2D NMR Analysis of Ethylcellulose - American Chemical Society

Chapter 25

Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on December 5, 2014 | http://pubs.acs.org Publication Date: December 10, 2002 | doi: 10.1021/bk-2003-0834.ch025

2D NMR Analysis of Ethylcellulose 1

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Qiuwei Xu , M a r k Brickhouse , and Huiming Wang

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Merck Research Laboratory, P.O. Box 4, WP78-107, West Point, PA 19486 Hercules Incorporated, Research Center, 500 Hercules Road, Wilmington, DE 19808

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2-D NMR was used to characterize native and acid hydrolyzed ethylcellulose (EC), a Hercules product widely used as a film-former in ink and coatings applications and as a binder and filler in pharmaceutical applications. An important parameter in controlling the properties of ethylcellulose is the degree of substitution (DS) of ethyl functionalities on the cellulose backbone. NMR is one technique that was used to determine both the total and positional DS (ethylation at the 2,3 and 6 positions of the anhydroglucose unit (AGU)). This analysis requires complete hydrolysis of the sample, and an improved acid hydrolysis technique was developed for this application. Two-dimensional(2-D)NMRtechniques were used to confirm peak assignments related to positional DS determinations that were previously made by comparison with standards. In addition, 2-D NMR methods were used to evaluate positional DS of native ethylcellulose prior to acid hydrolysis. A comparison of the analytical results for the acid hydrolysate and native polymer will be discussed.

© 2003 American Chemical Society

In NMR Spectroscopy of Polymers in Solution and in the Solid State; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

325


326 Ethylcellulose a is cellulose ethyl ether. It is a widely added ingredient to formulations of varnishes, inks, lacquers and adhesives. Its elasticity enables it to form films, foils and plastics (1). Owing to its inert chemical properties, it can be used for food-contacting packaging, drug tablet coating and binding. Microencapsulating drugs with ethylcellulose can controls drug release in vivo (2,3,4). In βΐ—>4 linked D-glucose, there are three accessible hydroxyl groups for substitutions, the C2, C3 and C6 positions (Figure 1). Ethyl substitutions at these positions are measured as degree of substitution (DS) and positional degree of substitution (PDS). The former indicates the averaged number of ethoxy groups on each glucose residue. The latter shows the distribution of substitution over the C2, C3 and C6 positions. The physical properties of ethylcellulose are determined by these structural parameters. N M R has played an important role in determining the chemical structures of industrial and bacterial polysaccharides (5,6,7,8). Typical polysaccharides show fingerprint region on ID proton and carbon spectra. Anomeric protons or carbons usually appears in the downfield region; however, ring protons tend to be crowded in a narrow region of chemical shift (~lppm). Despite the better chemical shift dispersion of C N M R , its low sensitivity and low natural abundance makes it undesirable to acquire direct carbon spectra, especially of rigid or associated polysaccharides. The extension of I D proton N M R into proton-proton 2D N M R has shown an improvement in resolving overlapping proton peaks in the upperfield region while maintaining a proper sensitivity. Inverse-detected H - C spectra were acquired and used to investigate the structural differences of ethylcellulose polymers and their acid hydrolysates. 1 3

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l 3

Experimental Preparation of acid hydrolyzed E C was performed exclusively with disposable glassware. ~500 mg of E C is dispersed in 4-5 ml of a trifluoroacetic acid (TFA)/water solution (prepared gravimetrically, 74.47 grams T F A in 32.52 grams deionized water). The dispersion was prepared in a loosely capped 17 ml vial and heated on a Thermolyne Type 16500 DriBath @ 75-80C for 24-45 hours. The samples were heated sufficiently long to minimize the glycosidic Ο Ι carbon peak @ 102 ppm (from intact polymer and oligomer). However, care was taken to prevent caramelization of the samples. It was typical for the samples to turn light brown under these conditions; overheating caused the samples to turn black. After heating, the samples were allowed to air-dry in a disposable evaporating dish for at least two hours with mild heating. The resulting solids were dissolved, with mild heating, in ~ 4ml d6-DMSO, which contains 0.04M Cr(acac) as a relaxation agent. The native E C polymer was dissolved i n C D C 1 at ~40mg/ml. Most N M R measurements were made on a Bruker Avance 500MHz instrument with a 5mm pulse field gradient (PFG)-Broadband Inverse(BBI) 3

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In NMR Spectroscopy of Polymers in Solution and in the Solid State; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2002. 3

Figure 1. Annotated Proton N M R Spectrum of Ethylcellulose in CDC1 with Generic Structure of Ethylcellulose of DS=3

