for financial support of this study. They thank J. ST. Amy, Department of Chemistry, Purdue Unirersitj-, for his modified design and construction of the scanning attachment. LITERATURE CITED
(1) Boltz, D. F., llellon, M. G., AXAL. CHEM.20, 749 (1948). (2) Brimley, R. C., Nature 163, 215 (1949).
(3) Brown, J. A,, Marsh, 1cI. M., ANAL.
CHEW24, 1952 (1952). (4) Bull, H. B., Hahn, J. Vi’., Baptist, V. H.. J . Am. Chem. Soc. 71. 550 (1949): (5) Damon, J. RI. O., “Direct Spectrophotometric Determination on Filter Paper of Germanium, Phosphorus, and Arsenic,” Ph. D. thesis, Purdue University, Lafavette, Ind., 1958. (6) Fisher, R . B., Parsons, D. S., llorrison, G. A , , Satzire 161, 764 (1948). (7) Gilman, H., Ingham, R . K., Gorsich, R. D., J . -4m. Chem. Soc. 76, 918 (1954). (8) Kitson, R . E., RIellon, >I. G., IND. ESG.CHEY.,AXAL.ED. 16, 128 (1944). (9) Lacourt, -1.) Sommereyns, Gh., Claret, SI.,Mikrochemae ter. Mekrochzm. Acta 38, 444 (1951). (10) Ladenbauer, I., Hecht, F., X i k r o cham. Acta 1954, 397. (11) Mellon, 11. G., Record Cheni. Progr.
(Kresge-Hooker Sei. Lib.) 11, 177 (1950). (12) Mykolajewycz, R., AXAL. CHEM. 29, 1300 (1957). (13) Olson, J. AI., “Factors Bffecting Separation of Silicate, Germanate, Phosphate, and Arsenate by Filter Paper Chromatography,” M. S. thesis, Purdue University, Lafayette, Ind., 1955. 114) Parke. T. V.. Davis. IT. W..A X V ~ L . CHEM.24, 2019 ’(1952).’ (15) Redfield, R. R., Guzman Barron, E. S., d r c h . Bzochem. Bzophys. 35, . 443 (1952). 116) Rockland. L. B.. Underwood.’ J. C.. ANAL. C H E ~28. . 1679 11956). (17) Vaeck, S. V.,’Anal. ‘Chi&. Acta IO, 48 (1954). (18) Vaeck, S. V.,Ibid., 12, 443 (1955). (19) Kinslow, E. H., Liebhafsky, H. A., AYAL.CHEJI.21, 1338 (1949). RECEIVEDfor review Xarch 7, 1958. Accepted June 19, 1958.
S pe c ific Ide nt ific at io n of Hy d roxya mino Ac ids on Paper Chromatograms of Protein Hydrolyza tes DANIEL P. SCHWARTZ Agricultural Research Service,
U. S.
Department of Agriculture, Washington,
b Individual methods are described for the specific identification of serine and threonine on paper chromatograms. The test for serine is based on the condensation of the formaldehyde, liberated by periodic acid oxidation, with acetylacetone, in the presence of ammonium salts to form the yellow, highly fluorescent, 3 3 diacetyl-1, 4-dihydrolutidine. Hydroxylysine, found only in a few proteins, also gives a positive response. Threonine is specifically detected in the presence of all amino acids b y a test based on the reaction of acetaldehyde with sodium nitroprusside and piperidine, following oxidation of the amino acid with periodic acid.
