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ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
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the three-stage thermoelectric cooling module. This concentration corresponds to a total of 3 x 10' iodine molecules within the sampled intracavity vapor cell volume (-0.4-mm laser beam diameter and 5.0-cm path length). The isotopic selectivity of iodine intracavity absorption was measured by using the intracavity l Z i I 2 vapor cell and the external fluorescence signal from the lZ912monitor cell. The normalized fluorescence signals from the external lm12cell were measured as a function of the intracavity '"12 concentration. These curves, shown in Figure 3, were measured simultaneously with those presented in Figure 2. Figure 3 shows that the isotopic selectivity of iodine intracavity absorption is relatively insensitive to both the laser output power and the intracavity absorber concentration. The small amount of lZ7I2impurity in the 12912 fluorescence monitoring cell is expected to fluoresce less intensely in Figure 3 when the l2;IZconcentration inside the cavity is high. This decrease is not observed, however, because an offsetting fluorescence signal is produced by the slight increase in laser power at the and 12i112sIabsorption wavelengths a t high intracavity 1z'12concentrations. The power dependence of the latter effect produces the scatter in the data shown in Figure 3. The isotopic selectivity of the intracavity absorption method allows the simultaneous and independent measurement of a number of iodine isotopes. This was demonstrated by placing an iodine-129 vapor cell (which also contained iodine-127) inside the laser activity. The measured fluorescence signal from an identical external iodine-129 cell is plotted as a function of I? number density (see lower curve in Figure 4). The response of an external iodine-127 cell is shown as the upper curve. Both curves were normalized to unity a t the lowest measured intracavity iodine number density. T h e relative concentration of "'I2 in the intracavity vapor cell is given by
[lz'I,]/[TOTAL 12] = N l / N , where N , is the total intracavity iodine number density ('*'I2 + 127112sI+ lZ7I2)a t a given point on the lower curve in Figure 4 in its linear region, and N 2 is the apparent "'I2 number density a t the same external fluorescence signal level on the upper curve. When the data are normalized a t a point for which the external fluorescence signals are independent of the intracavity iodine concentration, this equation is independent of the relative response of the external fluorescence detection system. From this equation, the isotopic abundance of
iodine-127 in the intracavity iodine-129 vapor cell is given by (N1/N2)1/2.Using Figure 4, we determined the isotopic abundance of iodine-12'7 to be in the range 24-2870. This is in agreement with the estimated isotopic abundance of iodine-127 in this cell. In conclusion, we have demonstrated the high sensitivity and large dynamic range of iodine intracavity absorption spectroscopy with a CW dye laser. Because the method is isotope specific, lZ9I2can be measured in the presence of fission-produced stable "'I2 and naturally occurring lZiIzin the atmosphere. The sensitivity of this technique can be increased by using a larger laser beam and a long sample cell. For example, with a 4-mm laser beam diameter and a 50-cm long intracavity sample cell, we expect the sensitivity of this measurement technique to be increased by a factor of -1000. This would permit a measurement of lZgI2a t levels below the maximum permissible concentration.
ACKNOWLEDGMENT The authors express their appreciation to M. E. Wilkins for providing technical assistance in making these measurements.
LITERATURE CITED Y. Wang, Ed., "CRC Handbook of Radioactive Nuclides", Chemical Rubber Co., Cleveland, Ohio, 1969, p 622. A. P. Baronavski and J. R. McDonald, " A Radioiodine Detector Based on Laser Induced Fluorescence", Nav. Res. Lab. (U.S.) Rep., 3514 (1977). R. L. Brown and W. Klemperer, J . Chem. Phys., 41, 3072 (1964). J. L. Steinfeld and W. Klemperer, J . Chem. Phys., 42, 3475 (1965). N. C. Peterson, M. J. Kurylo, W. Braun, A. M. Bass, and R . A. Keller, J . Opt. SOC.Am., 61, 746 (1971). R.J. Thrash, H. von Weyssenhoff, and J. S.Shirk, J . Chem. Phys., 55, 4659 (1971). R. A. Kelier, E. F. Zalewski, and N. C. Peterson, J . Opt. SOC.Am., 62, 319 (1972). T. W. Hansch, A . L. Schawlow. and P. E. Toschek, If€€ J . Quantum Electron., qe-8, 802 (1972). R. A. Keller. J. D. Simmons. and D. A. Jenninas. J . Oot. SOC.Am.. 63, 1552 (1973). W. J. Chiids, M. S. Fred, and L. S.Goodman, Appl. Opt., 13, 2297 (1974). W. Brunner and H. Paul, Opt. Cornmun., 12, 252 (1974). W. G. Schweitzer. Jr.. E. G. Kessler. Jr.. R. D. Deslattes. H. P. Laver. and J. R. Whetstone, Appl. Opt., 12, 2927 (1973). W. F. Giauque, J . Am. Chem. Soc.,53, 507 (1931). C. A. Goy and H. D. Pritchard, J . Mol. Spectrosc.. 12, 38 (1964). K. Tohma, J . Appl. Phys., 47, 1422 (1976).
