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ANALYTICAL CHEMISTRY, VOL 51, NO 7 , JUNE 1 9 7 9
500 ppm) to 500 mL of the water, resulting in phenol concentrations ranging from 1 ppm to 20 ppb, the minimum detectable concentration Figure 4A shows the spectrum that is obtained from a 200-ppb solution of phenol in pond water. Natural water itself does exhibit a considerable fluorescence, due to substances already present in the water and mostly from organic sources. This can be observed in the spectrum as a strong rising of the base line and as an increase of the spectral noise. After the necessary background subtraction and base-line correction. followed by a soft spectral smoothing, the spectrum of the derivatized phenol appears quite clearly (see Figure 4B). The minimal detectable concentration of phenol in the pond water was estimated below 100 ppb. In the University tap water, the minimum detectable concentration was below 50 ppb, but higher than the level. obtained for distilled water. I t has however to be emphasized that the minimum detectable concentration depends greatly on the characteristics of the water source studied. In samples, collected from the River Scheldt, crossing an industrial area, and treated in the same wag as the above cited pond and tap water samples, we were not able to detect added phenol below 300 ppb. I t must be stated that the above results are obtained for phenols added to natural water samples. Generalization toward naturally occurring phenol concentrations may be done only when more elaborate studies are done on this subject Furthermore, routine applications can be taken into consideration only if the naturally occurring fluorescence is reduced to levels that approach the distilled water situation. ACKNOWLEDGMENT T h e authors thank Chris W. Brown for his valuable discussions on this subject. We are also indebted to Jozef Janssens for technical assistance.
LITERATURE CITED ( 1 ) Fountaine, J. E.; Joshipura, P. 6.: Keiiher, P. N.; Johnson, J. D. Anal.
Chem. 1974, 4 6 , 62. (2) Afghan. E. K.; Belliveau, P. E.:Larose, R. H.; Ryan, J. F. Anal. Chem. 1974, 77, 355. (3) Koppe, P.; Dietz, F.; Traud, J. fresenius' 2 . Anal. Chem. 1977, 285, 1. (4) Goulden, P. D.: Brooksbank, P.; Day, M. B. Anal. Chem. 1973, 45, 2430. (5) Gales. M. E., Jr. Analyst(London) 1975, 700, 841. (6) Friestad, H. 0 . :O t t J E.: Gunther, F. A. Anal. Chem. 1969, 4 1 , 1750. ( 7 ) Chriswell, C. D.; Chang, R. C.: Fritz, J. S. Anal. Chem. 1975, 4 7 , 1325. (8) Kleverlaan. N. T. M. Chem. Weekbi. 1975, 2, 13. (9) Coburn, J. A.: Chau, A. S. Y. J . Assoc. O f f . Anal. Chem. 1976, 59, 862. ( l o ) Chau. A . S. Y.; Coburn, J. A. J . Assoc. Off. Anal. Chem. 1974, 57, 389. (11) Kawahara, F. K. Environ. Sci. Technol. 1971, 5 , 235 (12) Bhatia, K. Anal. Chem. 1973, 4 5 , 1344. (13) Renberg. L. Anal. Chem. 1974, 46. 459. Porthault, M. Bull. Soc. Chim. (14) Audouard. Y.: Suzanne, A,: Vittori, 0.: F r . 1975, 130. (15) Irish, D. E.; Chen, H. Appl. Spectrosc. 1971, 25, 1. (16) Bradley, E. B . ; Frenzel, C. A. Water Res. 1970, 4 , 125. (17) Baldwin, S. F.; Brown. C. W. Water Res. 1972, 6, 1601. (18) Braunlich, G.; Gamer, G. Water Res. 1973, 7, 1643. (19) Ahmadjian, M.; Brown, C. W. Environ. Sci. Technol. 1973, 7, 452. (20) Cunningham, K. M.; Goldberg, M. C.: Weiner. E. R . Anal. Chem. 1976, 4 9 , 70. (21) Van Haverbeke, L.; Lynch, P. F.; Brown. C. W. Anal. Chem.. 1978, 50, 315. (22) Van Haverbeke, L.: Brown, C. W. "Modern Techniques for the Detection and Measurement of EnvironmentalPollutants"; Toribara, T. Y.; Ed.; Plenum Press: New York. 1978. (23) Thibeau, R. J.: Van Haverbeke, L.; Brown, C. W. Appl. Spectrosc. 1978, 32, 98. (24) Van Haverbeke. L.: Goldfarb, D.; Brown, C. W. Anal. Chem., submitted for publication. (25) Van Haverbeke, L.; Brown, C. W. A m . Lab., 1978, July. (26) Snell, F D.: Snell, C. T.; "Colorimetric Methods of Analysis"; Van Nostrand Reinhold Co.: New York, 1967: Voi. I V and IVa.
