ANALYTICAL CHEMISTRY
1998 hydroxide were precipitated with sodium bicarbonate while the reduced iron remained in solution and was discarded in the filtrate. The bromate oxidation method is somewhat mqre difficult than the usual persulfate oxidation. However, it was selected after repeated trials using persulfate gave erratic results for vanadium. Results are given in Table 11. LITERATURE CITED
(1) Cain, J. R., J . I v d . Eng. Chem., 3,476-81 (1911); 4, li-19 (1912).
(2) Englis, D. T., and Wollerman. Louis, .%BAL. CHEM.,24, 1983 (1952). (3) Goddu, R. F., and Hume. D. K.,Ibid.. 22, 1313-17 (1950). (4) Kolthoff, I. M.. and Sandell. E. B., IND.ENG.CHEM.,ANAL.ED., 2, 140-5 (1930). (5) Miles, J. W., “Applications of Vltraviolet Spectrophotometry,” Ph.D. thesis, University of Illinois, 1953. (6) Willard, H. H., “Laboratory Alanual for the Course in Advanced Quantitative dnalysis.” 5th ed,, Edwards Brothers, Ann Arbor, llich., 1920. RECEIVED for review July 9 1 9 3 5 . Accepted September 14 1955
Microscopic Identification of Microgram Quantities of 1-Arabinose and 1-Fucose Direct Synthesis of Crystalline 2,4-Dinitrophenylhydrazone and 1,l-Diphenylhydrazone Derivatives by Solvent Diffusion Technique GERALDINE E. SECOR and LAWRENCE M. WHITE Western Utilization and Research Branch, Agricultural Research Service,
The previously described solvent diffusion technique for the identification of D-fructose has been extended to the identification of L-arabinose using 2,4-dinitrophenylhydrazine and pyridine, and L-arabinose and L-fucose using 1,l-diphenylhydrazine and 70% ethyl alcohol. The sugar hydrazones are formed by direct synthesis at room temperature and are observed and identified microscopically. As little as 1 to 10 y of the pure or 5 to 15 y of the chromatographically separated pentose gives the test.
A
TEST has been described for identifying microgram
amounts of some pure and chromatographically separated pentoses and hexoses, particularly n-fructose, by direct synthesis and microscopic observation of their 2,Pdinitrophenylhydrazones ( 8 ) . The test given by L-arabinose under these conditions was so insensitive that it was considered as an interference in the test for D-fructose. This paper reports an improvement in the sensitivity and specificity of the 2,4dinitrophenylhydrazine test for L-arabinose and describes a similar test for identifying microgram amounts of pure and chromatographically separated L-arabinose and L-fucose as their crystalline 1,l-diphenylhydrazones. Information obtained by the combined use of these tests and the test previously published ( 3 ) can be a valuable aid in confirming the identity of an “unknown” sugar.
U. 5.
Department o f Agriculture, Albany 70, Calif.
n-Butyl alcohol-ethyl alcohol-water (10: 1: 2 ) . Duolite A-4 and Amberlite I R 120-H resins (3). METHODS AND OBSERVATIONS
Using the reagents indicated below for the various tests, prepare and assemble the diffusion cell, and observe the progress of the diffusion as previously described (3). Compare the appearance of any reaction product with that of products formed under the same conditions by authentic sugars, with blanks prepared with the hydrazine reagents, and with the sugars only on the slide. ZI4-Dinitrophenylhydrazine-Pyridine Test for cArabinose. Wet the dried sugar spot with four or five applications of the 2,4dinitrophenylhydrazine-ethyl acetate reagent, using a small glass rod. Apply sufficient reagent to leave some undissolved after diffusion is complete. Place 0.5 to 0.6 pl. of pyridine in the well of the culture slide and immediately assemble and seal the cell. 1,l-Diphenylhydrazine-Ethyl Alcohol Test for L-Arabinose and cFucose. Cover the dried sugar spot with an excess of the 1,ldiphenylhydrazine-sodium acetate reagent. Invert the slide and tap it to remove reagent not adhering to the sirup. Remove reagent remaining outside the sugar area with a fine camel’s-hair brush. Place 1 pl. of 70% ethyl alcohol in the well of the culture slide. Assemble and seal the cell immediately. Table I gives a brief description of the prodOBSERVATIOVS. ucts formed in the various tests, the time required for the reaction products to appear, and the approximate amount of sugar required to give a visible test. DISCUSSION
APPARATUS AND REAGENTS
Apparatus as previously listed (3). hficropipet, approximately 0.5-pl. capacity. Usual equipment for descending paper chromatography. Equipment for eluting sugars from chromatograms ( 3 ) . Pure L-arabinose, L-fucose, D-ribose, and D-galactose. 2,4-Dinitrophenylhydrazine-ethylacetate reagent ( 3 ) . Pyridine, reagent grade. I, 1-Diphenylhydrazine reagent. Add 0.25 millimole (21 mg. ) of powdered anhydrous sodium acetate, reagent grade, to 0.25 millimole (55 mg.) of finely ground 1,1-diphenylhydrazine hydrochloride in a glass weighing bottle. Mix by gently rotating the container for a few minutes and keep in a desiccator when not in use. The reagent gradually becomes sticky, owing to absorption of moisture and liberation of the free base. I t should be replaced with a fresh mixture when it becomes sticky. Ethyl alcohol, 70%. Mineral oil, heavy. Filter paper, Whatman KO.1 sheets. Ethyl ether, anhydrous, reagent grade.
