1628
A N A L Y T I CA L C H E M I S T R Y
for their threonine and serine content: casein, squash seed globulin, dentinal protein, and edestin, which were prepared in this laboratory; egg albumin, obtained from J. B. Allison of Rutgers University; granular gelatin, U.S.P.; human globin hydrochloride from Sharp & Dohme; and alkali-precipitated zein from the Corn Products Refining Co. The average of the results, expressed as per cent, and based on 1670 nitrogen appear in Table I. The values obtained are compared with the ranges of values for similar proteins as given by Block and Bolling (1, 2 ) . DISCUSSION
The use of colorimetric methods for the estimation of the acetaldehyde and formaldehyde produced by periodic acid oxidation of threonine and serine permits the determination to be made upon smaller samples than those originally employed by Shinn and Nicolet (8, 10) and in a shorter period of time. By the colorimetric methods only a single oxidation is necessary. The results obtained upon samples of known concentrations of both amino acids have been graphed, and show that an average of four values falls upon a straight line, although there is a deviation of from 6 to 9% for single determinations a t each concentration. The values given for a number of proteins are in agreement with the literature values for the same proteins using the original methods of Shinn and Kicolet. The same criticism of the serine method originally noted by Nicolet and Shinn ( 8 ) )that hydroxylysine will interfere, still holds with the colorimetric method. Among the proteins analyzed by the authors, as far as present knowledge goes, only gelatin contains hydroxylysine. Inskip ( 7 ) has not found any of the
other proteins to contain this amino acid. I n determining the amount of serine present in a gelatin hydrolyzate a correction hae been made for the hydroxylysine present. The alternate method for the determination of threonine, in which the acetaldehyde is aerated directly into the color reagent, p-hydroxybiphenyl, a8 suggested by Block and Bolling (Z), requires less time and there is somewhat less variation in the colorimetric readings a t each concentration than in the procedure first described. LITERATURE CITED
(1) Block, R. J., and Bolling, Diana, "Amino Acid Composition of Proteins and Foods," 2nd ed., Springfield, Ill., C. C Thomas Publishers, 1951. (2) Block, R. J., and Bolling, Diana, "Determination of the Amino Acids," Revised ed., Minneapolis, Minn,, Burgess Publishing Co., 1940. (3) Block, R. J., and Bolling. Diana, J . Biol. Chem., 130, 365 (1939). (4) Boyd, H. J., and Logan, AI. A., Ihid.,146,279 (1942). (5) Eegriwe, Edn-in, 2. anal. Chem., 95, 323 (1933). (6) Eegriwe, Edwin, Ibid., 110, 22 (1937). (7) Inskip, L. W., J . Am. Chem. Soc., 73,5463 (1951). (8) Kicolet, B. E., and Shinn, L. 9..J . Bid. Chem., 139, 687 (1941). (9) Rees, hI. IT., Biochem. J.,40, 632 (1946). (10) Shinn, L. A, and Kicolet, B. E., J . Riol. Chem., 139, 687 (1941).
HECEIVED for review M a y 9, 1952. Accepted June 14, 1952. These studies were substantially aided by Contract K R 181-817 between the Office of Naval Research, Department of the S a w , and Georgetown University, Material taken, in part, from a thesis submitted by the senior author t o the Graduate School of Georgetown University in partial fulfillment of the requirements for the degree of master oi science.
Rapid Method for Paper Chromatography of Organic Acids F. W. DENISON,
JR., AND
E. F. PHARES
Biology Disision, Oak Ridge National Laboratory, Oak Ridge, Tenn.
OST methods for the paper chromatography of organic acids employ slom-traveling solvents and long-drying procedures (1.3,6)requiring many hours for completion of the chromatogram, The method described in this paper using ether, acetic or formic acid, and water, requires less than 2 hours for satisfactory development of the chromatogram. The speed of development is slightly faster than the chloroform and iso-octane solvents described by Stark, Goodban, and Owens ( 3 ) . Only 10 to 15 minutes are required to free the paper of siiamping acid. The acids may bP applied as the free acids in ivater or organic solvents, or as a solution of the sodium salts, acidified with sulfuric acid either before or after application to the paper. The general technique is similar to that of Lugg and Overell ( 1 ) but offers advantages in simplicity and rapidity. The method should be of value for a rapid survey of unknown acids and especially for more positive identification when used in conjunction with other solvent systems (1, S), and mith partition columns, on uhich the relative positions of the acids are changed considerably ( 2 ) . METHOD
Approximately 0.0002 meq. of the organic acids in nater or organic solvent x a s applied to 40 X 57 em. sheets of Whatman No. 1 paper in spots 4 em. apart and 3 em. from the bottom of the paper. The spots were kept to a diameter of less than 1 em. by the repeated application of less than 5-microliter amounts, the drying process being aided by a stream of warm air from a small hair dryer. The paper was stapled into the form of a cylinder, leaving a small space between the edges of the paper, and placed in a glass jar, the bottom of which was covered to a depth of about 1 cm. with the solvent solution. The top of the jar was covered with a weighted glass plate, using a neoprene gasket to effect an air-tight seal. FVith a temperature of 22" C. the solvent front traveled about 15 cm. above the initial spots
in 1.5 hours. At the end of the development period the paper was suspended in a hood. -2 steam manifold, 60 em. in length with holes about 1 cm. in diameter and spaced about 10 em. apart, was placed about 25 em. in front of the paper and a strong flow of steam tc as maintained over the entire surface of the paper. An infrared lamp was focused on each half of the paper from a distance of about 40 em. K i t h this treatment the acetic acid was removed in 5 to 10 minutes and formic acid in 15 minutes. The chromatogram was then sprayed M ith neutral or slightly alkaline bromocresol green, 400 mg. per liter of 95% ethyl alcohol. R, values were measured fiom the center of the acid spots. The Rj values obtained by applying solutions of sodium salts (Table I, column E ) were lower than those from the free acids. This \\as overcome by acidification of the solution with sulfuric acid to a pH of about 1. Considerably higher concentration of acid did not appear harmful. Spots of sodium salts were also acidified on the paper by addition of small amounts of dilute
Table I. HI X 100 for Organic Acids Acid A B C D E .. 9 .. Tartaric .. 12 11 19 43 0 Oxalic 71 08 Citric 16 08 06 24 78 Malic 13 20 25 16 71 51 36 a-Ke toglutaric 14 82 43 45 49 Glycolic 28 82 32 40 Malonic 73 52 78 45 91 21 and 56 45 Pyruvic 70 87 87 Lactic 72 58 93 75 8.5 Succinic 62 76 90 94 98 Fumaric 84 90 Oxalacetic 46 A = 13 parts ether, 3 parts glacial acetic, 1 part water. B = 14 parts ether 1 part glacial acetic, 1 part water. C = 1 4 partq ether' 0.2 part formic 1.8 parts water. D = 6 partsacetone 6 parts ether 3 parts glacial acetic, 1 part water. E = Same as A , but'sodium salts k e d for spotting with no sulfuric acid.
