differences in the density of the spots of the same concentration were noted. Density Values. A 10-minute heat treatment a t 105' C. was chosen because i t gave t h e highest density readings for lactose a n d t h e monoses when 5-, lo-, a n d 15-minute heating periods were tested. A No. 440 filter is used to read the spot densities, because it gives maximum absorption and the highest density readings of a number of filters tested. ACKNOWLEDGMENT
The author extends his sincerest
appreciation to Wesley Bucek for his chromatographic assistance. LITERATURE CITED
(1) Alcock, Margaret, Cannell, J. S., Nature 177, 327 (1956). (2) Aronson. M.. Arch. Biochem. and Biophys. 39,370(1952). (3) Counsell, J. I., Hough, L., Kadman, W. H., Research 4, 143 (1951). (4) Hough, L., Jones, J. K. N., Wadman, W.H., J . Chem. SOC.1950, 1702. (5) McFarren, E. F., Brand, K., Rutkowski, H. R., AXAL. CHEX. 23,1146 (1951). ( 6 ) Opienska-Blaut, H. J., Xadecka-
.,
Borkowska, I., Borkowski, h'ature 169, 798 (1952).
T.,
(7) Pazur, J. H., Science 117, 355 (1953). (8) Porter, W. L., Hoban, Nancy ANAL. CHEM.26, 1846 (1954). (9) Roberts, H. R., McFarren, E. F., J. Dairy Sci. 36, 620 (1953). (10) Trucco, R. E., Verdier, P., Rega, A,, Biochem. et Biophys. Acta 15, 582 (1954). (11) Tu, C. C., Ward, K., Jr., J . Am. Chem. SOC.77, 4938 (1955). (12) Wallenfels, K., Naturwissenschaften 38, 306 (1951). \ - - - - I -
RECEIVED for review December 18, 1956. Accepted April 4, 1957. Division of Agricultural and Food Chemistry, 130th Meeting, ACS, Atlantic City, N. J., September 1956.
Rapid Procedure for Separating C, to C, Volatile Fatty Acids by Horizontal Paper Chromatography at Elevated Temperature HENRY R. ROBERTS and WESLEY BUCEK Research laboratories Division, National Dairy Products Corp., Oakdale, long Island,
b Acetic, propionic, butyric, valeric, and caproic acids are separated as their ethylamine salts in 1 hour on paper chromatograms by using watersaturated butanol at 50" C. A horizontal solvent development using 12 X 17 cm. strips of Whatman No. 1 filter paper separates the acids as round compact spots. The acids appear as stable purple spots on a yellow background when the chromatogram i s dipped into a chlorophenol red indicator solution. Preliminary studies indicate that this rapid procedure could be made quantitative by either the area or maximum spot density method.
D
of a rapid horizontal paper chromatographic procedure for the determination of lactose in lactose hydrolyzates a t a n elevated temperature (8)led to the application of this technique to separation of the C B to Cs volatile fatty acids. Horizontal solvent development a t an elevated temperature not only increases the solvent f l o ~rate, but, of greater importance, i t achieves extremely rapid separation of coniponents a t a great reduction in developing time. The use of rectangular strips of filter paper, rathcr than filter paper disks, in combination with a horizontal solvent flow as first described by Meredith and Sammons (7) permits separation of the components as round compact
spots rather than circular bands. The round spots lend themselves to quantitation by measuring either the color intensity or the area of the spots. Aside from the work of Hough, Jones, and K a d m a n ( 6 ) , Counsell and associates ( S ) , and Alcock and Cannel1 ( I ) , little has been done with paper chromatography a t elevated temperatures because of the difficulty of maintaining adequate equilibrium a t these temperatures in conventional descending and ascending chromatographic apparatus. Horizontal paper chromatography provides a way of resolving this problem. EXPERIMENTAL
EVELOPVEKT
Khatman S o . 1 filter paper, 12 x 17 cm., is used. The origin is 2.5 cni. from one end. d 1.5-cm. tab, folded a t a right angle to the paper, is inserted into the solvent trough. The opposite end of the paper iq serrated by using pinking shears, and :I 1.0-em. tali is folded a t a
FILTER PAPER I
TROUGH
G L A S S ROD
Figure 1. Position of filter paper during horizontal solvent development
