Spectrophotometric Assay and Identification of Monosaccharides LAURENCE H. FROMMHAGEN Virus Laboratory and Department of Biochemistry, University o f California, Berkeley, c a l i f .
Accurate and rapid assay of monosaccharides is made possible by the highly reproducible rate of periodate oxidation determined spectrophotometrically. Furthermore, the fact that most of the monosaccharides possess distinctive rates of periodate oxidation permits their simultaneous identification and assay. The method is also applicable to eluates of paper chromatograms.
RE""
NT work on the characterization of the polysaccharide associated with influenza virus ( 3 )required an assay with a sensitivity in the 1 to 10 y range for each of the constituent monosaccharides separated by paper chromatography. The majority of the conventional microanalyses for sugars mere found to have lower limits of sensitivity somewhat above this range, Rominent among these is the periodate method of Flood and Hirst ( B ) , in which a determination is made of the formic acid produced when the sugars are oxidized with sodium periodate for 20 minutes a t 100" C. Dixon and Lipkin ( 1 ) recently reported a method for thc quantitative determination of vicinal glycols based upon thv spectrophotometric measurement of periodate utilization a t 220 mp, the absorption maximum of periodate. It was apparent that this method, if it could be applied to the analyses of monosaccharides, would possess not only the requisite sensitivity and accuracy but also ease and speed. The initial results were disappointing, in that periodate oxidation of the sugars under the conditions of the method of Dixon and Lipkin is slow, indeed requiring days a t room temperature The application of heat, while greatly speeding up the reaction, led under no circumstances to the calculated utilization of periodate. This is understandable in terms of the many subtle sidc reactions of periodate which could occur in such a system. There was, however, remarkable reproducibility in the curvp6 obtained by plotting periodate consumption (in terms of the ahsorbance of the reaction mixture) against the initial concentration of a given monosaccharide under constant conditions of time and temperature. Not only was excellent agreement obtained in replicate determinations, but also analyses made on different days agreed exceptionally well. It thus appeared possible to obtain highly accurate assays of monosaccharides by reference to standard curves in which the consumption of periodate during an arbitrary time interval was plotted against concentration of sugar. Furthermore, the fact that such curves showed characteristically distinct slopes for different monosweharides suggested that this might be a supplementary method for the qualitative identification of sugars. The lack of stoichiometry encountered in the attempt to modify the procedwe of Dixon and Lipkin is explained and resolved by the recent spectrophotometric studies by Marinetti and Rouser ( 4 ) of the periodate oxidation of ribose-5-phosphate. By employing the spectral range of 280 to 310 mp, which permitted the use of buffers, these workers were able to obtain a stoichiometric consumption of periodate with several sugar phosphates and with glucose. However, the method presented here appears to offer the advantages of greater simplicity, shorter assay period (3 hours rather than 23), and a considerably greater sensitivity by virtue of using the absorption maximum of periodate a t 220 mp. On the other hand, limitations are placed on the
method by the fact that phosphate and acetate buffers, as well as other inorganic substances and organic materials, absorb in this region. APPARATUS
Beckman spectrophotometer, Model DU. Quartz cells (standard 3-ml. size) of 1-cm. light path. The assays are carried out in ordinary 30-ml. test tubes which are thoroughly cleaned by immersion in chromic acid cleaning solution, followed by adequate rinsing with tap and distilled water. Rubber stoppers may be used, provided they are not in intimate contact with the contents of the assay tube. REAGENTS
Sodium Metaperiodate, IO-*:M. This solution can conveniently be prepared by simply dissolving 21.4 mg. of anhydrous sodium metaperiodate in 1 liter of water. A solution containing 5 ml. of the fresh periodate solution plus 1 ml. of water is commonly found to have an absorbance reading of ea. 0.804 a t 227 mp. However, in a matter of 24 hours this drops to about 0.795, a plateau value which remains constant for about a week. Fresh periodate solutions are allowed to age for 24 hours and the standard curves are based on the latter absorbance value. .e .E
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2
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; 0
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6
10
14
20
Concentration i n y Figure 1. Periodate utilization curves (absorbance vs. concentration) of several monosaccharides 1. 2. 3. 4. 5.
D-Glucose D-Galactose D-Mannose D-Ribose D-2-Deoxyribose
L-Fucose L-Sorbose D-Sorbitol D-Fructose 10. D-Glucosamine 6.
7.
8. 9.
