Microscopic Identification of Microgram Quantities of
D-Fructose Direct Synthesis of Crystalline 2,4=Dinitrophenylhydrazone by Solvent Diffusion Technique LAWRENCE M. WHITE
and
GERALDINE E. SECOR
Western Utilization Research Branch, Agricultural Research Service,
A simple test is described for identifying as little as 0.5 y of pure or 10 y of chromatographically separated Dfructose by direct synthesis and microscopic observation of crystalline D-fructose 2,4-dinitrophenylhydrazone dioxane solvate (4). Less sensitive and less rapid tests are also given by L-fucose, D-galactose, D-mannose, and L-arabinose. Other common pentoses, hexoxes, and reducing disaccharides do not give a visible 2,4-dinitrophenylhydrazone under the conditions described. All of the reacting sugars can be reliably distinguished from one another by the characteristic appearance and properties of their 2,4.-dinitrophenylhydrazones.
C
HRONATOGRAPHIC studies can be used t o obtain preliminary information regarding the identity of sugars in plant materials; however, isolation and characterization of the pure sugar or a derivative are required for final proof of the presence of the sugar. Since sugars normally appear on the paper chromatogram only in microgram quantities, a very sensitive test is required for such studies. The recently described reaction of 2,4-dinitrophenylhydrazinewith o-fructose in acidified, moistened dioxane t o form D-fructose 2,4-dinitrophenylhydrazone dioxane solvate ( 4 ) proved to be sufficiently sensitive and rapid to he used for the positive identification of chromatographically separated D-fructose. This paper describes the technique used to identifv both pure o-fructose and n-fructose eluted from paper chromatograms. Certain pentoses and other hexoses also give characteristic, hut less sensitive tests under the conditions described. The characteristic properties of all the 2,4-dinitrophenylhydrazones formed in the test are given to show how D-fructose may he distinguished from other sugars tested. APPARATUS
Stereoscopic microscope, 25 and 100 X, with artificial light source for viewing by reflected and transmitted light. Micro culture slides, 75 X 25 mm., with cylindrical \yell 3 mm. deep by 15-mm. diameter. Microslides, 75 X 25 mm. Syringe, glass, 5 ml. Micropipets, 1- and 2 4 . capacity. Microbeakers, round-bottomed, 5 ml. Petri dishes, 100 X 15 mm. REAGENTS
.
Pure D-fructose, L-fucose, o-mannose, D-galactose. and Larabinose. 2,4-Dinitrophenylhydrazine reagent. Add 5 ml. of ethyl acetate to 0.1 gram of 2,4-dinitrophenylhydraxine, cover, and heat on the steam bath until solution is complete. Allow to cool and use the supernatant solution without filtering Dioxane reagent. 4 d d 0.3 ml of dilute hydrochloric acid (1 volume of hydrochloric acid, specific gravity 1.19. plus 9 volumes of water) to 9.7 ml. of reagent grade 1,4-dioxane. Pyridine, reagent grade. Ethyl ether, anhydrous, reagent grade. Ion exchange resins. A mixture of 2 parts by weight of 35- to GO-mesh activated Duolite A-4 and 1 part by weight of 35- to 60-mesh Amberlite IR 120-H. Wash resins thoroughly with water and air dry before mixing. Paraffin oil, heavy
U. S. Department o f Agriculture,
Albany 10, Calif.