V O L U M E 2 4 , NO. 10, O C T O B E R 1 9 5 2
1629
sulfuric acid until phenol red indicator assumed its acidic red color. The latter procedure had the disadvantage of tending to spread the spots on the paper. Rj values of acids treated in this manner were identical with those obtained with the free acids. Since the sulfuric acid spot gave no detectable migration with these solvents, there was no interference with the organic acids of low R, values. DlSCUSSIOlY AND RESULTS
Table I shows the R, values obtained with various solvent systems. Some of the acids show similar R, values. However, the use of more than one solvent system is considered necessary for
15 cm
a a 0
0
GLYCOLIC 0
0
u KETOGWTAR IC
0
0 0
SUCCINIC 0
I I
0
FUMARIC 0
I
the identification of any acid. The Rj value of oxalic acid increases somewhat with increase in concentration. The solution found most useful in all respects was ether-acetic acid-water in the ratio of 13 to 3 to 1 (column A). This gave sharp spots with very little tailing and sufficient migration of even tartaric acid to move it away from the initial spot. Less acetic acid (column B) caused more tailing and gave less resolution of the acids of low R, values. Formic acid with ether and water (column C) gave smaller spots and good resolution of all acids except succinic and lactic. Ether-acetone-acetic acidwater gave very small, sharp - spots - with no tailing. but resolution was poor owing t o high R, values. Ether-valeric acidwater, ether-water, and ether-glacial acetic acid gave only st,reaks of all acids. The system ether-chloroacetic acid-water was used, but the paper could not be freed of chloroacetic acid, and it was necessary to spray with dilute potassium permanganate. All acids used, except succinic, gave a light spot against a purple-brown background, having Rj values similar to those with formic and acetic acids. Even this strong swamping acid failed to eliminate the tailing of oxalic acid. Solvent systems A and D were single phase solutions. Systems B and C made two phases, which were allowed to separate for a few minutes after mixing. The ether phase was decanted into the developing jar and the aqueous phase discarded. The problem of esterification (3) did not occur in these systems because of the absence of alcohols, and frequent fresh solutions were not necessary. The treatment for removal of swamping acids, although rapid, does not appear to decompose the more labile acids such as oxalacetic and a-ketoglutaric. The temperature of the paper does not exceed 40' C., and the paper is never dry. The process is actually a steam distillation. If the steaming process is carried out for a sufficient length of time, the spots appear well defined immediately upon spraying, showing no improvement with further drying. Of the common inorganic acids, only nitric interferes seriously, obscuring most of the acids with R, values below 0.7 when present in large amounts. Phosphoric acid in concentration8 greater than O.lyowill obscure spots below 0.2 R f units. Hydrochloric acid does not migrate in these solvents. S n increase of 10' C. in the temperature from 22" C. sloivs the rate of solvent travel about log;',, while a decrease of 10" C. increases the rate by a similar amount. I t appeared that an absolutely tight chamber was essential for maximum speed of development a t any teniperature. Figure 1 shows the characteristic size and shape of the various acid spots using ether-acetic acid-water (13 to 3 to I). Pyruvic acid gives the characteristic lower spot only when treated with sulfuric acid. These chromatograms also have a very distinct line marking the solvent front after spraying, making other designation of the front unnecessary. LITERATURE CITED
(1) Lugg, J. IT. H.. and Overell, B. T., A u s f r a h n J . Sci.
0 W
Q
Figure 1. Paper Chromatogram of Acids Using Ether-Acetic Acid-Water (13 to 3 to 1)
(I. Commercial sample b . Sample prepared according t o Umbreit, Burris, and Stauffer ( 4 )
Research, 1, 98 (1948). (2) Phares, et al., ANAL.CHEM.,24, 660 (1952). (3) Stark, J. B., Goodban, -4.E., and Owens, H. S., Ibid., 23, 413 (1951). (4) Umbreit, 117. K.,Burris, R. H., and Ytauffer, J. R., "Manometric Techniques and Tissue Metabolism," pp. 210-11, Minneapolis. Burgess Publishing Co., 1949. (5) Williams, R. J., and Kirby,Helen, Science, 107, 481 (1948). RECEIVED for review September 4. 1951. Accepted July 2 , 1952. Work performed a t Oak Ridge S a t i o n a l Laboratory under contract No. W-7405-Eng-26 for the Atomic Energy Commission.
