of silver. Under these circumstances, the accuracy and precision of the thioacetamide method are approximately
+=0.37,. In the case of the fixing baths (Table
111), the precision of the digestion method was not entirelj- satisfactory, and a comparison between the two methods to establish the accuracy of the thioacetamide method is not useful. The precision of the thioacetaniide method is approximately +1%. The samples analyzed are representative of the diffcrent types of fixing baths in common usage, INTERFERING ELEMENTS
Any metal which forms an insoluble sulfide potentially may interfere in this method. Flaschka (5) has shown that E D T A prevents the precipitation of the sulfides of a number of metals, including copper, cadmium, zinc, cobalt, and nickel. Studies made in these laboratories have shown that, in the presence of EDTA, none of these metals (as well as lead and ferric iron) interferes in the titration of silver with thioacetamide. Among the common metals, only mercury and ferrous iron interfere in the presence of EDTA. Mercury forms a n insoluble sulfide and ferrous iron reduces silver ion to silver metal. The use of E D T A is recommended whenever metal contaminants may be present, and these data were obtained with E D T A present in the solutions being titrated. None of the common anions, including the halides, interferes. Although cyanide does not prevent the formation of silver sulfide, it seriously reduces the
initial silver ion concentration in the solution and, therefore, markedly diminishes the size of the potential break at the end point. Accordingly, only small amounts of cyanide can be tolerated. Oxidizing agents are variable in their behavior and may cause difficulty. Permanganate and chromate are reduced by alkaline thiosulfate to manganese dioxide and chromic hydroxide, respectively. and do not interfere. Free iodine is also reduced, but the oxidation products have a deleterious effect on the end point. Ferricyanide is reduced slowly, and in these experiments, its presence led to low results for silver. APPLICATION OF METHOD TO OTHER METALS
Thioacetamide can be used for the direct titration of mercuric ions in solution. I n this case, the thiosulfate is omitted and E D T A is used to prevent the precipitation of mercuric oxide from the alkaline qolution. The silver sulfide electrode conies to equilibrium rapidly with this solution and no waiting period is necessary between additions of the reagent. Figure 3 shows a typical titration curve for mercury. The curve for the titration of silver in thiosulfateEDTA mixture is included for comparison. I t is apparent that the mercury curve shows a substantially larger potential break a t the end point. Curve C represents the titration of a mixture of silver and mercury in thiosulfate-EDTA mixture and the end point corresponds to the sum of the tn-o titers. There is no indication of an intermediate end point which would differentiate between these ions in a mixture.
It appears probable that the thioacetamide method might be extended to the potentiometric titration of a number of the heavy metals. The problem in this connection \Todd be the discovery of suitable indicator electrodes. Copper, in ammoniacal solutions, has been successfully titrated using a mercuryplated platinum electrode described b y Siggia, Eichlin, and Rheinhart (10). It has been the experience in these Laboratories that this electrode requires frequent regeneration. LITERATURE CITED
(1) Barber, H. H., Grzeskowiak, Edward,
ANAL.CHEM.21,192 (1949). (2) Barber, H. H., Taylor, T. I., “Semimicro Qualitative Analysis,” rev. ed., Harper, New York, 1953. (3) ~, Bowersox. D. F.. Swift. E. H., ANAL. CHEY.30. f289 (1958). ’ (4) Butler, ’E. A.; Peters, D. G , Swift, E. H., Zbid., 30, 1379 (1958). (5) Flaschka, H., 2. anal. Chem. 137, 107 (1952); Chemist Analyst 44, 2 (1955). (6) Iwanof. F. W.. Chem. Zentr. 106, 883 ’(1935 (1935 11): ((7) 7,) Kolthoff. Kolthoff, M., Furman. Furman, T. H.. H., ~~~~~-~~ I. M.. “Potentiometric Titrations,” “Potentiomet,ric Titrations,”’ 2nd ed.; ed., p. 171, Wiley, New York 1949 1949. (8) Peters, D. G., Swift, h. H ., Talanta 1, 30 (1958). 19) Sebborn. W.. Kodak Harrow Research Laboratories, ‘personal communication. (IO) Siggia, Sidney, Eichlin, D. W., Rheinhart, R. C., Ax.4~.CHEM27, 1745 (1955). (11) Swift, E. H., Butler, E. -I., Ibid., 28, 146 (1956). \
RECEIVED for review Sovember 3, 1958. Accepted April 20, 1959. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 1958. Communication No. 1986 from Kodak Research Laboratories.
