Anal. Chem. 1980, 52, 913-916
913
Proton Nuclear Magnetic Resonance Spectrometry for Determination of Substituents and Their Distribution in Carboxymethylcellulose Floyd F.-L. Ho" and Daniel W. Klosiewict Research Center, Hercules Incorporated, Wilmington, Delaware 79899
Carboxymethylcellulose (CMC) was hydrolyzed in aqueous sulfuric acid at -90 OC to yield substituted glucoses which were analyzed by proton nuclear magnetic resonance. A procedure for the measurement of the degree of substitution (DS) of CMC was developed. Agreement of the DS determlned in thls procedure with that obtalned from a standard ASTM method is generally good. Additionally, the average distribution of the carboxymethyl substituents in CMC was also measured from the same spectrum. This result showed that the reactivity of the hydroxyls in cellulose toward carboxymethylation varied in the order OH(2) > OH(6) > OH(3), where the numbers In parentheses represent the carbon posllions in the anhydroglucose unit.
Carboxymethylcellulose (CMC) is an important industrial polymer which finds wide application in detergents, textiles, paper, food, drugs, and oil well drilling operations, among others. In 1974, over 75 million pounds were produced in the United States alone ( I ) . Many of the properties of CMC in actual applications depend t o a large extent on two key structural parameters, namely, the degree of substitution (DS) of the hydroxyl groups on the anhydroglucose unit and the distribution of the carboxymethyl substituents. T h e DS of CMC can be determined by a number of analytical techniques which were critically reviewed by Timokhin and Finkelshtein ( 2 ) ,Grosse and Klaus ( 3 ) ,and more recently by Schleicher e t al. ( 4 ) . T h e most common procedures in use include titrimetry (potentiometry (5) and conductometry (6)), gravimetry (copper (7) and uranyl nitrate (8) assays), and colorimetry (using 2,7-dihydroxynaphthalene(9) and disulfine blue (10) reagents). In these analyses, multiple steps and lengthy sample preparation are frequently encountered. In addition, all of these procedures yield only the DS value, without providing information on the distribution of the carboxymethyl substituents. A rapid proton nuclear magnetic resonance (NMR) procedure is presented in this paper, which is capable of providing both the DS and the average distribution of the substituents.
EXPERIMENTAL Sample Preparation. The CMC samples with various DS
levels which were examined in this work were purified experimental or commercial materials from Hercules Incorporated synthesized by reacting alkali cellulose with sodium monochloroacetate under rigidly controlled conditions (11). No further purification was carried out. In a typical experiment, about 150 mg of sample was weighed into a 5-mL glass vial. Subsequently, 1 mL of D 2 0 was added and the CMC allowed to swell. Following this, -1 mL of a 50/50 (v/v) mixture of D 2 0 + D2S04was introduced slowly, to disperse the CMC sample as completely as possible. This slurry was heated at -90 "C for a given period of time, with occasional shaking, to help break up the gel. The actual heating time required depends on the crystallinity and the DS of the sample, but is generally between 0.5 to 2 h. A t the end of the hydrolysis, effectively all of the samples examined had
-
0003-2700/80/0352-0913$01 .OO/O
dissolved and formed a homogeneous solution of low viscosity having a slight yellow color. A portion ( -0.4 mL) of this solution was filtered directly into an NMR tube for measurement. NMR Measurement. NMR spectra were obtained on a Varian EM-390 spectrometer operating a t 90 MHz and at an ambient probe temperature of 25 "C. A typical set of spectra are shown in Figure 1 (spectral width = 5 ppm). In these spectra, a small peak at -2.0 ppm is due to acetic acid, which was introduced as an internal reference. The main group of signals in the region between 3-4 ppm arises from the six C-H protons, associated with the C2to C6 carbons in an anhydroglucose ring (Figure 2). The portion of the spectrum, below 4 ppm, contains important information regarding the substitution in these carboxymethylated cellulose samples. An expanded spectrum (spectral width = 2 ppm) of this region from a CMC sample with DS = 0.8 is shown in Figure 3. NMR measurements at elevated temperatures were carried out on a Varian A-60 spectrometer operating at 60 MHz with a variable temperature probe. Spectra obtained a t 32 and 80 "C are shown in Figures 4a and 4b, respectively. In these figures, the strong signal arising from acid protons and residual water is not shown. This signal is generally observed a t a much lower field (-8 ppm). A pair of spinning side bands from this signal are always present and usually show peak heights equal to about 1-2% of the main peak. The magnitude of this spinning side band is applied to correct the integral of the carboxymethyl region (4.0-4.5 ppm; see Figures 1 and 3). This correction is required because of a contribution to the integral value from the spinning side band of the main C-H peak at -3.8 ppm. To increase accuracy, the spinning side band should be minimized by carefully adjusting the homogeneity controls on the spectrometer and by using a high quality NMR tube.
