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 butanolrich amine rich 0.19 0.20 0.20 0.21 0.23 0.24 0.31 0.35 0.35 0.43 0.45 0.45 0.57 0.57 0.54 0.69 0.70 0.59 0.76 0.75 0.65 0.83 0.79 0.68 0.88 0.81 0.71
Table 1.
+
Acid Formic A4cetic Propionic n-Butyric n-Valeric Caproic n-Heptanoic Caprylic Pelargonic
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
1449
Table I.
Mobilities of Several Groups of Compounds after Irrigation, Reactions with the Godin Reagent, and Fluorescing Reactions
Compound Inositol Dulcitol Sorbitol Mannitol Xylitol Adonitol Arabitol Polyglycerol Glycerol Erythritol Cellobiose Maltose Lactose Trehalose Sucrose Sorbose Glucose Fructose Ribose Xylose Mannose Fucose Arabinose Rhamnosed Diacetone fructose Raffinose
R/ Value 0.09 0.35 0.36 0.36 0 .38b 0.42 0.47 0.51
0.61
...
0.17 0.17 0.19 0.22 0.23
0.30 0.31 0.37 0.39
0.40 0.42 0.42
0.44 0.47 0.84
Fluorescence Reaction with Godin Reagent Before After Daylight" Ultraviolet" irrigation irrigation ... w Xone None None None dL P Pk None None L Kone Sone ... dL
...
None
B1
...
...
bL
None
None
pW
r\;one
G 1'Gr G
d spot" Y - T
None
...
rione None
... ...
... ...
G -'Gr Y 1 Gr PI-
d spotC sty-F st Cr - F W pW - F
... PY
CG
...
Gr with Pk rim Gr with Pk rim G - Gr PY G-Y dG
0.34
...
0.44
P Pk Cr
...
Melibiose
...
Melezitose Galactose Lyxose Turanose Glucosamine n-Acet ylglucosamine cu-?rIethyl-n-mannoside a-5Iethj-1-D-glucoside p-illethyl-n-xyloside Ascorbic acid Galacturonic acid i\lethvl-D-galacturoide Chlorogenic acid Methyl ester of methylD-galactosiduronic acid Malic acid Citric acid Caffeic acid p-Coumaric acid Acetyl-p-coumaric acid
... ... ...
0.52 0.53 0.60 ... 0.12
YY
...
Gr
0.40
Pks
0.45
...
0.57
B1
F
w
vir
w
Gre
,..
...
...
... ...
...
Sone
...
...
T-F T d apotc
...
, . .
... ...
... ...
PF
...
...
d spotC st W - F YtY-F Blk B1 - F Pk - L sty-F pK -F
... ...
, . .
...
... ...
w
Y Y 1BI-F
L
Pk then Ble Pk then B1
...
lTone iY
0,641
0.63 0.67 0.77 0.77
...
Sone ?;one B1
Pk i,ith B1 center B1 - Gr B1 - Gr
kdde
st B1 - F L
R
... None I
.
.
h
h
, . .
B1
...
None None
Kone None
None IF-
None
... ...
...
Kone None Sone
Sone Kone Sone
...
... ...
...
... ...
...
w
...
...
p-Methoxycinnamic acid 0 83 Pke B1 - F b B1 b B1 Syringic acid Streaked L None Pke None h-one G None Oxalic acid Streaked Trimethoxybenzoic acid ., Sone None Pk Phloroglucinol 0 74 Brick - Re ... ... dR Phlorizin Pk - P6 0 75 ... ... b Pko L Pyrocatechol 0 81 ... ... Veratraldehyde 0 84' Sone Sone Chalcone 0-Y 0 ... ... Dihydroxyacetoned 0.55 IT - F ... ... Symbols used. Br = brown Cr = cream L = lavender R = red Blk = black F = fluorescence 0 = orange T = tan B1 = blue G = green P = purple W = white C = colorless Gr = gray Pk = pink Y = yellow h = bright 1 = light s t = strong d = dark p = pale * Single determination. Dark spots under ultraviolet occurred whenever molecule contained a fructose residue. Concentrations as low as 10 y easily visible under ultraviolet. e Fades rapidly. f Did not react with Godin reagent. Spot was developed with ammoniacal AgXOa. 0 Spot spreads over area of approximately 9 sq. em * ?*layhave contained fluorescing impurities. I
1450
.