Standard Monosaccharide Solutions (20 y per ml.). One hundred milligrams of the carefully dried monosaccharide is made u to 10 ml. in a volumetric flask; 1 ml. of this solution is then difuted to 500 ml. The following sugars were used: Dglucose, Merck, C.P.; D-galactose, D-mannose, and D-ribose, Nutritional Biochemical Corp., ~-2-deoxyribose, California Foundation for Biochemical Research, C.F.P. ; kfucose, Mann Research Laboratories, C.P. ; L-sorbose, recrystallized from a 1202
V O L U M E 28, NO. 7, J U L Y 1 9 5 6 commercial source; and D-sorbitol, D-fructose, and D-glucosamine, Pfanstiehl Laboratories, C . P . -411 the sugars were chromatographically pure. QUANTITATIVE PROCEDURE
One milliliter of the solution containing the standard or the unknown (0 to 20 y ) is pipetted into 5 ml. of 10-4M sodium periodate solution. I n the case of standard curve determinations or assays of solutions containing only one pure sugar, the zerohour absorbance reading mal be assumed to be that of the periodate blanks (5 ml. of 10-4M periodate and 1 ml. of water) which ai e run concurrently. However, when other organic substances. which may contribute to the absorbance of the assay mixture, are suspected to be present, it is necessary to determine the absoi bance immediately after addition of sample to the periodate solution. This is particularly true of chromatographic eluates Absorbance readings are taken a t 227 nip (slit width, 0.8 to 1.2 nim.) a t 3 hours. The contents of the assay tubes are simply poured into a cuvette which has been standardized against distilled water. I n this manner the standard cuives of absorbance us. concentration of a series of ten important monosaccharides were determined (Figure 1). The logarithmic scale of the ordinate in Figure 1 was used in order to demonstrate certain characteristics of the curves. Of greatest interest is the linearity of the 3-hour period in the range of 1 to 10 y for certain sugars-e.g., riboseand 1 to 20 y for other sugars-e.g., glucose. Many of the data in Figure 1 are drawn from duplicate runs, each a t the 2-, 6-, lo-, 1 4 , and 20-y levels, on three separate days a t room temperature (about 24“ C.) and daylight illumination. The range of absorbance readings seldom deviates from the mean by more than 10.004 absorbance unit a t the 3-hour period. It is obvious from these data that an accuracy in excess of 98% can be expected a t this time period. The range, however, becomes somewhat broader a t the 24- and 48-hour periods, which are more susceptible to temperature fluctuation than the 3-hour period. Four disaccharides-maltose, cellobiose, lactose, and sucrosehave also been found to possess distinctive and highly reproducible periodate oxidation curves under the same conditions. Under room conditions marked by drastic daily fluctuations of temperature, it would be necessai y to reinvestigate the constancy of the standard curves. QUALITATIVE IDENTIFICATIO3
The approach to this problem will depend upon whether or not the exact concentration of the monosaccharide is known. An unknown monosaccharide assayed a t a known concentration within the conditions of the general procedure will yield an absorbance value which will fall upon its standard curve a t that concentration. It will be necessary to obtain absorbance readings a t both the 3-hour and 25-hour periods in order to permit differentiation of the 3-hour “family” curves. Of the ten monosaccharides shown in Figure 1, all the sugars having the same periodate oxidation curve a t the 3-hour pcriod possess different curves a t the 25-hour period. The convergence of the %hour curves a t low concentration dictates a lower limit of about 4 y for this method. The upper limit is governed by the upper region of the standard curves. Many of the sugars-e.g., glucose-can be analyzed a t concentrations as high as 20 to 30 7 ; however, this great excess of other sugars-e.g., ribose-would use up the periodate before the 3-hour assay period. The following example illustrates the identification of a monosaccharide a t known concentration. One milliliter of a solution containing 10 y of the unknown monosaccharide yielded an absorbance reading of 0.609 a t the 3-hour period and 0.375 a t the 25-hour period. These absorbance values a t the 10-7 level are shown marked with small circles in Figure 1. The 3-hour read-