TtIETHOD
Preparation of Diffusion Cell. PURESL-GAR SOLUTIONS. Place an aliquot of a pure sugar solution, covering a minimum area (20 to 50 sq. mm.) in the center of a microslide and allow to air dry a t room temperature. When dry, wet the sugar spot mith 2,4-dinitrophenvlhydrazine-ethyl acetate reagent by making 4 to 5 applications with the tip of a g1a.s rod about 1 mni. in diameter. The amount should be sufficient to leave undisEolved reagent present after diffusion is complete. Wet the ground area around the yell of the culture slide using mineral oil in the glass syringe. Place 1 p l . of dioxane reagent in the well. Immediately invert the test dide over the culture slide with the sugar spot centered over the well, and press firmly in place to make a n airtight seal. As an alternative procedure, cover the dried sugar spot with finely powdered 2,4-dinitrophenylhgdrazine, invert the slide and tap i t sharply to remove any reagent not adhering to the sirup, Remove the reagent outside of the sugar area with a fine camel’shair brush. The remainder of the procedure for preparing the diffusion cell is the same as described. More reliable and uniform results are obtained using the 2,4-dinitrophenylhydrazinein solution, particularly when the amount of sugar is near the limit of sensitivity of the test. CHROMATOGRAPHED SUGARSOLUTIONS. Rinse the chromatogram with anhydrous ethyl ether immediately on removing it from the tank and locate the sugar spot by means of guide strips. Cut out an area approximately 1.75 X 2.5 inches having the sugar spot in the center. Cut off and discard two ‘/z X 7 / g inch triangles from one of the short edges to form a blunt point. Elute the sugar from the excised area by the folloming simple modification of Dent’s procedure ( 1 ) . Make a ‘/*-inch fold on the edge opposite the point and insert i t between the ends of two microslides. Place a 1 X ’14 inch piece of filter paper between the other ends of the slides to ensure capillary feed to the chromatographed strip and put these ends in a Petri dish filled with aater. Cover with a relatively airtight enclosure to provide humid conditions and elute the sugar, allowing only one drop of eluate t o flow from the tip into a 5-ml. round-bottomed beaker. Add 5 mg. of mixed resins and evaporate nearly to dryness a t room temperature under a stream of filtered air. Transfer the sugar residue to the slide with a micropipet, using several small portions of water, not more than 6 p l . in all, to make the transfer and to wash the resin and beaker. Avoid the transfer of the resin particles. Care should be taken to keep the area of the transferred sugar solution to a minimum, 20 to 50 sq. mm., hut water-repellent coatings should not be used because they tend to give erratic and unreliable results. .411ow the sugar solution to air dry a t room temperature and proceed as for pure sugars. Observations during Diffusion Period. Observe the progress of the solvent diffusion a t 25 and 100 X using both reflected and transmitted light. Some sugar 2,4-dinitrophenylhydrazones begin to form almost a t once, others after 1 hour or more. With very small amounts of sugar, the reaction product appears only after several hours or overnight. Much ran be learned regardin the amount and kind of sugar present by noting the time require8 for the formation of the reaction product, its rate and habit of growth, and its general appearance. Frequently the sugar area will not become completely wetted by the dioxane. I n this case, tilt the cell so t h a t the dioxane pool on the test slide will cover the entire area of the sugar spot. Behavior of Reaction Product over Pyridine. After the reaction appears to be complete, open the diffusion cell by lifting off the slide with a razor blade, and allow to air dry. Place 1 pl. of pyridine in the dry well of the culture slide and reassemble t h e cell. Note the rate of solution of the product and the appearance of any new crystalline phase that may be formed. OB S ERV AT1ON S
D-Fructose. o-Fructose 2,4-dinitrophenylhydrazonegrows a s clusters of fine yellow needles having sharp, tapered ends, growing rapidly from a common center (Figure 1, -4).Dendritic growth
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V O L U M E 27. NO. 6, J U N E 1 9 5 5
Figure 1. Photomicrographs (36 X) of sugar 2,Cdinitrophenylhydrazones after synthesis in diffusion cell Photozraphed with reflected and transmitted lishf