Z14-Dinitrophenylhydrazine-Pyridine Test for L-Arabinose. Cnder the conditions described for this test only three of the sugars listed in Table I-L-arabinose, L-fucose, and D-galactosegive a visible reaction product. L-Arabinose is readily separated from the other reacting sugars by chromatography with n-butyl alcohol-ethyl alcohol-water (10:1:2), so the test on an eluate from a chromatogram is specific for L-arabinose. The present test using pyridine is about ten times as sensitive for L-arabinose as was the previous one (3) using acidified moistened dioxane, and the product is characteristically crystalline instead of gellike. However, the reaction product appears only after 16 to 24 hours instead of in a few minutes to 3 hours as in the previous test. The use of pyridine as the solvent also eliminates the possibility of acid hydrolysis of oligosaccharides, although this may occur in certain cases as a result of the resin treatment. L-Fucose and D-galactose do not give satisfactory tests with 2,Pdinitro-
V O L U M E 2 7 , NO. 12, D E C E M B E R 1 9 5 5 Table I.
Appearance of Reaction Products, Reaction Time, and Sensitivity of Sugars to Tests
2,4-Dinitrophenylhydrazine-Pyridine Sugar"
Usual appearance of product
~ - B r a b i n o s e Fine, trichitic, yellow needles in clusters or sheaves [Fig. 1. F (3) i L-Fucose Clusters of yellow, flat, narrow blades with ragged terminations D-Ribose ............ D-Galactose
O
1999
Yellow, clear, nondescript gel. may become crystalline on aging or with large amounts
Time, hr.
Test Sensitivity, Y ChromatoPure graphedb
16-24
5
15-25
16-24
50
...
16-24
C
...
16-24
25
...
1.1-DiphenylhydrazineEthyl Alcohol Test Sensitivity, Y Usual appearance Time, Chromatoof product hr. Pure graphed b Small, white, dense clusters of radiating filaments-"moldlike" growth Segmented, dense, white clusters of fine needles Loose clusters of blunt-ended, colorless bars or may he plumelike
............
One hundred micrograms of each of t h e following sugar8 gave no visible reaction product after 16 hours with either test:
0 02- 2
1-3
5-10
0.08- 2 . 5
10
15-25
-
75
...
e
...
0.1
2
16-24
D-xylose.' D-xylose, L-rhamnose.-
H20, D-glucose, D-mannose. D-fructose, a n d L-sorbose. b e
Amount of sugar applied t o paper. KO visible reaction product with 100 y of pure sugar.