.
ANALYTICAL CHEMISTRY
reported, they are outlined here for reference purposes. I n addition, use of the Godin reagent, prepared by adding 1 volume of 1% vanillin in ethanol to 1 volume of 3% aqueous perchloric acid just before use (Z), has been extended for the detection of numerous compounds not previously reported as reacting and foi the compounds reported in the paper For qualitative examination of the common mono- and disaccharides, glucose. fructose, maltose, sucrose, and inositol, this solvent combination was particularly effective in bringing about separation in 16 to 21 hours a t 23.5" C. with Schleicher & Schuell 602 ED paper in a Chromatocab on large qheets or in a 12-inch jar on strips. Mobilities for the rarious classes of compounds investigated are listed in Table I. Each value represents the average of a t least three determinations unless othernise stated. The position. of the spots Tvere detected with the Godin reagent ( 8 ) and checked with amnioniacal silver nitrate on separate chromatograms. The mobilities are the result. of irrigating with the solvent mixture for 21 hours a t 25.5" C. The distance of travel for each spot \vas measured from the starting line to the centei of the spot on its long axis. This solvent mixture n orked n ell as the initial irrigant in tIvo-dimensional chromatography n-here it n as follon ed by nater-saturated phenol as the second irrigant. It was equally effective for quantitative determinations of the simpler sugars but required four repeated irrigations with fresh solvent interspersed with drying periods of 1 to 2 hours between each irrigation for the complete separation of glucme from fructose. The ultraviolet lamp n a s an i d i + pensable tool during these inr-estigations and was used to examine yiots before and after irrigation aq me11 RS after development 1%ith the Godin reagent ( 8 ) (Table I). Strips and sheets were spotted m-ith a Gilmont ultramicroburet, the spots being approximately 2 to 4 mm. in diameter and 3 cm. apart. Each microliter of solution contained approximately 100 y of the compound being tested. All spots n ere air-dried rrithout heat prior to irrigation. After spraying n i t h the Godin reagent, all chromatograms were heated for 10 minutes a t 85" C. S o background colors developed. The r h o matogranis could be reheated to bring back those colors that faded rapidly. Overheating caused the paper to become brittle and background cdors to appear nonuniformly. Such compounds as inositol, dihydroxyacetone, glucose, galactose, mannose, arabinose, xylose, fucose, and others reported by Godin ( 8 ) not to react could he seen readily under ultraviolet light. Xan-
nciae, :ir:iljiiiose, and xylose were dificultly visible under daylight. These coiiipouncls may have been present' in conc>eiitrations higher than those used by Godin. giving rise t o a n apparent dis:igreement wit,h Godin's report' (6). To determine the quality of the reaction :it lower concentrations, some of the foregoing compounds, along with others t h t seemed bo give rather pale reactions, were examined a t lower concentrations (Table 11). These were unirrigated spots placed on the paper with the ultr:iiiiicrol,uret. The small spots 11-ere enlargetl to an area of approximately 1 sq. cni. 13-: superimposing a droplet of tlistilled water and alloir-ing the chromatogram to dry thoroughly before q)r:i>-ing. All the spots could be seen untler ultrxriolet light a t a concentration (Jf 25 y and two compounds could be picked out a t a concentration as Ion- :is 10 y. Color reactions observed untler daylight for dihydroxyacetone and fi1co.e may be due to the presence of impurities.
Table
II.
