acids and other substances is also contemplated. ACKNOWLEDGMENT
The authors wish to thank Jerome F. Saeman and Gilbert IT.Castellan for a helpful discussion of this problem. LITERATURE CITED
(1) Bate-Smith, E. C., Westall, R. G., Biochim. et Biophys. Acta 4, 427 (1950). ( 2 ) Benson, A. -4.,Bassham, J. A., Calvin, M., Goodale, T. C., Haas, V. A,, Stepka, K., J . Am. Chem. Soc. 72, 1710 (19.50).
(3) Cassidy, H. G., L,i+ndamentals of
Chromatography, p. 145, Interscience. New York. 1957. (4) Closs, K., Haug, hl., Chem. &: Ind. (London) 1953,103. ( 5 ) Consden, R., Gordon, A . H., Martin, A. J. P., Biochem. J . 38, 224 (1944). (6) Fowden, L., Ibid., 48, 327 (1951); Fom-den, L., Penney, J. R., LVature 165,846 (1950). ( 7 ) Kotake, M., Sakan, T., Nakamura, K., Senoh, S., J . Am. Chem. SOC. 73, 2973 (1951). (8) Kowkabany, G. N., Advances i n Carbohydrate Chem. 9 , 339 (1954). (9) Lapp, R. E., rlndrews, H. L., “Nuclear Radiation Physics,” p. 456, Prentice-Hall, New York, 1954. (10) Martin, -4.J. P., Synge, R. L. hI., Bzochem. J . 35, 1358 (1941).
c.
W.J., Ant,. .V. Y . Acad. Sci. 49, 265 (1948). (12) Partridge, S. &I., Jatwre 164, 443
(11) hloore, S., Stein,
(1949‘). (13) Thompson, J. F., Steward, F. C., Plant Physiol. 26,421 (1951). (14) Woiwod, A. J., Biochem. J . 45, 412 (1949).
RECEIVED for review October 26, 1957. Accepted February 11, 1958. Presented in part, Division of Carbohydrate Chemistry, Symposium on Carbohydrate Chromatography, 128th meeting, ACS, hIinneapolis, Minn., September 1955. Taken from the thesis presented by C. K. Hordis, O.S.F.S., in partial fulfillment of the requirements for the degree of master of Fcience, The Catholic University of .Limerica.
Separation of Halogenated Acetic and Propionic Acids by Paper Chromatography JOHN W. CHITTUM, THOMAS A. GUSTIN, ROBERT L. McGUIRE, and JOHN T. SWEENEY Departmenf of Chemistry, The College o f Wooster, Wooster, Ohio
b There has been a need for improved separation of halogenated aliphatic acids. The paper chromatographic procedure described provides good resolution of acetic, mono-, di-, and trichloroacetic acids in mixtures of all four. Also, the bromo acids and acetic acid can b e separated from each other. Rl values are included for 2- and 3-chloro- and bromopropionic acids and for 2,3-dibromopropionic acid. This procedure offers greatly improved separation of propionic acid from these halogenated propionic acids.
A
study required a procedure for detecting and separating minute amounts of the individual halogenated fatty acids in mixtures which may include others of the group. The success of other workers (1-3, 5-10) in separating unsubstituted aliphatic acids by paper chromatography suggested this approach. Long, Quayle, and Stednian (8) reported Rl values for acetic, mono-, di-, and trichloroacetic, bromoacetic, and 3-chloropropionic acids, using Whatman S o . 54 paper. Their developer consisted of 80 parts of ethanol, 4 parts of ammonium hydroxide (specific gravity, 0.88), and 16 parts of water. Development was don-nward. However, their procedure does not separate acetic acid from monochloroacetic acid, and the resolution of other pairs is poor. Reid and Lederer (9) determined Rf values for the first nine normal fatty acids and some others, using Whatman S o . 1 paper and upward development. CURREXT
Their work has been extended by the authors to include 11 chlorinated or brominated acetic and propionic acids. Acetic and mono-, di-, and trichloroacetic acids show good resolution in mivtures containing all four (Table I). Acetic and the bromoacetic acids can be separated well. However, 2- and 3chloropropionic acids were not resolved, nor 11-ere 2- and 3-bromopropionic acids. The Rfvalues (Table I) are reproducible even in the presence of other acids in the mixtures. Successive runs with dichloroacetic acid alone gave 0.25, 0.26, 0.28, and 0.28 (average 0.27); in the presence of acetic and monoand trichloroacetic acids, the values were 0.27, 0.26, 0.26, 0.26, and 0.26 (average 0.26). Tribronioacetic acid, alone, gave five successive values from 0.31 to 0.32. R, values for di- and tribronioacetic acids changed slightly
Table I.
