ANALYTICAL CHEMISTRY
1216 The barrel of G is inserted into C from left to right, and the rest of the components are assembled. When all screws are tightened, G is cemented in place with sealing wax. The syringe is lubricated and sealed with stopcock grease.
spraying with the modified Fleury reagent it is possible to extend the usefulness of that reagent to the detection of other polyole such as adonitol, glycerol, mannitol, erythritol, sorbitol, and dulcitol. These alcohols appear as various shades of gray or The assembled buret is shown in Figure 2. black: Adonitol and erythritol appear first as black spots, followed by glycerol, mannitol, and sorbitol as gray spots. A groove IS cut in the baseboard to receive one of the guides, and the drive mechanism and buret are secured to the board by Dulcitol, also a gray spot, appears last. No background color brass strips and screws. The buret proper is a capillary provided develops with the Fleury reagent and, after p-anisidine phosphate with a side arm and delivery tip. The end of the capillary which is has been sprayed over it, a cream or light buff background apto fit the syringe is slightly flared with a torch and flaring tool, pears. As with p-anisidine phosphate alone, the intensity and the and the hemispherical end of a ‘/Finch brass rod is pressed into the hot flared tube to form a cavity. The cavity is ground, using definition of the spots are enhanced under ultraviolet light. It Carborundum and the brass rod as a tool. The end of the syringe is possible to detect under ultraviolet light an occasional alcohol is ground approximately to fit the cavity, and the glass surfaces spot that is not visible under daylight. are then ground together with Carborundum. Ca illary and Stock reagents for the Fleury test were prepared as outlined syringe are cemented together n i t h sealing wax. $he rubber tube attached to the side arm makes it easy to rinse the buret, (1). However, positive results were obtained repeatedly only so that titrants may readily be changed. (A pinch or screw when the concentrations of the reagents used in the sprays were clamp closes the rubber tube while titrations are in progress.) changed as follows: i , 5 % mercuric nitrate-water, 1 to 1, followed The whole buret assembly is mounted to slide parallel to the by heating for 10 minutes a t 90’ to 100” C.; 10% barium acetatebore of the capillary tube, so that the tip of the buret can be moved over or away from a “titration table” (2). glacial acetic acid, 1 to 10, followed by heating for 10 minutes The capillaries of the burets are 0.6 mm. in cross section; one to develop the inositol spot or 30 minutes to develop other revolution of the screw advances the meniscus 3.5 mm., and the polyols. If polyols other than inositol are known to be present position of the meniscus can easily be controlled as precisely as or suspected, it is advisable, to spray a second time with the i t can be read (0.1 mm., or 0.06 ~ 1 . ) . More delicate control could be attained by using smaller differences of pitch in the differbarium acetate-glacial acetic acid mixture and heat once more ential screw mechanism-Le., if pitches of 20 and 24 threads per for 10 to 30 minutes to bring out the spots. p-Anisidine phosinch were used, the precision of control would be doubled. phate may be sprayed over the paper only after the final spraying with barium acetate. The p-anisidine phosphate is applied aB LITERATURE CITED outlined by Mukherjee and Srivastava (4) (1) Conway, E. J., “Micxodiffusion Analysis and Volumetric Error,” Sensitivity tests were conducted, and observations made under 3rd ed., Van Nostrand, New York, 1951. daylight and ultraviolet light are outlined in Table I, along with (2) Kirk, P. L., “Quantitative Ultramicroanalysis,” Wiley, New the colors observed at each stage in the development of the spots. York, 1950 The unirrigated papers were spotted with an ultramicropipet. Spots were spread over a larger area by superimposing 1 droplet of distilled water delivered from a capillary pipet. The spot8, when developed, covered an area of approximately 1 cm. Multiple Spray Technique for locating Sugars The color of the inositol spot deepened a-ith time, while those and Polyols on the Same Chromatogram of the sugars faded slightly but were still readily observed under M. G. Lambou, Southern Utilization Research Branch, both daylight and ultraviolet light after 7 weeks. A measure of Agricultural Research Service, consistency was observed, in that all the sugars tested containing U. S. Department of Agriculture, New Orleans 19, La. a fructose unit appeared gold-colored under ultraviolet light. Overheating causes the whole strip to turn black. Repeated Y afcLTxPLE spraying it is possible to develop spots of sugars spraying with the Fleury reagents makes the paper brittle and and polyols on the same chromatogram. By spraying first causes i t to disintegrate readily. with modified Fleury reagent ( I ) , followed by p-anisidine phosOther sugar-detecting reagents such as resorcinol (6) and aniline phate (d), the characteristic orange spot for inositol is retained oxalate (S), and the Godin reagent (2) for polyols other than inoas well as the characteristic colors of the sugars reported to desitol were unsuccessful as substitutes for p-anisidine phosphate, velop with the second reagent. By prolonged heating after nor could the Fleury spray be used over these reagents. However, when resorcinol was audied over the ‘Fleury sprays and heated for 5 minutes at 105” C., it mas found to Table I. Color Reactions of 20 Sugars and Polyols with Modified enhance the color of the inositol spot. Fleury Reagent The spot changed from orange to a Sensitivity, brilliant rose-orange, so intense in color Modified Fleury Reagent p-Anisidine Phosphate (4) over Y _____ Heated after Last Spray Fleury Reagent Day- Ultrathat as little as 1 y of inositol could Compound 10 min. 30 min. Deylicht Ultraviolet light violet be detected. Inositol Orange Orange Orange Red The multiple spray technique outlined Adonitol .2a 1; Gray Black Lavender Glycerol ... Gray White‘ ’ ’ Lavender a 10 has been applied satisfactorily to chroMannitol Gray Dull yellow Yellow io 5 Erythritol Gray Black Lavender Purple 10 10 matographically separated sugars and Sorbitol ... Gray Tan Light brown 20 25 polyols extracted from plant materials. Dulcitol ... Gray Tan Yellow 25 20
B
__
Arabinose Fructose Glucose .Mannose Sucrose lMaltose Lactose Melibiose Cellobiose Trehalose R a ffin o 8 e Melezitose Glucosamine
Yeiidw
...
Yeiiow
...
... ... ...
Yeliow YeiiOw
Spots not visible in daylight. 6 Fades rapidly.
Tan Tan Black Pale t a n Brown Pale t a n Pale t a n Pale
la; ...
Tan Pale yellow Yellow
Brown Golden brown Dull yellow Pale t a n Gold Dull yellow Dull yellow Dull yellow Bright yellow Pale pink b Tan Tan Yellow
Brown Gold Dull yellow Yellow Gold Dull yellow Bright yellow Yellow Bright yellow White Gold Gold White
10 10
10 10
10
10 10 5 2: 10
10 10
10 10 10 10 10 10 10 20
:;
20 10 10
LITERATURE CITED
(1) Fleury, P. F., Courtois, J.-E., Malangeau, P.. Bull. SOC. c h i n . bwl. 35,
537 (1953). (2) Godin, P., Nature 174, 134 (1954). (3) Horrocks, R. H., Manning, G. B., Lancet 256, 1042 (1949). (4) Mukherjee, S., Srivastava, H. C., Nature 169, 330 (1952). (6) Rachinskii, V. V., Knyazyatova, E. I., Doklady Akad. Nauk S.S.S.R. 85, 1119 (1952).