Paper Chromatography of Sugar Phenylosazones as Their Borate Complexes BARBARiN ARREGUjN lnstituto de Quimica de la Universidad Nacional Aut6noma de MGxico, Ciudad Universitario, M6xico 20,
b Few papers have appeared on the chromatography of sugar osazones. In this work a paper chromatographic method was developed which is suitable for the separation of the osazones as complexes with potassium borate. The migration rates of these complexes are different, and mixtures of them separate well on paper.
S
the discovery by Fischer (3) that sugars react with phenylhydrazine \vith the formation of osaINCE
zones, this reaction has been frequently used for the identification of sugars. Recently these derivatives of monosaccharides have been successfully chromatographed using circular chromatography (1). Likejvise, in a n unpublished report ( 7 ) ,the separation by paper chromatography of four osazones was achieved by using filter papcr impregnated with formamide. The reaction of boric acid with glycols and polyhydroxy compounds requires a cis-orientation of the hydroxyl groups. or that they be placed in a semirigid system which prevents the rotation of
D. F.
the hydroxyl groups to a lesb favorable trans-orientation. The behavior of boric acid with polyhydroxy compounds has been reviewed ( 2 ) and the borate complexes of sugars have been separated by paper partition chromatography using boric acid ( 8 ) . I n this connection sugars or sugar derivatives which in themselves have few structural differences, show different complexing abilities towards borate ions, which in turn is reflected in their properties. resulting in different mobilities in chromatographic studies. I n other Tvork with sugars and plienoVOL. 31, NO. 8, AUGUST 1959
1371
Table
I.
Paper Chromatography of Osazones of Sugars
Osazoiies of
.kral>inose Xylose Glucose Fructose Rhamnose G:Llactose Sorbose Cellobiose Nelil)ioee i\la1tosr Lactose Dioacin sugars" Glycosideb from BrickeIia p e n -
.
75codioxane, water, 1,2dichloroethylen e 54/40/6 3 hr. 17" C.
Rf
Rf
0.65-0,65 0.28-0.30 0.07-0,08
0.92-0.93 0.75-0.77 0.30-0.34 0.31-0.33 0.82-0.83 0.54-0.55 0.37-0 40
0.08
0.36-0 37 0.12-0 13 0.09-0 09 0.02-0 05 0.05-0.06 0.04-0.05
0.05-0.05
0.01
0.04
0.01
0.06-0.09
0.39
dida a
7594 dioxane, hexane, water 60/60/30 6 hr. 21OC. RZ 1.80-1.90 1.00-1.00 0.40-0.41 0.40-0.42 1.20-1.28 0.53-0.55 0.43-0.45 O.lP0.15 0.06-0.07 0.04-0.08 0.31-0.34 0.40 1.25
Solvents, V./V. 757, dioxane, hexane, water, benzene 55/50/25/20 3 hr. 21" C.
Glucose and rhamnose ( I O ) . Glucose ( 4 ) .
lic coiiipounds ( l a ) , good resolution was obtained with borate buffer Ribosides have been separated free froin deoxyribosides, purine, and pyrimidine bases by paper chromatography (9) using butyl alcohol-borate as the solvent mixture. Ion exchange resins have been used ( 5 , 6) for the separation of borate coniplrses of mono- and disaccharides. The introduction of two phenylhydrazine groupings in a carbohydrate molecule changes the solubility. I n the reaction of the sugar osazones with borate molecules, different sugar osazones probably condense with a number of borate molecules forming different complexes which, on paper, show mobilities sufficiently different to permit their separation. The qualitative chromatographic resolution of various phenylosazones of mono- and disaccharides is described below. EXPERIMENTAL
The method consisted in forming the osazones (11) of known single sugars and of mixture of sugars obtained from glycosides by hydrolysis. The filter paper used was Schleicher & Schuell Co. S o . 2040, impregnated with a 0.1M solution of potassium tetraborate to give a p H of 9.3, and dried in a ventilated oven a t 60". The osazones were then applied to sheets of paper 17 cm. wide and 45 cm. long, a width which permitted five circular areas of application, about 3 mm. in diameter. The paper was equilibrated for 20 minutes prior to the beginning of the unidimensional descending chromatography.