nhen mixed with acetic and the other bromoacetic acids (Table I). After this work was begun, Hashmi and Cullis (4) reported the Rf values for several monobrominated and monoiodinated fatty acids, n-ith downward development. Using l-propanol-ammonia as the developer, they obtained Rf values for acetic and monobromoacetic acids having the same numerical spread as obtained in this work with 1-butanol-ammonia. However, the per cent spread is much greater in the presrnt work and the order is reversed. The present work s h o m a greater difference between the R values for propionic and 2-bromopropionic acids. Hashmi and Cullis (4) found no difference in R , \?alum for 2- and 3-bromopropionic acids. K i t h the present procedure, there is some difference between both the isomeric bromo and chloro acids.
Comparison of R, Values of Acids
R I Values Authors Acid ;Icetic Monochloroacetic Dichloroacetic Trichloroacetic Monobromoacetic Dibromoacetic Tribromoacetic Propionic 2-Chloropropionic 3-Chloropropionic 2-Bromopropionic 3-Bromopropionic 2,3-Dibromopropionic
Long
Hashmi and et al. (8) Cullis ( 4 ) 0.52 0.39 0.52 ... 0.60 ... 0 70 0 51
0 45
0 56
0 48
Single acids 0.08 0.14
0.27 0 0 0 0
45
02 10 31
0 08 0 23
In mixtures 0.08 0.14 0.26 0 46 0 03 0 14 0 27
0
VOL. 30,
NO. 7, JULY 1958
1213
Tailing and spreading of spots prevented clean-cut resolution of either pair. By resorting to multiple development or downward displacement, a resolution may be possible with the butanolammonia system, particularly if a more delicate detecting device could be found to permit the use of smaller amounts of acids. The 1-butanol-ammonia system is available for better resolution in specific cases, or as a different solvent when two-dimensional development seems desirable. EXPERIMENTAL
The acids were dissolved either in water or in acetone, the latter being preferred when prolonged storage was involved, even with refrigeration. Samples were treated with equal volumes of concentrated ammonium hydroxide and promptly applied to the paper (Whatman KO,1). A sample of 1 pl., containing from 40 to 80 y of each acid present, was used.
The rest of the procedure was essentially that of Reid and Lederer (9). The developer was 1-butanol saturated with aqueous 1.5N ammonium hydroxide. The spray was 0.04% (wt. per vol.) bromocresolpurple in a 1 to 5 (vol. per vol.) dilution of 35 to 40% formalin in ethanol, adjusted finally to pH 5.0. The spots were revealed by intermittent exposure to the vapors above concentrated ammonium hydroxide and, because of their transient nature, were marked as soon as they were established. The usual precautions were taken to maintain a saturated atmosphere in the chromatographic chamber, and the temperature mas maintained a t 25.0" zt 0.1 O c. ACKNOWLEDGMENT
Needed equipment was obtained through grants from the Sigma Xi RESA Research Fund and the William H. Wilson Fund a t The College of TTooster.