S
NETHODS have been described for detecting and confirming the identity of hydroxyamino acids on papergrams. The procedure suggested by Synge (6) involves the detection, with Nessler’s reagent, of ammonia liberated by periodate oxidation. This method will not differentiate b e h e e n serine and threonine, and also requires that the paper be free from any ammonia that may have been employed in the solvent. The methods suggested by Bucharian, Dekker, and Long ( I ) , and by Ptletzenberg and Mitchell (6) are also utilizable, but these too will not distinguish between serine and threonine. I n the procedure described,
EVERAL
D.C.
serine is specifically detected in the presence of all commonly occurring amino acids including threcnine. IDENTIFICATION OF SERINE
The test is based on the fact that serine liberates formaldehyde when oxidized with periodic acid ( 8 ) . The formaldehyde set free in the reaction is then condensed with acetylacetone in the presence of ammonium salts to form t h e yellow, highly fluorescent, 3,5-diacetyl-1,4-dihydrolutidine( 7 ) . The finished chromatogram, dried a t 50” C. or below, is sprayed carefully with a 0.035M solution of periodic acid in methanol containing 2 vol. % redistilled y-collidine. K h e n the sheet is visibly dry, i t is sprayed with a solution containing 15 grams of animonium acetate, 0.3 ml. of acetic acid, and 1 ml. of acetylacetone per 100 ml. of methanol. The latter solution is a modification of the reagent described by Nash (7) for the quantitative estimation of formaldehyde. The paper is allowed to develop a t room temperature until the yellow spot indicating the position of serine is visible. This usually requires from 1 to 4 hours, depending on the concentration of serine. Under ultraviolet light, however, the spot is usually visible within 30 minutes after the application of the second spray. The approximate lower limit of detection for serine is 2 y per
sq. cm. in daylight, and 1 y per sq. em. under ultraviolet light, after chromatography on SThatman No. 1 paper in the solvent system, methanol$7-ater-pyridine (80 to 20 t o 4) (9). The only other amino acid which gives a positive test under these conditions is b-hydroxylysine, The limits of detection for this amino acid in 17-hite, and ultraviolet light, reFpectively, following chromatography under these conditions, are 1.5, and 1.0 y per sq. em. Under similar conditions, L- or DLthreonine gives no spot in white light even in high (35 y per sq. em.) concentration. If the paper is examined under ultraviolet light after 24 hours, threonine is visible as a dark zone which cannot be confused with the yellow fluorescent serine spot. The dark threonine zone is due presumably to the fcrmation of diacetyldihydrocollidine from the condensation of acetaldehyde with the reagent ( 7 ) . Hom-ever, approximately 15 to 20 y of theonine are needed for detection under ultraviolet light, which renders its detection impractical with paper chromatography. A much more sensitive reaction for threonine is described. The following compounds were examined, both in n hite and ultraviolet light, in concentrations ranging from 35 to 50 y per sq. cm. All of the common amino acids, o-phoephoserine. o-phosphothrconine, o-phosphoethanolamine, methionine sulfone and sulfoxVOL. 30, NO. 1 1 , NOVEMBER 1958
0
1855
ide, citrulline, cyetathione, p-aminoisobutyric acid, p-alanine, taurine, yamino-n-butyric acid, methylhistidine, thiolhistidine, sarcosine, cysteic acid, asparagine, glutamine, lanthionine, ornithine, and a-amino-n-butyric acid were negative under these conditions. Compounds other than serine and hydroxylysine which responded to the test, and the approximate lower limit of detection (micrograms per square centimeter) in white, and ultraviolet light, respectively, were ethanolamine, 1.0 and 0.8; glycerol, 0.5 and 0.2; and L-a-phosphoglyceroethanolamine, 2.7 and 1.8. SPECIFIC IDENTIFICATION
OF THREONINE
The test depends on the fact that threonine liberates acetaldehyde when oxidized with periodate ( 5 ) . The acetaldehyde is detected by applying a slight modification of t h e sensitive reagent described by Feigl and Stark (3). The dry, finished chromatogram is sprayed n4th the periodic acid-collidine
reagent described for serine. TThen visibly dry, the sheet is sprayed carefully with a solution made by mixing, just before use, 1 volume of 5% methanolic solution of sodium nitroprusside ’ piperidine with 1 volume of 20 vol. % in methanol. The threonine spot is indicated by a blue zone within 15 minutes. The sensitivity of the test under these conditions, which may not be optimal, is 1.5 y of L- or DL-threonine per sq. em., after chromatography under the conditions used for serine. All compounds except threonine which have been mentioned are negative in concentrations ranging from 35 to 50 y per sq. cm. Higher concentrations were not tested. The reactions described are not necessarily limited to the compounds reported which give a positive response. A review of the periodate reaction by Jackson (4) should reveal a number of compounds which would be expected to give a positive reaction on chromatograms containing biological materials other than protein hydrolpzates.