-
RECEIVED for review November 20, 1978. Accepted March 19, 1979. This work was supported by the United States Department of Energy.
Determination of Three Phenolic Compounds in Water by Laser Excited Resonance Raman Spectrometry Laurent Van Haverkeke and MCdard A. Herman" Rijksuniversitair Centrum Antwerpen, Laboratorium voor Anorganische Scheikunde, Groenenborgerlaan 171, B 2020 Antwerpen, Belgium
Three phenolic compounds are determined in aqueous solutions by resonance Raman spectrometry via the derivatiration with the diazonlum salt of 4-nitroaniline. The detection level of phenol in distilled water was 20 ppb. I n natural waters, the detection level of added phenol was 50 to 300 ppb, depending on the characteristics of the water sample. A calibration curve is set up and concentrations are measured within 10% or less. The spectra of three different phenols have been recorded to indicate the identification capabilities of the technique.
Several methods for the detection of phenols in water a t low concentrations have been described in the literature. The majority of these methods can be divided in two series. First,
there are the spectrophotometric analyses; the most commonly used coloring reactions are the ones that use 4-aminoantipyrine (1-4) and 3-methyl-2-benzothiazolinone hydrazone (3-6). The other important class of methods involves gas chromatography (7-11). For this method, the pollutants must, however, be taken out of their aquatic medium and transferred into an organic phase by an extraction procedure. Among the many other methods that have been proposed, we mention polarography and ion-exchange liquid chromatography (12-14). T h e above cited methods all have least one factor that inhibits their use in routine applications. Either the technique used is applicable only in nonaqueous media and, therefore, the pollutants have to be extracted into an organic layer, or
0003-2700/79/0351-0932$01.00/0 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
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Table I. Specific Properties of t h e Four Methods
method sulfanilic acid 4-nitroaniline 4-amino antipyrine 3-methylbenzothiazolinone-
absorbance maximum of the dye, nm
stability of the dye in the laser beam
438 477 506 526
good good degradation good
RRS intensity
fluorescence intensity
low high
high medium-low
low
low
-
-
2-hydrazone (MBTH) t h e method is definitely applicable in water but fails on t h e specificity requirement, and only the total amount of phenolic material can be detected. Our goal was t o develop a method for analysis of phenolic compounds in water t h a t satisfies t h e following three requirements. (i) T h e method must be applicable in aqueous medium. (ii) T h e sensitivity must be realistic, Le., concentrations below the 1-ppm level have t o be detectable. (iii) A distinction between different members of t h e phenol family must be possible. Since the revival of Raman spectrometry in the mid-sixties, this technique has been used in analytical situations. Irish and Chen (15)adequately reviewed the analytical aspects of Raman spectrometry. In t h e early seventies, Raman spectrometry was also applied t o pollution studies (16-20). For t h e past three years, however, we have been trying to apply the resonance Raman effect. In our studies on industrial fabric dyes (21,22),we were able t o detect the compounds a t 30-50 ppb levels and identify them at concentrations between 75-175 ppb. Using pre-resonance Raman spectrometry, we were able t o detect some pesticides in water below the ppm level (22, 23). Quantitative measurements are easily carried out ( 2 4 ) and the method can also be applied in a continuous flow setup (25). In t h e present contribution this technique is applied t o phenolic pollutants, after they have been transformed t o appropriate colored derivation products.