phenylhydrazine when pyridine is the diffusing solvent. L Fucose gives a crystalline product, but relatively large amounts of the sugar are required to give a test. D-Galactose reacts at moderate levels, but the product is usually an uncharacteristic gel. After 24 hours, or when a large amount of D-galactose is present, the field may show a mixture of the gel, sugar crystals, and well-defined clusters of fine, long yellow needles. These two sugars will react with 2,4-dinitrophenylhydrazineto give a visible product more rapidly, at much lower levels, and form more characteristic derivatives if dioxane reagent (3) rather than pyridine is the diffusing solvent. The test for barbinose may be inhibited if other sugars are present, the extent depending on the sugar and relative amount. D-Fructose, D-glucose, >sorbose, and D-xylose are only slightly inhibitory when present in an amount equal to or less than that of L-arabinose, whereas n-galactose and D-mannose are markedly inhibitory. D-Fructose and n-mannose would be the only sugars present with L-arabinose after chromatographic separation with the n-butyl alcohol-ethyl alcohol-water solvent. The advantage of the 2,bdinitrophenylhydrazine-pyridine test over the 1,l-diphenylhydraeine-ethylalcohol test for L-arabinose is that the crystalline arabinose 2,4dinitrophenylhydrazone formed is much more characteristic in appearance than is the arabinose 1,l-diphenylhydrazone. The disadvantages are that it is only approximately one third as sensitive and that it takes about 16 hours longer to obtain a positive test. 1,l-Diphenylhydrazine-Ethyl Alcohol Test for L-Arabinose and L-Fucose. 1,l-Diphenylhydrazine has been known for many years as a sensitive reagent for the identification and separation of barabinose (1, 8 ) and L-fucose ( 1 ) from other sugars as their 1,l-diphenylhydrazones. The insolubility of Larabinose and L-fucose 1,l-diphenylhydrazones and the slight influence of impurities on their crystallization make these derivatives well suited t o the reaction conditions of the solvent diffusion test. Under the conditions described for this test only three of the sugars listed in Table I-Larabinose, L-fucose, and D-ribose-give a visible reaction product, although other sugars are known to react with this reagent under other conditions ( 1 ) . The tarabinose and L-fucose 1,l-diphenylhydrazone crystals are similar in appearance and can be distinguished from one another only by careful comparison with tests made using authentic sugars. &Ribose 1,I-diphenylhydrazone crystals are sufficiently characteristic in appearance to be easily dzerentiated from those of L-arabinose or bfucose 1,1-diphenylhydrazone. Table I shows that the times for the appearance of the reaction products of the three sugars are short and essentially the same. The sensitivity of this test for Larabinose and L-fucose is satisfactory; however, it is so poor for D-ribose that the test is of little value for this sugar, particularly in eluates from chromatograms.
The 1,l-diphenylhydrazine-ethyl alcohol test is specific for barabinose on eluates from chromatogram irrigated with n-but11 alcohol-ethyl alcohol-water (10: 1:2), since L-fucose and D-ribose move much faster than does barabinose. L-Fucose and D-ribose move together in this solvent, so interference by D-ribose is encountered in the test for L-fucose. The extent of the interference depends on the relative amounts of the sugars. In general, the sensitivity of the L-fucose test is decreased about threefold rind the time required for the product to appear is increased by large amounts of D-ribose. Sugars that do not give a visible product with 1,l-diphenylhydrazine may prevent, delay the appearance of, or diminish the amount of the L-arabinose or >fucose 1,l-diphenylhydrazone. The extent of the inhibition depends on the sugar and its relative amount. The sugars giving visible products with 1,l-diphenylhydrazine react over a wide range of ethyl alcohol-water proportions. LArabinose and bfucose gave the best tests a t about 70% ethyl alcohol concentration, while the D-ribose gave the most sensitive test at 25 to 50% ethyl alcohol. The 70% ethyl alcohol concentration was chosen t.0 eliminate interference from D-ribose as far as possible and t,o avoid atypical forms of L-arabinose and Lfucose 1,l-diphenylhydrazones. The 1,l-diphenylhydrazine hydrochloride, Eastman Kodak No. 954 White Label, was used without further purification. Tests made with a purified crystalline preparation ( 4 ) showed less darkening and deterioration on long standing than did those made with the unpurified reagent ; however, the purified reagent has the disadvantage that its crystal form superficially resembles that of D-ribOPe 1,l-diphenylhydrazone and it might be confused with this product. Other hydrazine derivatives and solvents are being investigated for their utility in identifying microgram quantities of pentoses, hexoses, and other compounds containing carbonyl groups by the solvent diffusion technique. ACKNOWLEDGMENT
The authors thank Francis T. Jones for his helpful interpretations of the morphology of the hydrazone derivatives. LITERATURE CITED
(1) H a a r , A. W.van der, "dnleitung Bum Xachweis, aur Trennung und Bestimniung der Monosaccharide und Aldehydsauren," pp. 131, 178-81, 234, Gebruder Borntraeger, Berlin, 1920. (2) Keuberg, C.. and Wohlgemuth, J., Z. physiol. Chem., 35, 31 (1902).
(3) White. L. SI.,and Secor, G . E., AxaL. CHEW., 27, 1016 (1956). (4) Wise. L. E., and Peterson, F. C., Ind. Eng. Chem., 22, 362 (1930). RECEIVED for review July 18, 1955. Accepted August 24, 195.5. T h e mention of products does not imply t h a t they are endorsed or recommended by t h e Department of Agriculture over others of a similar nature not mentioned.