ACKNOWLEDGMENT
The author gratefully acknowledges a sample of L-a-phosphoglyceroethanolamine monohydrate from Erich Baer, University of Toronto. LITERATURE CITED
(1) Buchanan, J. G., Dekker, C. A., Long, A. G.. J . Chem. SOC.1950. P. 3162. (2) Consden, R., Gordon, H . , Martin, A. J. P., Biochem. J . 40,33 (1946). (3) Feigl, F., Stark, C., Chemist-Analyst 4 5 , 3 9 (1956). ( 4 ) Jackson, E. L., L‘Organic.Reactions,” Vol. 2. D. 341. Wilev. New Tork. 1944. ( 5 ) Marti;, A. ’J. P.,”Synge, R. L. M., Biochem. J . 35, 294 (1941). (6) hfetzenberg, R. L., hIitchell, H. K., J.Am. Chem. SOC.76,4187 (1954). ( 7 ) Kash, T., Biochem. J . 55, 416 (1953). (8) . , Nicolet. B., Shinn, L.. J . Am. Chem. s o c . 61, 1615 (i939j. (9) . . Redfield, R., Biochim. et Biophus. Acta io, 344 (1953 j.
RECEIVEDfor review March 25, 1958. Accepted June 19, 1958.
Densimetric Analysis and Graphical Readjustment of Composition for Recovered Ternary Chromatographic Solvents DWIGHT F. MOWERY, Jr. New Bedford lnsfitute of Technology, New Bedford, Mass,
b An approximate but rapid densimetric method for analyzing recovered ternary chromatographic solvent mixtures is described, and details are given for the construction of a chart allowing quick graphical readjustment to a predetermined composition. The method has been applied successfully to the 1-butanol-pyridine-water ( 1 03-3) system and the ethyl acetate1-propanol-water (5-3-2) system.
W
the advent of large partition columns for quantity separations (9,3,6, 7 ) and routine use of analytical partition columns (1, 6), chromatographic solvent recovery is important. Khere the stability of the components makes it feasible, recovery has become a necessity for economy as well as safety. B y recovery and re-use of solvent, the amount which must be purchased and kept on hand can be reduced considerably. For this re-use, a rapid method for analysis and readjustment of the 1856
ITH
ANALYTICAL CHEMISTRY
composition is desirable. This paper describes an approximate densimetric analytical method utilizing simple and inexpensive equipment. The method depends upon measurement of the quantity of water needed t o saturate the solvent mixture and determination of the density of the resulting saturated solution. Refractive index, commonly used in conjunction with density for the analysis of ternary mixtures (4, 5 ) , was of little use for the two solvent systems investigated. Details of the construction of a chart for quick graphical correction of 1-butanol-pyridine-vater mixtures to a 10-3-3 composition by volume as well as a similarly constructed chart for adjustment of ethyl acetate-l-propanolwater mixtures to a 5-3-2 composition are presented. A composition differing slightly from that for which a chart was designed can be handled easily by first adjusting to the chart composition and then to the required composition. Although a t present applied to only two solvent mixtures, the method should
prove useful with many ternary chromatographic solvent systems. The method consists in saturating 250 ml. of the solvent mixture at 25’ C. with water and determining the density of the resulting solution with an alcohol hydrometer having a narrow range. The hydrometer reading reveals the ratio of the two nonaqueous solvents, and the quantity of water required for saturation yields the water content. The composition of the ternary mixture is thus fixed and may be plotted on a triangular coordinate system. The position of the point allows easy graphic determination of the quantities of components which must be added t o produce a predetermined compcsition. Because it is unnecessary to know the actual percentage composition for this graphic procedure, a simplified diagram results by elimination of the triangular coordinate system after construction of the chart. EXPERIMENTAL
Equipment and Procedure.
The