Effect of Decreasing Concentration"
Concentration 100 y Day. UV
Compound Diacetone fructose G Br Dihydroxyacetone Pk-T Y-F Fucose T IT-F Rhamnose T IT-F W-F Ribose T Sorbose XJ lose IT-F a-Methyl-omannoside T IT-F a Symbol. same as for Table I.
pz
50 Day. LV 1G Y-F Pk T-F pT W-F pT W-F
pz
The Godin reagent - could be used to develop spots of carbohydrates suecessfully after chromatograms had been exposed to ninhydrin or iodine crystals, or both. However, the color of the suot was not alviavs identical with that developed on a chroniatogram not previously sprayed with ninhydrin or exposed to iodine. I
TV-F T IT-F
Day.
UV
pT
T-F
25 y
10
UV T-F
Day. T-G Pk
Y-F W-F IT-F IT-F T K-F
G
IT-F
"/
IT-F
K-F
LITERATURE CITED
( 1 ) Albon, N., Gross, D., L 4 7 W s t 77, 410 (1952). ( 2 ) Godin, P., S u t u r e 174, 134 (1954). (3) Lambou, 11. G,, unpublished manuscriut. RECEIVED for review Februarv 23. 1957. Accepted RIay 6, 1957. The "mention of
trade names does not imply their endorse-
ment by the
L
)
of Agriculture ~
over similar products not mentioned.
Separation and Quantitative Determination of Methyl Mannosides in an AutomaticaIIy Controlled Cel Iulose Partition Column DWIGHT
F. MOWERY,
Jr.]
Ripon College, Ripon, Wis.
bA
reliable quantitative analytical method for nonreducing carbohydrates utilizes an automatically controlled cellulose powder column and the nonspecific anthrone color reaction. The method has been tested b y analysis of mixtures of known composition containing methyl oc-D-mannopyranoside, methyl @-D-mannopyranoside, methyl a-E-mannofuranoside, methyl /3-Dmannofuranoside, and D-mannose. In most cases the percentage of a component found i s within 2 units of that actually present over the range from 1 t o 5770, even though the components present in small quantity are immediately preceded and followed in the column b y relatively heavy bands of material.
A
many successful methods h a w been developed for the paper chroniatographic separation and identiL~HOCGH
Presmt address, X e x Bedford Institute of Technology, K e a Bedford, Mass.
fication of nonreducing carbohydrates (3, 4,6, 9, 1 1 ) , the quantitative determination of these substances has proved more difficult. Direct densitometric measurements on paper chromatograms have been advocated b y some chromatographers (11, I S ) , but nonreducing, unlike reducing sugars, suffer from lack of good nonspecific spray reagents. This means that for reasonable accuracy known standards bracketing the quantity of each unknown present must be chromatographed simultaneously with the unknon-n mixture. K i t h mixtures of five unknowns, IT hen percentages may vary over nide limitq. the labor of standard preparation, spotting, and adjustment to the correct range for each unknown can become considerable. Methods requiring spot elution and microanalysis ( 1 , 7 , 11, 12, l 7 ) , although allowing greater freedom in choice of analytical reagents and eliminating the need for simultaneous processing of known standards. are generally laborious and require considerable care for
good results. A starch column was utilized by Gardell (8) for the separation of pentoses. niethylpentoses, and aldohexoses. The column effluent n a s collected in small fractions, the sugar in each fraction was determined colmimetrically, and the quantity of rach sugar in the original mivture n a s calculated by a process of summation. The main disadvantnge of this nietliod n a s the necesqity of packing a fresh starch column for each dctrrminf'1 t'ion. The present article describes a cellulose column n-hicli has been used for an indefinite number of separation; of mannose and methyl mannosidcs by the flowing technique and anthrone analysis of each automatically collected fraction. The chromatographic column is shown in Figure 1. Percentages of individual methyl niannosides can msily be determined from the relati1-c areas under the peaks in a n absorbance plot. Anthrone in 9570 sulfuric acid was selected as the best nonspecific analytical reagent for hexoses and methyl hexoVOL. 2 9 , NO. 10, OCTOBER 1957
1451
~