N. Y. right angle to the paper to permit easy flow of solvent off the paper strip. The chromatographic chamber and the manner in which the filter paper strip is supported in a horizontal position (Figure 1) have been described (8). The Cp to Cg volatile fatty acids are separated as their ethylamine salts ( 5 ) . Aqueous solutions of the acids are adjusted to p H 8.0, by using 33y0 aqueous ethylamine. The ethylamine salts are applied to the paper in 0.5-pl. volumes by either a Gilmont ultraniicroburet or a platinum loop. The developing solvent, 1-butanol saturated with water, is prepared at 22' C. in a temperature-controlled room. The atmosphere within the chamber is made alkaline by making the water-rich layer 0 . 1 s with respect to ethylamine. This layer is placed in the bottom of the chamber. After spotting, the filter paper strip is placed in the chamber, which is in an oven, and solvent development occurs a t one of two temperatures, 50" or 60" C. The oven provides equal heating of the chamber and eliminates the formation of condensate. The lower fatty acids from acetic to caproic are separated in 1 hour a t 50" or 60" C., compared to 20 hours by the conventional descending technique. Following solvent development, the chromatogram is air-dried for 1 hour and then dipped into a 0.2% solution of chlorophenol red in 95% ethyl alcohol. The ethylamine salts of the acids appear as purple spots against a yellow background. These spots ale very stable and even after a month are still visible. If the Chromatograms are to VOL. 29, NO. 10, OCTOBER 1957
1447
,
be filed for future reference, preservation with a clear plastic spray is recommended. RESULTS AND DISCUSSION
The chromatograms obtained when the normal Czto C6acids are chromatographed a t 50" and 60" C. for 1 hour in water-saturated butanol as the developing solvent are shown in Figures 2 and 3. The ethylamine salts are clearly separated. Chromatographing at 60" C. is not advantageous when a mixture contains only Cs to Cg acids. At 60' C. caproic acid (C,) can be separated from heptanoic (C,), Figure 3, but not in 1 hour at 50" C. Heptanoic acid cannot be separated from caprylic (C,) and pelargonic acid (C,) in this short period. The ethylamine salts of formic and acetic acids are not separated by using water-saturated butanol as the developing solvent, with either a horizontal or a descending solvent flow. Formic acid can be easily separated from acetic acid by chromatographing the acids as their hydroxamate derivatives (4). At the beginning of this study the 0.1N ethylamine was present in the developing solvent, water-saturated butanol, rather than ili the water-rich phase in the bottom of the chamber. The separations were identical, but the amine introduced a n undesirable effect. The entire upper portion of the chromatogram had a purple color comparable to the color of the acid
separation of normal
CZto C, volatile
ACETIC
PROPIONIC
BUTYRIC
VALERIC CAPROIC
Figure 2. Horizontal poper chromatogram showing separation of normal Cz to Cs volatile fatty acids Aher 1 hour in water-iotwated butanol ot 50'
spots. This "shadow," which extended just above the acetic acid spot, waa eliminated when the amine was removed from the developing solvent and placed in the bottom of the chamber
fatty acids
After 1 how in woter-roturoted bulmol a1 60" C.
1448
ANALYTICAL CHEMISTRY
C.
with the water-rich phase. It could also be eliminated by retaining the amine in the developing solvent and replacing the water-rich phase in the bottom of the chamber with mater-
Figure 4. Horizontal paper chromatogram showing separation of CZto Cg volatile fatty acids After 2-hour development a t 50° C. in woler-rafuroted butanol
Rf Values of Ethylamine Salts of Fatty Acids at 50" C. Solvent System Trough butanol-rich Trough butanol-rich, Trough butanol-rich + amine, bottom waterbottom water-rich amine, bottom butanolAcid rich amine rich Formic 0.19 0.20 0.20 A4cetic 0.21 0.23 0.24 Propionic 0.35 0.31 0.35 n-Butyric 0.43 0.45 0.45 n-Valeric 0.57 0.57 0.54 0.70 Caproic 0.69 0.59 0.75 n-Heptanoic 0.76 0.65 0.79 Caprylic 0.83 0.68 Pelargonic 0.81 0.88 0.71 Table 1.