is common. The product is D-fruCtOse 2,4-dinitrophenylhydrazone dioxane solvate (8, 4). The lengths of the crystals usually are governed by the siae of the droplet of dioxane in which the cluster is growing. The crystals me relatively insoluble in dioxane hut are very soluble in pyridine and dissolve rapidly. Moderate amounts of pure D-fructose usually start to react within 2 to 4 minutes, and 0.5 y within 1hour. The test for chromatographed D-fructose is positive within 1 hour when 10 y or more of the sugar me applied to the paper. I-Fucose. >Fucose 2,4dinitrophenylhydrazone grows as very long, streight, ION needles with blunt ends (Figure 1, B ) . Growth usually begins after the slide is well wetted and thereaiter is extensive and rapid. The needles are laid down in a random pattern and are usually not confined by the siae of the dioxane pool. The product is >fucose 2,4dinitrophenylhydra.one dioxane solvate (6). At low levels, hair-like dendritic crystals is more soluble form. >Fucose 2,4-dinitrophenylhyd~a~~n~ in dioxane but far more insoluble in pyridine than is o-fructose 2,4-dinitrophenylhydrazone. Moderate amounts oi pure I,fucose start to react after ahout 15 minutes snd chromatographed >fucose aiter ahout 30 to 60 minutes. Pure >fucose gives B positive reaction within 1 hour a t the 5-y level and ehromatographed >fucose at the 25.7 level. D-Mannose. n-Msnnose 2,4dinitrophenylhydraeone usually grows in clusters of short, wide, flat, yellow blades with obtuse ends (Figure 1, C). The product is n-mannose 2,4dinitrophenylhydrazone monohydrate and the angle st the obtuse end is 123" (5). Edge views of the D-mannose 2,4-dinitrophenylhydraeone blades superficially resemble the D-fructose 2,4dinitrophenylhydrmone needles. The obtuse ends on the blade8 are heat observed at lOOX and become more apparent as the crystals age (3 to 18 hours). Growth of the crystals usually begins on a well wetted slide aiter about 60 minutes and once started it is very slow. Ample reagent should he used to provide growth centers for the reaction product. Pure and chro-
1017 matographed n-mannose gives positive tests within 1 hour a t the 25- and 125-y levels, respeotively. When placed over pyridine in the cell, the original crystals dissolve by a "peeling off" process with the marked formation of gas bubbles. When the crystals dissolve ephemeral "fucose 2,4-dinitrophenylhydraeone-like" needles may appear before the slide becomes well wetted with pyridine. A further charactenstio property of D-mannose 2,4-dinitrophenylhydrasone is its behavior on placing the air-dried pyridine solution of the product back over 1 PI. of dioxane reagent in the diffusion cell. Almost immediately after exposure to the dioxane, a new gel-like phase appears which is converted an aging a short time into scattered expanding rosettes with central craters (Figure 1, D ) . These are composed of very fine needles which gradually grow over a, period of several hours on a well wetted slide into the typical D-mennOSe 2,4dinitrophenylhydraaone monohydrate. D-Galactose. A t the 50-7 level, colorless crystals of D-gdaetose tend to form as the dioxane wets the sugar sirup on the slide. n-Galactose yields a characteristic bright yellow, clear gellike 2,4-dinitrophenylhydrmme which first forms as small globular pstehes (Figure 1, E). These may coalesce in time if the amount S The reaction product is very insoluble over of sugar N ~ high. pyridine. Moderate amounts of D-galactose begin to reaet after about 10 minutes. Pure and chromatographed D-galactose give positive tests within 1hour a t the 5- and 50-7 levels, respectively. LArabinose. Colorless crystals of &arabinose may form in the dioxane solution a t the 50-7 level. >Arabinose yields a bright yellow, nondescript gellike hlanket of the 2,4dinitraphenylhydrazone which usually covers the whole field. The test for >arabinose is relatively insensitive, 25 to 50 y of the pure sugar or 125 t o 150 y of the chromatographed sugar being required to give a positive test within 1 hour. When placed over pyridine, the L-arabinose 2,4-dinitrophenylhydraaone gel dissolves rapidly and if a few crystals of solid reagent are present in the field (these may be added while the slide is air drying), yellow, small, very fine hairlike needles with pointed ends best visible a t lOOX are formed within 2 to 4 minutes (Figure 1, F ) . These usually grow from a particle of reagent in the form of sheaves or lobse clusters. These crystals are L-arabinose 2,4-dinitrophenylhydraeane pyridine solvate ( 6 ) .