RESULTS AND DISCUSSION Spectral Analysis. Although NMR has been utilized extensively for compositional analysis as well as structural characterization of polymers (12, 13), its application t o industrial cellulosic polymers has been rather limited (14-18). This limitation results primarily from the relatively low solubility and high viscosity of solutions of these materials used for NMR analysis. Often, sample preparation must be aided by a molecular degradation (17, 18). Acid hydrolysis of cellulose is one method which is generally rapid and complete (19). From studies done on CMC, the complete hydrolysis to substituted monomeric glucose, with no effect on the carboxymethyl groups, has been reported (20,21). Time11 (20) carried out the hydrolysis of CMC in 72% sulfuric acid a t room temperature for 48 h, followed by dilution and refluxing of the aqueous solution. In our work, the hydrolysis of CMC was conducted in a moderately concentrated aqueous sulfuric acid (acid strength -20%) a t -90 "C for approximately 1 h. Samples with a higher degree of substitution require a longer period of heating. T h e progress of the hydrolysis was conveniently monitored by observing an increase in the intensity of the NMR signals from protons a t the reducing end (C,) of the degraded sugar. This group of signals arises as a result of the cleavage of the glycosidic linkage in cellulose, and is observed in Figures 1 and 3 as two sets of doublets in the spectral region between 4.5 and 5.5 ppm. The 1980 American Chemical Society
914
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6 , MAY 1980
Figure 1. NMR spectra of acid hydrolyzed CMC samples at 90 MHz: = 0.4, (B) DS = 0.8, (C) DS = 2.0
(A) DS
I , , , , I
I
,
,
,
l,,,,l8,,,l,ll
5.0 4.0 3.0 Figure 4. NMR spectra of a CMC sample (DS = 1.4) at 60 MHz obtained at (A) 32 OC; and (8) 80 OC OH
Figure 2. An anhydroglucose ring ,6'
Figure 3. NMR spectrum of a CMC sample with DS = 0.8 showing the expanded portion of the spectrum between 4.0-5.5 ppm (S = substituted and U = unsubstituted at C,) at 90 MHz
doublet splitting is caused by coupling with the single proton at Cz. T h e set of doublets at the lower field can be assigned to the proton at C1 of the a-anomer, while the set of doublets a t t h e higher field was attributed to the proton at C1 of the @-anomer(22). The S and U identifications of the doublets in Figure 3 refer to substituted and unsubstituted hydroxyl group a t C2. T h e carboxymethylation of the hydroxyl group a t C2 produced a substantial downfield shift in the proton at C1. This observation was established by a comparison of the intensity of the signals attributable to the proton a t C1 as a function
of the degree of substitution (see Figures 1(A), (B), and (C)). In a completely hydrolyzed sample, the ratio of the integral value of signals from protons at the reducing end to the other C-H protons of the glucose unit (signals between 3-4 ppm) was generally measured a t 1: 6 as expected, indicating that the hydrolysis is quantitative. T o have an accurate measurement, a complete hydrolysis is necessary. Most significantly, protons from the carboxymethyl (-OCH,COO-) group were observed alone in a spectral region between 4.0-4.5 ppm (see Figures 1 and 3). This provides a new analytical approach for the determination of D S , a measurement of the extent of carboxymethylation of the cellulose. I t was observed that similar spectra taken from glucose (22) or from a hydrolyzed cellulose sample, prior to carboxymethylation, do not contain signals in this region. Furthermore, the carboxymethyl CH2group gives rise to four sharp and intense signals in this region, thus permitting an analysis of the average distribution of the substituents. This measurement yields some information on the relatzue reactiuity of the three hydroxyl groups in an anhydroglucose unit. Applications of these two areas of analysis will be discussed separately. DS Determination. From the above discussion on the spectral analysis of CMC, it is clear that the DS measurement in this NMR procedure is simply the measurement of a ratio of two spectral integrals, A / B , where A is one half of the integral of the carboxymethyl signals in the region between 4.0-4.5 ppm, and R is the integral representing an area of one proton in an anhydroglucose unit. The value of B can be obtained either from the direct summation of the two sets of
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980
Table I. DS Determination of CMC Samples sample no.