+
saturated butanol. The only difference between the developing solvent and the phase in the bottom of the chamber was t h a t the developing solvent contained 0.1N ethylamine. Although such a system was adequate for separating the C2 to Cs acids, it was difficult to separate caproic (C,) from heptanoic (C;)acid. The elimination of a water-rich phase from the chamber lowered the Rf values of the ethylamine salts. Tl'ith more water present in the system, partitioning was more efficient and separations were improved. Examination of the Rf values of the ethylamine salts of the acids in the three solvent systems studied (Table I) illustrates the desirability of having a water-rich phase present. The desirable resolutions minus the shadow effect were obtained when the amine was placed in the water-rich phase in the bottom of the chamber. The chlorophenol red indicator reagent is recommended because of the stability of the spots and the sharp
+
contrast between the purple spots and the yellow background. It can detect as little as 0.25 y of acid after solvent development. Two other color reagents have proved satisfactory. A solution of 0.2% of bromophenol blue in 95% ethyl alcohol gives bright blue spots against a lighter yellowish blue background. The spots and background of the chromatogram after treatment u-ith this reagent are also stable for a number of weeks. As little as 0.25 y of a n acid can be detected with the bromophenol blue indicator reagent. A third suitable reagent iq 0.1% ninhydrin in chloroform ( 2 ) . After the chromatogram has been dipped in this reagent, heated for 10 minutes a t 60" C., and stored in the dark for 24 hours, the ethylamine salts appear as purple spots against a pale blue background. This reagent is the least sensitive and is discussed here to emphasize the over-all advantages of chlorophenol red indicator. Khile a 60-minute solvent develop-
ment a t 50" or 60" C. satisfactorily separates the C1 to C6 acids for qualitative identification, a 2-hour development a t either temperature separates the acids completely from one another for quantitation (Figure 4). S o quantitative procedure has been developed for using this rapid separation scheme, but preliminary studies indicate t h a t i t could be made quantitative by either the maximum spot density or area method. I n dealing with only the CI t o Ce acids, 50" C. is preferred, because t h e higher temperature (60" C.) offers no advantage in increased separations. When acids higher than caproic (C,) are present, the 60" C. developing temperature gives sharper separation between caproic and heptanoic acids. LITERATURE CITED
( I ) Alcock, Margaret, Cannell, J. S., S u t u r e 177, 327 (1956). (2) Buyske, D. A., Wilder, P., Jr., Hobbe, E. XI AXAL.C H E ~ 29, I . 105 (1957). (3) Counsell, J. ?;., Hough, L., Kadman, W.H., Research 4, 143 (1951). (4) Fink, K., Fink, R. 31,, Proc. Soc. Ezptl. Biol. and J!ed. 70,654 (1949). ( 5 ) Hiscox, E. R., Berrldge, X. J., h'ature 166, 522 (1950). (6) Hough, L., Jones, J. IC. S . ,Wadman, IT. H., J . Chem. SOC.1950, 1702. (7) Meredith, P., Sammons, H. G., Analyst 76, 416 (1952). (8) Roberts, H. R., AAAL.CHEJI.29, 14.1:3 (1957). RECEIVEDfor review March 28, 1957. Accepted June 20, 1857. 3Ieeting-inMiniature, Metropolitan Long Island Subsection, Sew York Section, .4CS, Brooklyn, S. Y., February 15, 1957.
Mobilities of Some Polyols, Sugars, Acids, and Other Compounds Reactions on Paper with the Godin Reagent M. G. LAMBOU Southern Utilization Research Branch, Agricultural Research Service,
b The solvent combination ( 1 -propanol -ethyl acetate-water, 7 to 1 to 2) has been used successfully to determine carbohydrates qualitatively and quantitatively in deionized extracts from raw and dehydrated sweet potatoes. Among a group of a t least seven developing sprays, the Godin reagent was one of the more sensitive. It compared favorably with ammoniacal silver nitrate with the added advantage
U. S.
Department of Agriculture, New Orleans 7 9, l a .
of color differentiation
among the spots and the possibility of detecting minute quantities of materials under ultraviolet light.
T
HE SOLVEKT MIXTURE-1-prOpanOl -ethyl acetate-water, 7 to 1 to 2used by Albon and Gross for the determination of raffinose in raw sugars extracted from beets (1) has been particularly effective in an investigation of
the sugars extracted from raw and dehydrated sweet potatoes ( 3 ) . I n the course of this investigation it was necessary to determine the mobilities of a large number of compounds using this solvent mixture a t a temperature somexhat higher than that reported by Albon and Gross. As mobilities as a function of this solvent combination for the majority of the compounds investigated have not been previously VOL. 29, NO. 10, OCTOBER 1957
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