EXPERIMENTAL Derivatization Methods. 4-Lvitroani/ine.REAGENTS.(a) 4-Nitroaniline, 0.69 g, (Aldrich 18531-0)is dissolved in 155 mL of 1 N HCl and diluted with distilled water to 1 L. (b) NaN02, 5 g, (UCB 1759) is dissolved in distilled water to 100 mL. (c) A buffer solution is made with 53 g of Na2C03.10H20(UCB 1719) and 15 g of EDTA2Na.2H20 (Titriplex 111,Merck 8418) dissolved in distilled water and adjusted to 1 L. The first two solutions are stored in a refrigerator at +4 "C. PROCEDURE. The diazonium salt is made by adding 1 mL of the nitrite solution to 100 mL of the 4-nitroaniline solution with vigorous mixing. This diazonium salt solution may he used up to 24 h after preparation if it is stored a t 4 O C . To 500 mL of the phenol solution, 50 mL of this diazonium salt solution are added. After vigorous mixing, 100 mL of the buffer solution are added and the solution is mixed again. The characteristic color of the diazo dye appears almost immediately. S u l f a n i l i c Acid. REAGENTS.(a) Purified sulfanilic acid, 4.8 g, (UCB 4425) is dissolved in distilled water and diluted up to 1 L. (b) NaN02, 1.7 g, (UCB 1759) is dissolved in distilled water and diluted up to a 1-L solution. (c) A 4 N H2S0, solution is made from a concentrated solution by appropriate dilution with distilled water. (d) 1 N NaOH solution. The first two solutions are also stored in a refrigerator a t +4 "C. PROCEDURE. By mixing 60 mL of each of the sulfanilic acid and NaNOz solutions and adding 10 mL of the H2S04solution, a solution of the corresponding diazonium salt is obtained. A 20-mL portion of this solution is then added to 500 mL of the phenol solution. After adding 100 mL of 1 N NaOH solution and vigorous shaking, the color of the corresponding diazo dye is formed. 4-Arninoantipyrine. REAGENTS.(a) 4-Aminoantipyrine, 2 g, (Aldrich A3930-0) is dissolved in distilled water and diluted up to a 100-mL solution. (b) Ammonia, 15 mL, (25%) (UCB 4747) is diluted with 85 mL of distilled water. (c) K3Fe(CN), 2 g, (UCB
1599) is dissolved in water and brought up to 100 mL. PROCEDURE. To 500 mL of the phenol solution, 3 mL of the 4-aminoantipyrine solution, 10 mL of the ammonia solution and 10 mL of the K3Fe(CN), solution are added. Vigorous mixing is necessary after each reagent is added. The colored derivative is formed almost immediately. 3-Methyl-2-benzothiazo~~none Hydrazone ( M B T H ) . REAGENTS. (a) Solution of 0.5 g 3-methyl-2-henzothiazolinone hydrazone hydrochloride (Aldrich 12973-9)per liter is made with distilled water. (b) (NH4)4Ce(S04)4.2H20, 0.2 g, (Merck 2273) is dissolved in distilled water; 0.4 mL concentrated sulfuric acid is added and the solution diluted to 100 mL. (c) A buffer solution is made of 2 g EDTA2Na.2H20 (Titriplex 111, Merck 8418), 8 g H3B03(Merck 165), and water up to a 500-mL solution. PROCEDURE. To 500 mL of the phenol solution, 100 mL of the MBTH solution is added and mixed vigorously. After 5 min, 50 mL of the (NHJ4Ce(S0,), solution is added and mixed well again. After another 5 min, 30 mL of the buffer solution, which is previously diluted to 50% with ethanol, is added and the solution is mixed again. The characteristic color of the azo dye is formed. Instrumentation. All Raman spectra were recorded with a Coderg PHO Raman spectrometer. Excitation was provided by a Spectra Physics model 164 Argon gas laser. The 488.0- and 514.5-nm laser lines were used with nominal power of 1 W a t the sample position. The optical slit width used was 13.5 cm-' and the applied scan speed for analog spectral recording was 50 cm-'/min, at a time constant of 1 s. Because of the inertia of the digital recording system, a lower scan speed of 25 cm-'/min had to be used, which implemented a time constant of 2.5 s. The detection system consisted of a cooled EM1 S20 photomultiplier type 9658A and a Coderg CPH 200 photon counting unit. The analog spectrum was recorded on a Goerz Servogor RE 511 potentiometric recorder. Digital information of the spectra was obtained by digitizing the analog output of the detection system by means of a Doric DS 100 integrating microvoltmeter. The sampling interval used was 1 data point per wavenumber. The digitized information was stored on paper tape by means of a Logicontrol Serializer and a Facit paper tape punch. All data manipulation was done on an IBM 1130 computer. The samples were positioned in the Coderg standard multiple-pass illumination chamber in two different ways. Either the standard Coderg 0.3-mL liquid cell was used or a flow-through system in a closed loop configuration, designed in this laboratory. Sample Solutions. Three stock solutions containing 500 ppm of phenol (Riedel-DeHaen AG 160171, o-cresol (Aldrich C8570-0) or rn-cresol (Aldrich C8572-7) were made with distilled water. Lower concentrations were made by appropriate dilution of these stock solutions. Simulated real world samples were made by adding a known amount of the stock solution to 500 mL of natural water. These natural water samples were obtained from the University water supply (tap water), from a nearby pond (pond water) and from the River Scheldt, going through the industrial zone of Antwerp (river water). After derivatization and prior to measuring the spectrum, each sample is filtered through a Millipore type SC 8-pm membrane filter.