DSby titrationu
DS bg NMR
1
0.44
2
0.75 0.88 1.38 1.80
0.53 0.80
3 4
5
‘
O
1
std. dev.
1.39
0.013 0.014 0.016 0.012
1.82
0.019
0.92
’
915
Titration by a standard acid wash procedure described in ASTM D 1439(78). The mean of measurements from 5 different specimens of a sample. doublets at the lower field (due to the single proton a t C 1 a t the reducing end) or by using one-sixth (l/,J of the total integral of the major C-H signals a t a higher field (between 3-4 ppm). In practice, the value of B is usually obtained from the average of these two individual measurements which are typically close in agreement. A typical set of data on DS determinations is presented in Table I. Included in Table I are the DS values obtained by this method on five samples, with each value representing the mean of five separate measurements. The standard deviation of these measurements is also included, and is approximately 2% of this mean. This deviation is acceptable and is primarily limited by the precision of the electronic integrator available in commercial NMR instrumentation. For comparison, DS values measured on these CMC samples using a standard ASTM (acid wash/base consumption) procedure ( 5 ) are also included in Table I. Although the agreement between values obtained from both techniques is generally good, the NMR results do tend to be slightly higher. In the titration procedure ( 5 ) , concern has been expressed ( 4 ) that some functional groups might not be accessible to the reagent, since a heterogeneous slurry with a swollen polymer is involved. In this case, difficulty may exist in obtaining a complete exchange of sodium ions and protons. A homogeneous system employing a combination of dimethyl sulfoxide/methyl amine/ ethyl amine as solvent has been utilized ( 4 ) . In the homogeneous system, a slightly higher result was also reported. However, the increases in DS values obtained from a homogeneous titration ( 4 ) and also from our work, are small and do not provide conclusive evidence on the question of the reagent accessibility problem. I t is significant to note that common impurities, such as water, NaC1, carbonates, and other inorganic salts will not interfere in this procedure. Weighing and drying of a sample is not necessary. Distribution of Substituents. Another significant result from this analysis is the measurement of the distribution of the carboxymethyl substituents. None of the other available analytical methods referred to previously for DS measurement can provide this information. T h e four sharp and intense signals from the carboxymethyl groups in the spectral region between 4.0-4.5 ppm (see Figures 1 and 3) can be assigned to carboxymethylation of hydroxyl groups at C3, C2u, C g , and C,, respectively, going from lower field to higher field. The assignment of the C2a and C d peaks is difficult, but is assisted by a variable temperature experiment. In Figure 4, a spectrum of a CMC sample a t elevated temperature, obtained at 60 MHz, is presented. At ambient temperature (Figure 4A), the spectrum is very similar to those obtained at 90 MHz (Figures 1 and 3). T h e two doublets a t -4.7 ppm, assigned to the proton a t C1p, are more evident a t 60 MHz (Figure 4A), since these doublets overlap in spectra taken at 90 MHz (Figures 1 and 3). Two peaks assigned to the C2a and C 2 p carboxymethyl groups are seen a t 4.3 and 4.4 ppm. However, a t 80 OC (Figure 4B), the rate of mutarotation between the a- and p-anomeric forms in this acidic medium is much faster. At
DEGilEE OF
SUBST!TUTION
Flgure 5. Calculated curves and experimental values of the distribution of substituents as a function of DS