DISCUSSION
During preliminasy trials with up to 500-7 quantities of D-ribose, ~-xylose, D-lyxose, L-rhamnose, D-glUCOSe, >sorbose, turanose, lactose hydrate, cellobiose, maltose hydrate, and melibiose hydrate, no microscopically visible 2,4-dinitrophenylhydrazone was formed. Each of the sugars which did react in the a h w e test-D-fruCtose, >fucose, n-galactose, D-mannose, and Irarahinosegives a 2,4-dinitrophenylhydrmone having a characteristic appearance or property so that any one sugar alone may be readily distinguished from the other reacting ~ugars, particularly if observations on authentic sugars, with and without 2,4-dinitraphenylhydra~inereagent, are carried on simultaneourrly. Most of the work reported here was done with D-frUCtOBe since it gave the most rapid and sensitive test. If two of the sugars that react with 2,4dinitrophenylbyd~~~ine are present together, the one that predominates is likely to he the dominant crystal form, in fact it may be the only form. The time required for the appearance of the reaction product and the amount of sugar required to give a positive test is also increased when two or more sugars are present. In cases of this kind, ohservations on mixtures of authentic sugars should always be made along with those on the unknown. With chromatograpbed sugars, D-fructose, D-mannose, and >arabinose usually overlap on the chromatogram and interfere in the test for one mother, whereas D-gdsctose and L-fucose run sufficiently behind and ahead of this group not to interfere.
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ANALYTICAL CHEMISTRY
The presence of sugars that do not react with 2,4-dinitrophenylhydrazine and impurities eluted from 4 square inches of Whatman S o . 1 paper that had been irrigated for 48 t o 88 hours in 1-butanol-ethanol-water (10 t o 1 t o 2) ( 3 ) also decrease the sensitivity of the test and increase the time required for the formation of the reaction product. I n very inipure solutions, the test is completely inhibited. The impurities eluted from the Whatman No. 1 paper decreased the sensitivity of the test for D-fructose by 10- to 20-fold. Therefore, the amount of the impurities \$as kept to a minimum by limiting the eluate to one drop and it was purified by treatment with ion exchange resin. These steps lead to ]owes of approximately 15% due to incomplete elution and approximately 50% due t o the resin treatment. Even with these losses, the test is sufficiently sensitive t o be readily useful for identifying D-fructose, L-fucose, and D-galactose. The use of other chromatographic papers, irrigants, or means of decreaqing the impurities probably would decrease these losses and make the teqt more sensitive for chromatographed sugars. I n the study of “unknown” unchromatographed sugai., it is desirable t o avoid the use of resins since they may cause hydroly*is of some oligosaccharides containing o-fructose to give a false positive test. The hydrochloric acid used in the above test also hydrolyzes sucrose and raffinose. I n order t o find conditions permitting the test t o be used for D-fructose in the presetwe of easily hydrolyzable sugars containing D-fructose, a number of solid organic acids and cation exchange resins were used in p l a c ~ of the hydrochloric acid to catalyze the reaction. Of thwe,. oxalic acid appeared to he the most satisfactory, considering sensitivity and time of reaction. When the use of oxalic acid is preferable, the sugar spot may be dwted with a mixture of equal parts by weight of powdered oxalic acid and 2,4-dinitrophenylhydrazine using 2 p1 of dioxane containing 3% mater in the \yell of the diffusion cell. Under these conditions, sucrose and raffinose give negative tests but pure D-fructose is positive at the 5-y level in 1 hour. When using o d i c acid and solid reagent, the field may contain many kinds and shapes of crystal. and carp must be taken not to confuv these with a reaction produc-t of the sugar. E\amination of several lots of commercial and recrystallized 2,4-dinitrophenylhpdrazine revealed a marked difference in the sensitivity of the various samples. For example, one unusually active sample gave a positive test n i t h 0.2 y of pure wfruct0.e