LITERATURE CITED
Brown, F., Biochem. J . 47, 598 (1950). Brown, F., Hall, L. P., Nature 166, 66 (1950). Burton, H. S., Ibid., 173, 127 (1954). Hashmi, M. H., Cullis, C. F., Anal. Chim. Acta 14, 336 (1956). Hiscock, E. R., Berridge, N. J., Nature 166,522 (1950). Isherwood, F. h., Hanes, C. S., Biochem. J . 55,824 (1953). Kennedy, E. P., Barker, H. A., ANAL. CHEW23, 1033 (1951). Long, A. G., Quag-le, J. R., Stedman, R. J., J . Chern. SOC.1951, 2197. Reid, R. L., Lederer, M., Biochem. J . 50,60 (1952). Renard, M., Bull. SOC. chim. belges 59,34 (1950). RECEIVEDfor review August 12, 1957. Accepted March 7, 1958. Based on the senior theses of Thomas A. Gustin, Robert L. McGuire, and John T. Sweeney, submitted in partial fulfillment of the requirementa for the BCS Certified B.A. degree and the Inde endent Study program at The College o f Wooster, June 1955, 1956, and 1957, respectively.
Compact Countercurrent Distribution Apparatus SAMUEL RAYMOND' College of Physicians and Surgeons, Columbia University, New York, N. Y.
b Countercurrent distribution apparatus of greater flexibility and compactness is needed, if this valuable analytical tool is to be used to its fullest extent in the average laboratory. The apparatus described includes both automatic drive mechanism and 100tube extraction train on a base 24 inches square. The extraction train includes tubes of special design which permit close mounting in compact racks. These tubes produce significantly less co-current flow than previous designs. The drive mechanism analyzes the necessary motions of the tube into two components, each separately driven b y its individual motor. The mechanism is controlled by electrical switches rather than mechanical devices, so that adjustment of parts of the cycle is rapid. With this apparatus, countercurrent distribution procedures can b e used routinely in the laboratory for separating mixtures quantitatively into their components.
F
optimum use, countercurrent distribution requires automatic apparatus with large numbers of tubes in the extraction train. The apparatus described is more compact than the OR
1 Present address, Graduate Hospital, University of Pennsylvania, Philadelphia, Pa.
1214 *
ANALYTICAL CHEMISTRY
apparatus of Craig and Post @), and has been used successfully in several laboratories. ARRANGEMENT
OF APPARATUS
The apparatus with automatic drive mechanism and 100-tube extraction train of 20-ml. capacity per tube is mounted on a base 24 inches square (Figure 1). The extraction train consists of 10 banks of 10 tubes, each mounted in a rack rotating on a main shaft. The shaft is driven by the automatic drive mechanism housed in a cabinet adjacent to the extraction train on the same base and supporting one end of the shaft. Additional racks of tubes can be installed on the same shaft by extending the base and main shaft; 10 inches of extension are required for each additional rack. Larger tubes can be installed, up to 35 tubes of 100-ml. capacity in the rack shown. Rack Mounting. The rack consists of two end plates of aluminum, 18 inches s q u r e , supporting cross bars a t each end of the banks. At each end, the individual tube is positioned in a notch on the cross bar on one side and separated from the cross bar on the other side by a rubber pad. Appropriate choice of dimensions in relation t o the diameter of the tube permits each cross bar (except the outer ones) to support two banks. The main shaft passes through flanges mounted at the center of each end plate
One flange is equipped with a locking device, which when released permits rotation of the rack independently of the shaft. Extraction Train. The extraction train in the standard 100-tube rack consists of a series of 100 tubes arranged in banks of 10. When seen from the front tube 1 is at the left, followed by tubes 2 to 10 from left t o right in the front bank. Directly behind tube 10 and connected to it by a ground joint is tube 11; tubes 12 to 20 follow from right t o left in the second bank, so that tube 20 is directly behind tube 1. The entire series is arranged in this alternating manner, ending with tube 100 as the left-hand member of the last bank. Tubes 1 and 100, respectively, carry socket and ball joint connections for various accessory tubes, providing operation as a single withdrawal or as recycle procedure. Between banks 5 and 6 there is a space for clearance of the main shaft and for mounting a reservoir bottle when automatic feed operation is required. A short tube bridging this space connects banks 5 and 6 and can be removed to divide the train into two trains of 50 tubes each. I n apparatus of more than one rack, the outlet tube of one rack is connected to the inlet tube of another by a special tube, which runs diagonally to connect the two racks. Extraction Tube. The tubes are a new design, shown in Figure 2. Each tube consists of a mixing cham-