this temperature, a complete spectral averaging took place between the C 2 a and C2p peaks to form a broadened singlet a t 4.35 ppm. It can be estimated (23) that this rate of exchange is approximately equal to 2r.A or 62.8 s-l, where .A is the separation between the C p and C2a and C2@signals (10 Hz) in the absence of fast exchange. At this rate of mutarotation, however, a complete signal averaging between the Clu and C,@signals (between 4.5 and 5.5 ppm in Figure 4) was still not possible, producing only greatly broadened signals in this region a t 80 “C. This is caused by the fact that the C l a and C 1 p signals are much farther separated than the C 2 a and C2/3 peaks. A faster rate of mutarotation is therefore required to produce a complete spectral averaging of the C l u and Cl@signals. The differentiation of signals between C 2 u and C@ is based on the fact that the latter is the larger of the two signals. Larger concentration of the @-anomerhad been reported (22) and was evident in this work by a comparison of the Cl@and the Cla signals in the lower field of the spectra (see Figures 1 and 3). The assignment of signals of carboxymethyl substituents a t C3 and c6 is much simpler because the former is always smaller in accord with the observations that the hydroxyl group at C 3 of the anhydroglucose unit in CMC is the least reactive one (18, 21, 24-26). The measurement of the distribution of the carboxymethyl substituents a t C2, C3, and c6 (based on the integral value of the respective signals) can be best obtained on a n expanded spectrum (spectral width = 2 ppm; see Figure 3). Care must be exercised with regard to eliminating interference from possible impurities. For example, sodium glycolate, if present, gives a singlet just to the right of the c6 signal a t -4.2 ppm. However, a glycolate impurity can be easily removed by a wash using aqueous methanol. The measurement of the distribution of the carboxymethyl substituents at the C p ,C B ,and c6 hydroxyl groups was applied to a large number of commercial and experimental CMC samples prepared under similar conditions. It is generally observed that there is slightly more substitution a t C 2 than a t Cg, with the least amount a t C3, indicating that the relative reactivity of the hydroxyls is in the following order: OH(2) > OH(6) > OH(3). A summary of these data is presented in Figure 5. Also, included in Figure 5 are calculated curves on the distribution of carboxymethyl groups as a function of DS employing the following two assumptions: (1)the carboxymethylation follows first-order kinetics, and (:!) the substituents have no influence on the rate of reaction of the unreacted hydroxyls. The set of computed curves shown in Figure 5 was calculated using a relative reactivity ratio of OH(2):OH(3): OH(6) = 2:1:1.5, which yields a “best fit” to the experimental data compared to curves calculated using other reactivity
916
Anal. Chem. 1980, 5 2 ,
Table 11. Relative Reactivity of Hydroxyl Groups in C~llulosetoward Carboxyinethylation authors Time11 ( 2 4 ) Ckoon/Purves (25)
Buytenhuysl Bonn ( 2 1 ) Ho/Klosiewicz Sonnerskog
k,:k,:k,
1:1:2 2:1:2.5
no interference strong interference
2.O:l :1.4a
no interferences
4.6:1:3.6b 2 : l : 1.5 (k,
(26)
Parfandry / Periin ( 18 )
interference of substituents
k,
+
k , ) / k , = 19:l
> k , >> k ,
a Mole ratio of water/cellulose = 7 . water/cellulose = 1 4 .
not investigated not investigated not investigated Mole ratio of
ratios. Although the fit with the experimental data is fairly good, some scatter in the results occurred. This is not surprising since the samples analyzed were not specifically prepared for the purpose of a precise reactivity ratio study. This would have required that all samples be synthesized under an identical set of reaction conditions. Nevertheless, the measured relative reactivity of the hydroxyl groups in cellulose toward carboxymethylation is generally in line with literature data, most of which was obtained from a complete chromatographic analysis of the hydrolyzed products (21,24,25). A comprehensive search of reported data on this subject is summarized in Table 11. Except for the early results by Timell (24) and Croon and Purves (25), it appears that the literature data agree that the reactivity of the hydroxyl group a t C2 toward carboxymethylation is slightly greater than that at C,. This can be explained by the higher acidity and greater accessibility of the hydroxyl at C2 compared to C6,even though the latter is a primary -OH group.