1372
ANALYTICAL CHEMISTRY
Dioxane was refluxed and then distilled, first over potassium hydroxide and then over metallic sodium. It was then made up to 75% with fresh distilled water. The hexane used distilled at 60-1" C. a t 580 mm. of mercury. The solvents contained water and the best results were obtained employing : A. 75y0 dioxane, hexane, water, 60/60/30 v./v. B. 75% dioxane, hexane, water, benzene, 55/50/25/20 v./v. C. 75% dioxane, water, 1,2-dichloroethane, 54/40/6 v./v. When the solvents were mixed, two layers formed. TT'ith solvents A and B, the upper layer was used as the mobile phase, the lower as the stationary one. With solvent C the lower layer was the mobile phase. T o obtain good separation with solvent A, it mas necessary to let the migrating solvent drain off the front of the chromatogram and, consequently, the RI obtained is reported as a function of xylose (Rz))taking the value of this sugar as unity. This method detects 15 to 25 y of various phenylosazones. The spots, vellow or faintly orange on the chromatogram, can be observed easily through a blue or blue-violet glass filter. The R , values reported correspond to phenylosazones of pure sugars or mixture of sugars, although in a few cases the sugars were obtained from natural glycosides by hydrolysis. I n the latter cases, the two aglycones were removed and the sugars reacted rvith phenylhydrazine to yield the corresponding osazones, followed by separation on the paper as borate complexes. Those glycosides which gave mixed osazones
chromatographed as well as those obtained from pure sugars. If too much osazone is applied there is a streaking tendency. RESULTS
The solvents employed effected separation of a number of phenylosazones of mono- and disaccharides. The results are presented in Table I. The figures show that xylose and arabinose are well separated from each other as well from the methylpentose, rhamnose. Glucose and galactose migrate differently and their RtJs are distinctly separated. The borate coniplexes of the disaccharide derivatives have, in general, very small R, values in all the solvent mixtures used. Arranging the monosaccharides by decreasing order of R, values the following series is obtained: arabinose, rhamnose, xylose, galactose, and glucose, n-hich holds for the three solvent mixtures used. By a comparison of the results obtained by others when sugars are chromatographed as borate complexes with the present results i t is possible to say, in general, that glucose and galactose which move almost a t the same rate, are well separated as osazones; rhamnose moves faster than arabinose, whereas with their osazones the opposite is true. Sugar anomers which yield the same osazones cannot be differentiated by this procedure. LITERATURE CITED
(1) Barry, V. C., Mitchell, P. W. D., J . Chem. SOC.1954,4020. (2) Boeseeken, J., Adi>ances in Carbo-
hydrate Chem. 4, 189 (1949). (3) Fischer, E., Ber. 17,579 (1884). (4) ~, Flores, S. E.. HerrBn. J.. Tetrahedron 2,308 (1958). ' (5) Khym, J. X., Zill, L. P., J . Am. Chem. Sor. 73. 2399 (19,511. (6,Ziid:,'74,-2090 (1952). (7) Muiioz, E., tesis profesional, Inst. Politecnico Nacional, RIBxico, D. F., 1955. (8) Rees, W. R., Reynolds, T., 'Vature 181.767 (1958). (9) Rbse, I: A,, Schweigert, B. S.,J . Ani. Chem. SOC.73, 5903 (1951). (10) Tsukamoto, T., Kawasaki, T., Yamauchi, T., Phnrm. Bull. (Japan) 4, 35 (1956). (11) Vogel, .4. I., ['Textbook of Practical Organic Chemistry," 3rd ed., Longmans, Green, New Tork, 1956. (12) Wachtmeister, C. A., Acta Chem. Scand. 5,976 (1951). I
,
RECEIVED for review February 3, 1958 -4ccepted April 13, 1959. Contribution No. 105 from the Instituto de Quimica de la Univewidad Nacional Aut6noma de RIBxico. Fellowship from the Instituto Nacional de la Investigaci6n Cientffica.