RESULTS AND DISCUSSION Our study was based on the four derivatization methods, investigated by Koppe, Dietz, and Traud (3). Their applicability in resonance Raman determinations was investigated by the following properties. (i) T h e magnitude of the resonance Raman enhancement, which is closely related to t h e molar absorption of t h e ab-
934
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979 I
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Figure 1. Resonance Raman spectrum of a 5-ppm solution of phenol after applying the 4-nitroaniline derivatization method
sorption band maximum and its distance from the available laser lines. (ii) The thermal stability of the formed dyes in the intense laser beam. (iii) The relative intensity of the resonance Raman bands vs. the 1640 cm-l water band. (iv) The occurrence and intensity of fluorescence of the formed dye. The results of these investigations are gathered in Table
I. Table I shows that one of the methods most used in spectrophotometric determinations, the 4-aminoantipyrine method, is of no use in resonance Raman determinations. The dye does not withstand the intense laser beam and decomposes readily, even when the exposure time is reduced to a minimum by using a continuous closed-loop flow-through system. Derivatization by the diazonium salt of sulfanilic acid gives a dye with an absorption maximum around 438 nm, which is relatively far from the available laser lines at 488 and 514.5 nm of our Ar+ laser. Furthermore, this dye shows considerable fluorescence, which makes it very hard to observe the resonance Raman frequencies. The two remaining methods have good stability in the laser beam and show absorption maxima close to one of the two available laser lines. The resonance Raman band intensities on the 4-nitroaniline diazo dye are however a lot better t h a n the ones of the M B T H method. Regardless of the slightly higher fluorescence of the former dye, the results that are obtained are better than for the MBTH adduct. Therefore, the derivatization method by the diazonium salt of 4-nitroaniline was considered to be the best method. The position of the absorption maximum indicates the 488-nm laser line as the better excitation frequency, which is supported by experimental measurements. The remaining part of this study is consequently based on this derivatization method. Whereas originally the spectra were recorded from solutions in the standard 0.3-mL cells, we used a closed-loop flowthrough setup for the rest of our experiments. In this setup, a large volume of the sample solution, usually 650 mL, is continuously flowed through the laser beam. Since the individual dye molecules are only momentarily exposed to the laser beam, any occurring decomposition will be reduced to a minimum, thus assuring more accurate measurements. T h e spectrum of a 5-ppm solution of phenol in distilled water and treated by the 4-nitroaniline derivatization method is given in Figure 1. As can be seen, there is a definite contribution from a fluorescence band of the dye, on which the resonance Raman spectrum is superimposed. The individual resonance Raman bands are, however, clearly observed. The broad, weak band around 1640 cm-’ is due to the deformation vibration of the water solvent molecules. When decreasing the concentration of phenol, some changes
J 1000
1400
1000
CY1
Figure 2. Resonance Raman spectra of a 50-ppb solution of phenol after applying the 4-nitroaniline derivatization method. (A) Original spectrum, (B) background spectrum, (C) spectrum obtained from the original spectrum after background subtraction
in the spectrum are noticed. Obviously, the 1640 cm-’ water band becomes relatively more intense. Secondly, the bands around 1120, 1180, 1340, and 1590 cm-l decrease much less in relative intensity than the remaining bands. This is clearly seen in the spectrum of a 50-ppb solution of phenol, as shown in Figure 2A. This effect can be explained by a contribution to this spectrum by the Raman spectra of the added reagents. Indeed, the components of the buffer solution that is added, N a 2 C 0 3and EDTA, are present in nonneglectable concentrations. Furthermore, if the concentration of phenol is relatively low, a large excess of the diazonium salt is present. At ambient temperature and in basic conditions, this diazonium salt is converted into the 4-nitrophenolate anion. Having a yellow color, this compound may also be apt to exhibit preresonance Raman spectra, as has been found for other members of this family (23). Since the Raman spectra of the buffer ccmponents and/or the 4-nitrophenolate anion may be relatively important, they may contribute to the total Raman spectrum, especially if the concentration of the dye in solution is very low. The combination of the former spectra may be considered as a “background spectrum” on which the spectrum of the dye is superimposed. T h e background spectrum is displayed in Figure 2B. It may be noticed that the most intense bands of this background spectrum coincide with the most intense bands of the spectrum of the 50-ppb phenol sample solution in Figure 2 A. T o obtain the spectrum of the dye, the spectra of the sample solution and of the background have to be subtracted from one another. Since our Raman spectrometer is a single beam instrument, this can be done only by additional data manipulation. The data of the two spectra are digitized and the background spectrum is subtracted from the sample spectrum, based on equal intensity of the 1640 cm-’ water band in both spectra. The result of this subtraction, after the necessary base-line correction and a soft spectral smoothing (3 times a triangular smoothing over 3 points), is displayed in Figure 2C. Comparison 3f this spectrum with the one in Figure 1 shows a remarkable agreement, except for the signal-to-noise,
A N A L Y T I C A L CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
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Table 11. Detection Limits for Phenol spectrophotometrically A. B. C. D.