1203 ing a t the 10--, concentration falls on the galactose-deoxyribose curve, while the 25-hour reading coincides only with the galactose standard curve. The use of both 3-hour and 25-hour absorbance values allows not only differentiation of “family” curves but also simultaneous determination of the concentration and identity of an unknown monosaccharide. I n all cases where the concentration is unknorvn, the following procedure is recommended. The solution containing the unknown sugar is either diluted or concentrated and run a t several concentrations according to the general procedure. The dilution having a 3-hour absorbance reading of 0.50 f 0.02 is read again a t 25 hours. The horizontal dotted line through the absorbance level of 0.50 a t the 3-hour period in Figure 1 bisects all of the standard curves, with the exception ot glucosamine and glucose (these are included by extension of the graph). The choice of this absorbance value thus ensures a coiicentiation within the limits of the method. The absorbance value a t the 25-hour period is marked on each of the 25-hour curves and the concentrations corresponding to each of the intersections with this absorbance value are referred to the 3-hour curve. The format of Figure 1 permits easy reference of concentrations between the 3-hour and 25-hour curves by simply laying a rule upon the diagram. The unknown will be identical to that standard upon whose curve of absorbance us. concentration both the 3-hour and 25-hour assay absorbance readings will fall a t the same concentration. For example, 1 ml. of an unknown is found to yield an absorbance reading of 0.503 a t the %hour period and 0.342 a t the 25hour period. Only sugar 7 , L-sorbose, will satisfy the requirement (see vertical dotted line of Figure 1 ) of possessing these two absorbance values a t the same concentration. At the same time its concentration has been fixed a t 11.2 y. An analysis of Figure 1 will reveal that a t the concentrations of mannose and fucose (Nos. 3 and 6 ) which yield 0.500 absorbance unit a t the 3-hour period there is also an equivalence in the absorbance values for the two sugars a t the 25-hour period. Therefore, this method cannot distinguish between mannose and fucose, although a t another absorbance level a t the 3-hour period they can be differentiated. The assay should be made in triplicate and the precision must be in the order of =k 0.004 absorbance unit a t the 3-hour period, in order to make a differentiation of curves which fall close togetheI. This method is particularly valuable in the case of a chromatographic eluate where another parameter of identification would be useful in addition to a quantitative assay of the monosaccharide. The method is also applicable to several disaccharides. It shows promise as a rapid method of differentiating gls cosy1 isomers-e g , maltose and cellobiose. APPLICATION TO PAPER CHROMATOGRAPHIC ANALYSIS
Two potentially disturbing factors affect the analyses when eluates of paper chromatograms are to be assayed. The first of these is the contribution to the absorbance of lingering chromatographic solvents and oxidative decomposition products of lignin present in many types of filter paper. Solvent contribution may be reduced to the vanishing point by steaming the chromatogram in an autoclave for 10 minutes a t 90’ to 100” C. before elution of the sugars. Care should be taken a t this point that the sugars are not affected. I n most cases, the absorbance due to organic substances derived from the paper is very small and variable; however, for the sake of accuracy it should be measured as the difference between the periodate blank and the zero-hour reading of the assay. A second source of interference may come from periodateoxidizable substances extracted from the paper. This effect is corrected for by eluting and assaying, in the same manner as the
ANALYTICAL CHEMISTRY
1204 Table I.
Recovery of Ribose and Glucose from Paper by Periodate Method
Calcd. Absorbance Correc- Corrected Absorbance RecovAssay Prepara- Amt.. R e a d i n g s tion Standard ery, Factor" Absorbance Curve y 0 hr. 3 hr. 3'% tion 0.657 0.650 98 Ribose 4 . 0 0,810 0 , 6 6 0 - 0 , 0 0 3 0.470 0.467 99 10.0 0 801 0 . 4 6 4 +0.006 0.757 0.758 100 Glucose 2 5 0 . 7 9 0 0.740 f0.017 5 0 0.796 0.713 + O 011 0.724 0.728 101 Paper blanks 0.811 0.799 Periodate blank 0.795 0.795 a Correction factor. =tDifference in absorbance reading of paper blank a t 0 and 3 hours. & Difference in absorbance reading of 0-hour assay and 0-hour periodate blank (see t e x t ) .
unknoivns and standards, blank sections of paper of the same size and from the same chromatogram for which the sugar spots were cut. This correction, expressed as the difference in absorbance between the paper blank at zero-hour and assay-hour pclriod, is added to the readings a t the various time intervals. The treatment of typical data is illustrated in Table I.