while another was sensitive only at the 5--/ level. This difference in sensitivity could not be ascribed t o differences in methods of recrystallization, particle size, moisture or ash content, ultimate analysis, or impurities detectable by infrared absorption, or hy x-ray powder photographs. However, the solution of any of the samples of 2,4-dinitrophenylhydrazine in ethyl acetate and the recrystallization of the reagent on the slide as described gave almost uniform sensitivity of the various samples and in most cases increased the sensitivity for pure or chromatographed D-fructose by 3- to 10-fold over that of the original powdered reagent applied dry. The use of an ethyl acetate solution of 2,1dinitrophenylhydrazine is also advantageous because it is difficult to get the powdered reagent t o adhere to the sugar ?pot when only a few micrograms of the sugar are present. The syntheses of crystalline L-fucose 2,4-dinitrophenylhydi~azone dioxane solvate, D-mannose 2,4dintrophenylhydrazone monohydrate, and L-arabinose 2,4-dinitjropheny1hydrazone pyridine solvate have not been reported previously. These compounds and crystalline D-galactose 2,4-dinitrophenylhydrazone pyridine solvate, which does not form under t,he conditions of the described tests, can be prepared on a macroscale by methods analogous to those used for the preparation of the D-fructote 2,4-dinitrophenylhydrazonesolvates ( 4 ) . ACKNOWLEDGMENT
The authors wish to thank Francis T. Jones for assistanc*r with the photonlicrographs and interpretation of the microscopical observations, and Kenneth J. Palmer and Glen F. Bailey for the x-ray and infrared examination of the 2,4-dinitrophenylhydrazine. LITER4TURE ClTED
(1) Dent, C. E., Biochem. J . . 41, 240 (1947).
(2) Jones, F. T., Black, D. R., and White. L. AI., in preparation. (3) White. L. AI., and Seroi, G . E., Arch. Riochem. and Riophgs.. 43, 60 (1 952). (4) White, L. AI., and Secor, G. E., J . A m . Chem. Soc., 75, f7343 (1953). ( 5 ) White, L. AI., Jones. F. T.. and Secor, G. E., unpublished results. RECEIVED for review August 20, 1954. Accepted December 4, 1964. T h e mention of a n y products does n o t imply t h a t they are endorsed or recon:mended b y the Department of .4griculture over others of a similar n a t w e not mentioned.
Continuous Determination of Oxygen in Gases J. T. CORCORAN Research Department, Standard
Oil Co. (Indiana), Whiting, lnd.
.in iniproied method has been developed for continuously determining small amounts of oxygen in gases. The apparatus described by Brady has been modified to measure reagent flow rate and to increase precision. For a series of like samples, oxygen content is determined directly from a correlation with optical transmittance.
A
SE.SSITI\7E method for determining small amount? of oxygen in gases has been described by Brad\ ( 1 ) . His method involves reaction between the oxygen in the sample and a n alkaline solution of sodium anthraquinone-@-sulfonate,a reagent that is colorless when oxidized and red when reduretl. The absorbance of the partially oxidized reagent is measured with a colorimeter. Continuou. analysis requires a n apparatuq for
hringing a fixed volume of gas in contact with a fixed volume of the reduced reagent per unit of time Biady’. method which covered the desired oxygen ranges was npplied t o the determination of traces of oxygen in ethylene. However. reference concentrations of oxygen generated in an electrolytic cell gave erratic colorimeter readings from day to day. Because the gas rate and oyygen concentration were knov n to be constant, the colorimeter fluctuations were attributed to changes in the reagent f l o ~rate through the equipment. T o allow satisfactory operation, modifications in the apparatus \I W P made. A4measuring device as added to the system, as shown in Figure 1, t o permit easy checking of flow rate.
It consisted of a graduated overflow tube connected to the g ~ , qeparator, D. Two stopcocks n’ere inqerted, one in the liquid lire