RECEIVED for review Xovember 9. 1978. Accepted February 2 2 , 1979.
Colorimetric Determination of Hexuronic Acids in Plant Materials Ralph W. Scott Forest Products Laboratory, Forest Service, U.S. Department of Agriculture, Box 5 130, Madison, Wisconsin 53705
A colorimetric reagent, 3-5-dimethylphenol, is selective for 5-formyl-2-furancarboxylic acid, a chromogen formed from uronic acids in concentrated H2S04at 70 'C. Addition of the reagent at 20 O C produces within 10 min, a chromophore absorbing at 450 nm. Selectivity is critical because of interferences from neutral sugar products and lignin when uronic acids are at 1-3% levels. D-Galacturonic and 4-0-methylD-glucuronic acids could be measured separately from Dglucuronic acid, by adding H,BO,. About 12% more chromogen was produced from D-galacturonic and 4-0-methylD-glucuronic acids of polymers than from the monomers. Analyses of wood containing 3 YO uronic anhydride gave 0.5 standard deviatlon per measurement. The time for dissolution, reaction, and color formation is 30 min for fast reactors, 60 min for glucuronic acid.
Analysis of structural plant material for the uronic acid group has often been done by heating with HC1 followed by measurement of the C 0 2 released from the uronic acids. Existing colorimetric methods for this analysis are limited by insufficient specificity for the 1-4% levels of uronic acids often encountered. The objective of this study was to develop a
colorimetric method very specific for hexuronic acid in the presence of neutral sugars, particularly glucose, mannose, and xylose. It was previously shown ( 1 ) that absorbances near 300 nm, resulting from the reactions of several uronic acids with concentrated H2S04,could be used for quantitative measurements. However, the presence of absorbances due to products from neutral sugars would prevent such measurements on mixtures. At that time it was noticed that phenol was a highly selective colorimetric reagent for the uronic acid product, 5-formyl-2-furancarboxylic acid, in the presence of both 5-hydroxymethyl-2-furancarboxaldehydeand 2-furancarboxaldehyde. The latter two derive from hexoses and pentoses in concentrated H2S04. Consequently, the phenolsulfuric acid method of Dubois et al. ( 2 ) could be modified to analyze for uronic acids by withholding the colorimetric reagent until after the completion of the reaction with sulfuric acid. Such a modification of the phenol-sulfuric acid method was developed. However, the 3-phenylphenol reagent introduced by Blumenkrantz and Asboe-Hansen (3)was a more sensitive reagent. Several other reagents were then tested by a modification described later. The choice of 3,5-dimethylphenol for the analysis was based primarily on its selectivity.