In these runs known amounts of the monosaccharides were spotted by micropipet on a sheet of Whatman No. 1 paper which had been run two-dimensionally in collidine: HOH (saturated) and BuOH :H.4c :HOH (4: 1:5 ) and dried. The paper n.as freed
of lingering solvents by the steaming procedure previously described. The areas corresponding to the deposits of sugars were rut out, folded, and immersed in 10 ml. of water for 4 hours with continued shaking a t 37' C. This method of elution has been found fully effective and convenient. The eluate was centrifuged in order to remove paper lint which might otherwise interfere with the absorbance reading. One milliliter of the eluate was then assaved according to the general quantitative procedure, vielding the data in Table I. Paper blanks and periodate blanks kere run simultaneously. At least one level of standard is always run, in order to br certain that standard conditions are prevailing. ACKNOWLEDGMENT
The author wishes to acknowledge gratefully the many helpful suggestions of C. A . Knight, E. K. Putman, and D. L. MacDonald, :dl of this university. This investigation was supported in part by a research grant, RG-4559, to C. A Knight from the Sational I n s t i t u t ~ sof Health, Public Health Service, and by grants from the Lederle Laboratories Division, American Cyanamid Co.. and the Rockefeller Foundation. LITERATURE CITED
(1) Dixon, J. S.. Lipkin, D., ANAL.CHEM.26, 1092 (1954). (2) Flood, A . E., Hirst, E. L., Jones, J. K. N., J . Chem. Soc. 1949,
1659.
(3) Frommhagen, L. H., Knight, C. A , , unpublished data. (4) Ilarinetti, G. V., Rouser, G., J . 4 m . Chem. Soc. 77, 5346 (1955).
RECEIVED for review December
27, 1955.
Accepted .ipril 13, 1956
Determination of Traces of Fatty Amines in Water ALBERT M l L U N and FRANCES MOYER Research Laboratories, General
Mills, Inc., Minneapolis, M i n n .
A method has been developed for determining traces of high molecular weight fatty amines in water. The procedure should be applicable to the control of amine concentration in steam condensate systems where fatty amines are added to inhibit corrosion. Amine concentration is determined by titrating with an anionic surface active agent to the disappearance of pink color due to an amine-eosin complex. A calibration curve is given for the concentration range of 0.5 to 10 p.p.m.
F
ATTY amines of high molecular weight have been used successfully for some time in steam condensate systems to inhibit corrosion. The efficient control of this treatment requires a constant check on the concentration of amine in the condensate waters at the 1 to 10 p.p.m. level. Therefore, a direct, rapid test for determining the concentration of fatty amine in water a t these low levels is desirable. This paper describes such a test, which should be applicable to steam condensates. Bouilloux ( 2 ) found that very dilute solutions of fluorescein, or its derivatives, in organic solvents are colorless, but form colors upon the addition of certain amines. Eosin, in particular, was very sensitive, forming a pink color in the presence of very small quantities of amine. He attributed the pink color to a quinoid-type structure resulting from the formation of an eosinamine salt. Prudhomme ( 4 ) used this color formation of amine with eosin to determine quinine in urine. A buffered sample solution containing eosin was extracted with chloroform and the chloroform extract containing the colored quinine-eosin salt was compared with the color of standard solutions. Harper, Elliker, and Mosely ( 3 )utilized the red color resulting
from the reaction of eosin and quaternary aninionium salts in a quantitative titration procedure for determining the latter at the 10 to 300 p.p.m. level. The titration was carried out with an anionic surface active agent which replaced the eosin in the quaternary-eosin salt and destroyed the color. The procedure described below is essentially that used by Harper, Elliker, and RIosely ( 3 )for quaternary ammonium salts. A buffered sample of water containing fatty amine is shaken up with a dilute solution of eosin in tetrachloroethane. The amine forms a pink tetrachloroethane-soluble compound with eosin. The resulting mixture is then titrated with a solution of sodium lauryl sulfate, previously calibrated against known quantities of amine, until the pink color in the nonaqueous layer has disappeared. Analyses by this method of known mixtures containing fatty amine in water in the range of 0.5 to 10 p.p.ni. indicate an accuracy within 0.5 p.p.m. of amine. REAGENTS AND APPARATUS
Indicator Solution. Dissolve 10 mg. of Eosin yellowish (sodium salt of tetrabromofluorescein) in 100 ml. of analytical reagent grade acetone. Add 10 ml. of the acetone solution to 90 ml. of tetrachloroethane. Remove the reddish color from the tetrachloroethane solution by adding 0.5 gram of citric acid and shaking for 1 minute. Filter through Whatman S o . 1 (or equivalent grade) filter paper. Buffer Solution. Prepare a 5% aqueous solution of citric acid and adjust to p H 3.5 with 0.1N sodium hydroxide. Add 1% tetrachloroethane t o prevent mold growth. Anionic Surface Active Agent Solution. Prepare a 0.01% aqueous solution of sodium lauryl sulfate. This solution should be recalibrated frequently and discarded when deterioration becomes evident. Test tubes, X 5 inches, are rinsed with alcohol and acetone and dried before use.