sulfanilic acid 4-nitro aniline 4-amino-antipyrine 3-methyl-2-benzothiazolinone hydrazone
34 PPb 14 P P ~ 31 ppb 17 PPb
by resonance Raman spectroscopy
4-ni troaniline
20 ppb
which is obviously less in the spectrum of the lower concentration. Using this spectra subtraction procedure, concentrations down to 20 p p b can be detected and identified. This result is based on the requirement that at least 3 bands must be distinguishable, Le., have a signal-to-noise ratio of a t least 3:l. T o avoid possible errors due to the subtraction procedure, only those bands that do not coincide with bands of the background spectrum (see above) may be taken into consideration. The 1340 cm-l band is still visible at concentrations of 10 ppb and less but, because of the above cited requirement, we did not consider these as valid detection and identification limits. This situation remained if we drastically reduced the amount of diazotized 4-nitroaniline added to the sample, thus also reducing the importance of the background spectrum. Since, however, the original procedure has a wider span of concentrations, we preferred it over the method with reduced diazonium salt. Table I1 gathers the detection limits obtained by this method and by the four spectrophotometric methods mentioned earlier in this work. For the latter ones, we assumed a 5-cm pathlength cell and a minimally detectable absorption of 0.004 absorbance unit and we required a signal-to-noise ratio of 3:l. As can be seen from the table, the resonance Raman technique has a detection level that is in the same region as the other four methods. We also carried out a preliminary investigation on the quantitative aspects of the method. For this purpose, we made u p a series of standard solutions ranging from 100 p p b to 7 ppm. We determined the height ratio of the 1590 cm-’ band over the 1640 cm-’ water band. The heights were estimated as the distances between the maxima of the bands and a straight base line, constructed through the minima around 1550 and 1720 cm-l. A plot of this height ratio vs. the concentration reveals a straight behavior over the whole region. In view of our previous study on quantitative measurements with resonance Raman spectroscopy with industrial dyes (21), we must indicate that this may be accidental. This straight line, however, does not go through the origin. This is due to the fact that the 1590 cm-’ band contains a contribution from the background spectrum. Furthermore, the error on the individual measurements becomes quite large when the higher concentrations are taken into consideration. This may be caused by the relatively small intensity of the water band for higher concentrations, and also by the rudimentary way in which the height ratio determinations are carried out. Nevertheless, this plot may be used as a calibration curve and concentrations can be determined within 10%. T h e qualitative aspects of the method were also investigated. This is done by applying the method on o-cresol and m-cresol solutions. In Figure 3, the spectra, obtained from 1-ppm solutions of phenol, o-cresol, and m-cresol are displayed from the bottom. The comparison of Figure 3A with Figures 3B and 3C clearly shows the differences in the spectra when a methyl group is introduced in the molecule. The comparison of Figure 3B with Figure 3C is even more significant. T h e difference between the two compounds is the position of the methyl group on the benzene ring. As the Figure shows, the spectral differences are very large. We may conclude that this method is not only capable of detecting the presence of a
,,-.
t I800
1400
1000
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Figure 3. Resonance Raman spectra after derivatization with diazotized Cnitroaniline of 1 pprn solutions of: (A) phenol, (B) ocresol, (C) rncresol
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Figure 4. Original ( A ) and background subtracted (B) spectra of a 200-ppb solution of phenol in pond water.
phenol, but is also capable of identifying the compound present. Unfortunately, the method is limited in its use, because only a certain number of phenols react with diazotized 4-nitroaniline to give colored derivatives. Koppe et al. ( 3 ) reviewed some 126 phenolic compounds and only 28 of them gave reasonably strong colored derivatives using the 4nitroaniline derivatization method. Those phenolic compounds that have a substituent in the 4 position and polyhalogen substituted phenols do not react or give only weakly colored derivatives. Finally, we tested the method for use in real world situations. Therefore, we used water from the University water supply tap and from a nearby pond. T o simulate the actual situation, we doped the obtained water sample by adding a certain amount of highly concentrated phenol solution (usually
936
A N A L Y T I C A L 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. Am. 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