This article not subject to U.S. Copyright. Published 1979 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL 51, NO. 7, JUNE 1979
EXPERIMENTAL Test Materials. Sugars were obtained commercially and used directly unless noted otherwise. D-Gdacturonic acid monohydrate was recrystallized from an acetone-water mixture after removal of cations by ion exchange. The isolation of an aldobiouronic acid, O-(4-O-methyl-a-~-glucopyranosyluronic acid)-(1-2)-D-xylose, and an aldotriouronic acid, 0-(4-0-methy1-a-D-glucopyranosyluronic ac~d)-(l-~)-~-(~-D-xy~opyranosy~)-(l-4)-D-xy~ose, and the preparation of 4-0-methyl-D-glucuronic acid have been reported ( I ) . Methyl (methyl-a-D-ga1actopyranosid)uronate was prepared as described by Jones and Stacey ( 4 ) except for the neutralization step, which was replaced by passage through a weakly basic ion-exchange column. The product was the monohydrate reported by Morel1 and Link ( 5 ) . It lost 1 mol of water when dried a t 78 OC as described ( 5 ) and then had the reported melting point (147-147.5 "C) and gave rotation, [@]20D +129.5" (c 1.9, H 2 0 ) , reported [.Iz0D + 1 2 8 O (c 1.0, H 2 0 ) ( 4 ) . Methyl @-D-glucofuranosidurono-6,3-lactone was prepared as described by Osman et al. (6). It had the reported melting point (139-140 "C) and gave rotation, [.]%D -57.9" (c 2.8, H@), reported -59" ( C 1.0,HzO). The 1,2-0-isopropylidene derivative of 0-L-idofuranurono6,3-lactone was supplied by Horton ( 7 ) and the methyl-3-Dmannofuranosidurono-6,3-lactone (mannuronolactone)by Laurens Anderson (Department of Biochemistry, University of Wisconsin, Madison, Wis. 53706). 5-Formyl-2-furancarboxylic acid (5FF) was provided by Feather and Harris (8). Polyguluronic acid was obtained from Arne Haug (Norwegian Institute of Seaweed Research, 7034 Trondheim-NTH, Norway). Enzyme lignins were donated by John Obst of this laboratory. Polymers were commercial products or were separated from natural sources by alkaline extraction. Their dry weights for analytical purposes were obtained by drying to constant weight under reduced pressure over anhydrous CaS04. Only two polymers gave additional weight losses at 105 OC: polygdacturonic acid 2.1%, aspen xylan 0.8% (not used for calculations). Of the phenols, only 3,5-diphenylphenol was synthesized. This was prepared by the procedure of Kenner and Shaw (9) except for the final decarboxylation step which was done by refluxing in dimethylaniline. Reagents. Concentrated HzS04(about 96%) and 2% NaC1 in water are the reagents for the general procedure. For the glucuronic acid determination, an additional solution was prepared containing 2 g NaCl and 3 g H,B03 in 100 mL of water. The colorimetric reagent was 3,5-dimethylphenol, 0.1 g in 100 mL glacial acetic acid. Apparatus and Technique. The general technique has been discussed ( I O ) . Samples were reacted with H2S04in 15 X 85 mm culture tubes held in heating blocks. The tubes were covered with loose plastic covers. All reagents were dispensed from syringe-type dispensers attached to stock bottles. Absorbances were measured with a double-beam spectrophotometer using 1-cm cells. Dissolution of Polysaccharides. Water-insoluble polysaccharides were dissolved by heating samples, containing 25 to 50 mg of carbohydrate, with 2-4 mL of 7 2 % H2S04at 50 "C for 10 min. Solid samples such as wood were 40 to 80 mesh. Powders which tended to clump were moistened with ethanol before the 7270 H2S04was added. Some samples may require trituration before and while heating or they may need longer heating periods. However, extended treatment should be avoided since tcsts with galacturonic acid showed about 1% loss for each 10 min of heating. Lignified samples were generally fully extracted in 10 min with some trituration, though lignin remained insoluble. After heating, the 72% H2S04solution was diluted nith water to a volume estimated to contain 20 to 80 kg/mL of uronic anhydride. (The term "anhydride" is used in this paper to indicate a molecular weight, 176 in the case of a hexuronic acid or 162 in the case of a hexose.) Solutions obtained from samples containing esterified uronic acid groups as in pectin should be made alkaline shortly before analysis. For example, after digestion with 2 mL of 7270 H2S04and dilution to 100 mL, pectin samples were further diluted 1:l with 0.6N NaOH. With the addition of alkali, small. but consistent, increases in uronic anhydride were observed in the analyses of wood and of some pulp samples.