ACKNOWLEDGMENT T h e authors thank J. D. Schlueter and C. A. Lewis for the
916-920
DS determination by the ASTM procedure and A. Z.Conner and R. W. Harrell for helpful discussions and encouragement. LITERATURE CITED (1) "Synthetic Organic Chemicals, U.S. Production and Sales", U.S. International Trade Commission, Publication No. 804: U.S. Government Printing Office: Washington, D.C., 1977. (2) Timokhin, I. M.; Finkelshtein, M. 2 . J . Appl. Chem., USSR (Engi. Transl.) 1963, 36, 394-400. (3) Grosse, L.: Klaus, W. Papier(Darmstadt) 1971, 25, 833-836. (4) Schleicher, H.; Dantzenberg, H.; Philipp, B., Papier (Damstad)) 1977, 37, 499-503. (5) ASTM D 1439-72 (78). (6) Eyler, R. W.; Klug, E. D.; Diephuis. F. Anal. Chem. 1947, 79, 24-27. (7) Conner, A. 2 . ; Eyler, R. W. Anal. Chem. 1950, 22, 1129--l132. (a) Francis, C. V. Anal. Chem. 1953, 2 5 . 941--943. (9)Calkins, V. P. Ind. fng. Chem., Anal. Ed. 1943, 15, 752-763. (IO) Mukhopadhyay, S.;Mitra, B. C.; Palit, S. R. Anal. Chem. 1973, 4 5 . 1775-1776. ( 1 1) Batdorf, J. B.; Rossman, J. M. "Sodium Carboxymethylcellulose" in "Industrial Gum"; Whistler, R. L., BeMiller, J. N., Eds.. 2nd ed.;Academic Press: New York, 1973. (12) Bovey, F. A. "High Resolution NMR of Macromolecules"; Academic Press: New York. 1972. (13) Pasika, W. M., Ed., "Carbon-I3 NMR in Polymer Science"; ACS Symposium Series No. 103, American Chemical Society: Washington, D.C., 1979. (14) Goodlett, V. W.; Dougherty, J. T.; Patton, H. W. J . Polymer Sci., Pad A - 7 1971, 9 , 155-161. (15) Ho, F. F.C.; Kohler, R. R.; Ward, G. A. Anal. Chem. 1972, 4 4 , 178-181. (16) DeMember, J. R.; Taylor, L. D.; Trummer, S.; Rubin, L. E.:Chikiis, C. K. J . Appl. Polymer Sci. 1977, 27, 621-627. (17) Clement, C. J. Anal. Chem. 1973, 45, 186-188. (18) Parfondry, A,; Periin, A. S. Carbohydr. Res. 1977, 51, 39-49. (19) McBurney L. F. in "Cellulose and Cellulose Derivatives", Ott, E. Spuriin, H. M., Graffin, M. W., Ed., Part I; Interscience Publishers: New York, 1954; p 130. (20) Timell, T. E. Sven. Papperstidn. 1952, 55, 649-660. (21) Buytenbuys, F. A.; Bonn, R. Papler (Darmstadt) 1977, 3 1 , 525-527, (22) Perlin, A. S.; Casu, 8 . Tetrahedron Lett. 1969, 2921-2924. (23) Pople, J. A.; Schneider, W. G.; Bernstein, H. J. "High-resolution Nuclear Magnetic Resonance"; McGraw-Hill: New York, 1959; p 223. (24) Timell, T. E. Sven. Papperstidn. 1953, 5 6 , 483--490. (25) Croon, I.; Purves, C. B. Sven. Papperstidn. 1959, 62, 876-882. (26) Sonnerskog, S. Sven. Papperstidn. 1948, 51, 50-51.
RECEIVED for review November 2,1979. Accepted February 7,1980. Hercules Research Center Contribution Number 1721.
Spectrophotometer Based on a Charge-Coupled Device Photoarray Detector Kenneth
L. Ratzlaff
Department of Chernisrry, The Michael Faraday Laboratories, Northern Illinois University, DeKalb, Illinois 60 1 15
A spectrophotometer is described in which a linear photoarray using charge-coupled technology is used as a detector. The device can access 260 nm of the visible spectrum in as little as 8 ms. Using the detector's integrating character, the exposure time is programmed by computer so that the signalto-noise ratio can be substantially Improved. A model for the variance as a function of wavelength and absorbance was developed and validated.
In the past several years, multichannel imaging devices have gained acceptance as detectors for visible spectroscopy. Thus far the vidicon ( I ) , a tube device, and the photodiode array ( 2 ) , a solid-state device, have been used extensively; a t the
same time, the solid-state charge-coupled device, although the most promising for commercial video imaging (31, has seen only a few spectroscopic applications ( 4 ) . Solid-state detectors have several geometric advantages including those of being easier to implement since they are self-scanned, of being easily cooled with solid-state thermoelectric devices, and of being geometrically precise. Among solid-state imaging devices, the photodiode array has been used for visible molecular absorption spectrophotometry to a limited degree. Applications have included systems for the study of kinetics ( 5 ) , for monitoring process water (6). for monitoring liquid chromatography effluent ( 7 ) , and for in-vivo measurement (8). We have previously characterized the charge-coupled device as a detector (9). It was found to be linear with intensity Q 1980 American Chemical Society