937
Secondary hydrolysates could be used directly without neutralization. These were produced by dilution of 72% HzS04 solutions to 3 or 4% H2S04followed by heating a t 120 "C for 1 h in an autoclave. First Procedure: Determination of Galacturonic and I-0-Methylglucuronic Anhydrides When Other Uronic Anhydrides Are Absent. (1) A 0.125-mL sample containing 20-80 pg/mL of uronic anhydride was mixed with 0.125 mL of 2% NaCl in a reaction tube. (2) Two mL of concentrated HzS04was added and immediately mixed with the water by shaking the slanted tube. The tube was placed in the 70 O C block for 10 min. It was then removed and cooled to room temperature by swirling in water for 20-30 s. (3) A 0.1-mL aliquot of the 3,5-dimethylphenol solution was added to the mixture with mixing as before and the tube was left at room temperature. (4) Between 10 and 15 min from addition of the colorimetric reagent, absorbances were read at 450 nm and 400 nm against a water reference having 100% transmission at 450 nm (an adequate reference with double-beam instruments). (5) Calculation of the uronic anhydride concentration was based upon the 450-400 nm difference. An average reagent blank for this difference was predetermined. D-Galacturonic acid monohydrate was a convenient standard suitable also for 4-0methylglucuronic acid since both compounds had the same molar absorptivity in this method. However, it was necessary to multiply the uronic anhydride absorptivity of this standard by the correction factor 1.12 to obtain the absorptivity of these uronic anhydrides when they were constituents of polymers. Second Procedure: Determinations of Galacturonic, 4-0-Methylglucuronic, and Glucuronic Anhydrides When Other Uronic Anhydrides Are Absent. Glucuronic acid gave a very weak response in the preceding procedure, but boric acid considerably increased that response without altering results from the other two anhydrides. The second procedure had two variants, the choice depending on whether or not glucuronic acid was the only uronic acid present. For the measurement of glucuronic anhydride as the only uronic component, the second procedure repeated the first with only two changes. The 2% NaCl was replaced by 0.125 mL of the NaC1-H3B03 solution in the first step and the heating period in the second step became 40 min. ~-Glucofuranurono-6,3-lactone (commercial D-glucurono~actone)was a convenient standard. When either or both of the other two uronic anhydrides were present with glucuronic acid the following steps applied. (1) A first sample was run by the first procedure. (2) A second sample was run concurrently, with the two changes from the first procedure as noted above. (3) The 450-400 nm difference of the first sample was subtracted from that of the second sample. This difference, due to the H3B03-inducedincrease in absorbance at 450 nm, was linearly proportional to the glucuronic anhydride content. Two samples of the D-glucuronolactone standard provided the differential absorptivity for determinations of glucuronic anhydride. (4)The determination of the galacturonic anhydride and/or 4-0-methylglucuronic anhydride on the basis of the first sample by the first procedure required either correction of the 450-400 nm difference before calculation or correction of the calculated concentration. The latter correction (to be subtracted) was about 670 of the determined glucuronic anhydride concentration. The first sample of the D-glucuronolactone standard provided the absorptivity for either correction. Alternative Procedures. Chloride and use of the 450-400 nm peak height measurement were used to reduce interference from neutral sugars and from lignin. Sensitivity was increased by elimination of either or both techniques, using instead 0.25-mL samples of standards and of unknowns containing over 5% uronic anhydride, and using the 450-nm maximum absorbance.
DISCUSSION A N D RESULTS The Color Reaction. Determining optimum conditions both for the hexuronic acid reaction with H 2 S O I and for the analysis of natural products depended upon the use of a sensitive and selective reagent for 5FF. Several reagents were tested by adding reagent solution t o t h e mixtures resulting
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 7 , JUNE 1979
Table I. Maximum Absorbance, Wavelength at Maximum Absorbance, and Selectivity Ratioa for Several Phenolic Reagents color reaction at color reaction at 20 ' C , 1 5 min 70 'C, 30 min reagents, phenol absorbance, wave- absorbance, wavederivatives length nm, ratio length nm, ratio phenolb 3,5-dimethyl3-phenyl3,5-diisopropyl2-carboxy3,4-dimethylb 3-methyl3,5-dimethoxy4-ethyl2-hydroxy-
0.39 1.15 0.72 0.82 0.85 0.75 0.70 0.80 0.70 0.41
472 448 525 452 478 446 463 440 440 474
1.6 8.8 12.0 5.9 1.0 1.2 7.0 3.2 8.7 8.2
0.76 1.30 0.55 0.66 0.85 0.71 0.70 1.00 0.71 0.47
472 2.2 448 7.0 525 1.7 452 5.9 479