A



328 probe. The data were acquired without sample spinning at 40°C for the polymer sample, and at 25°C for the hydrolysate. The phase sensitive spectra were acquired using the TPPI-States or TPPI scheme. The Hartmann-Hahn transfer in the Homonuclear Hartmann-Hahn ( H O H A H A ) experiments were realized with the composite pulses of M L E V 1 7 at 4.16kHz. The mixing time was set to 40ms. The 90 degree pulse of carbon high power was 10.5 usée. During proton acquisition in the Heteronuclear Multiple Quantum Coherence (HMQC) experiments, the carbon decoupling was applied with a field strength of 3.88 kHz. The duration of magnetization transfer from proton to carbon in H M Q C was set at 3.22ms (155Hz). The duration of developing long range protoncarbon correlation in the Heteronuclear Multiple Bond Correlation ( H M B C ) experiments was set at 70ms. For the polymer sample, the proton and carbon carrier frequencies were set at 500.1316568 and 125.7684284 M H z , respectively. The spectral widths of proton and carbon dimension were 5122.951 Hz (-lOppm) and 18865.393 Hz (150ppm) respectively. The 90 degree pulse of proton was 7.60 usee @ 5.0 dB power level. For the hydrolysate sample, the proton and carbon carrier frequencies were set at 500.132 and 125.768 M H z respectively. The spectral widths of proton and carbon dimension were 3501.40 Hz (~7ppm) and 18865.393 Hz (150ppm) respectively. Double Quantum Filtered Correlated Spectrocopy (DQF-COSY, 9,10,11) H O H A H A (12,13), H M Q C (14,15) and H M B C (16) spectra were acquired sequentially on each sample using multiple experiment automation on the Bruker 500 spectrometer. Between runs, lock was reset to 90% and Z l was re-shimmed without any sample spinning. The N M R spectrum shown in Figure 3 was acquired on a 600 M H z Bruker Avance N M R with a 5 mm PFG-TXI probe. The proton and carbon carrier frequencies were set at 600.1336081 and 150.9177568 M H z , respectively. The spectral widths of proton and carbon dimension were 8012.82 H z (-13 ppm) and 33557.015 Hz (222 ppm) respectively. The 90 degree pulse of proton was 6.90 usee @ -2.0 dB power level.

2-D N M R Analysis of Intact Ethylcellulose

Two structural features that affect the physical properties of E C are degree of substitution (DS) and positional substitution of ethyl groups. DS indicates the averaged number of ethoxy groups on each anhydroglucose repeating unit (AGU). Positional substitution is the distribution of ethyl groups at the three potential substitution sites, the hydroxyl groups C2, C3 and C6 (Figure 1). Historically, Hercules has used 1-D C N M R of acid-hydrolyzed E C to detennine positional DS (17). One of the goals of this work was to 1 3



329 determine i f 2-D N M R could be used to determine positional DS and positional DS of intact EC. The proton spectrum (Figure 1) of the polymer shows broad peaks, typical for a rigid polymer. The peaks on the left side of the spectrum (δ~ 4.3 ppm) are anomeric protons, the large peak on the right side are from the methyl protons of the ethyl groups ( δ - 1.2 ppm). The middle region contains the pyran ring and methylene protons of the ethoxy groups. The identification of proton correlation was assisted by D Q F - C O S Y , which identified adjacent protons by off-diagonal cross peaks. The assignments of the three pendant methylene proton substituents at 2,3 or 6 of the A G U ring were made through long range proton-carbon correlation (HMBC) as discussed below. The H M Q C and H M B C proton-carbon correlation experiments were used in combination to make the assignments of the ring protons as shown in Figure 2. The H M Q C and H M B C spectra are overlayed in Figure 2. H M Q C correlated proton with one-bond-linked carbon, and H M B C correlated protons and carbons that are typically 2-3 bonds apart. The contours in black were generated by H M Q C , and the ones in gray by the H M B C techniques, respectively. The ethyl linkages to the glucose and glucose-glucose linkages were revealed by the three-bond proton-carbon correlation. With the help of chemical shift dispersion along the carbon dimension, we could see that H4 overlaps with H6, so does H3 with H5. The cross peaks (H1-C4 and C1-H4) on H M B C arose through glycosidic linkages rather than through intra-ring coupling interactions. These cross peaks reflected the β1->4 linkage for cellulose backbone. The substitution position of ethoxyl groups was located based on the correlation peaks of methylene proton on ethoxyl groups and ring carbons. For example, the gray H M B C peaks labeled S3CH2-C3 indicates the three bond coupling between the sidechain methylene protons and the glycosidic carbon at position C3. The degree of substitution (DS) of the ethylcellulose sample described above was determined to be 2.7 by the Sealed-Tube Zeisel (STZ) technique. However, STZ DS determinations require the exhaustive acid degradation of polymer samples such as ethylcellulose. DS determinations of ethylcellulose were not usually performed on intact polymer samples. The peak separation from two-dimensional N M R spectra could conceivably help obtain DS values directly from intact polymer. Relative positional DS at C2, C3 and C6 could be obtained from the integration of cross peaks on H O H A H A (Figure 3). The adjacent proton coupling constants for the three ethoxyl groups were found to be similar (~15Hz) as measured from DQF-COSY. The three H O H A H A cross peak


In NMR Spectroscopy of Polymers in Solution and in the Solid State; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2002. l

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Figure 2. H - C 2-D Short Range H M Q C (black) and Long Range H M B C (gray) Correlation Spectra of Ethylcellulose in CDC1


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intensities were estimated to reflect the relative substitution percentages on these three positions. The overlapping methyl protons of the 1-D *H spectrum were resolved thanks to the chemical shift dispersion of the methylene protons. The relative positional DS values were determined by integrating the peaks labeled as C3, C2 and C6 on both sides of the diagonal line, and taking the average of these two sets of data.

4.4

3.6 Dl_

2.8 2.0 (ppm;

1.2

Figure 3. HOHAHA of ethylcellulose polymer, 40ms mixing The calculated amounts of relative ethyl substitution at C2, C3 and C6 are tabulated in Table 1 together with the results of the analysesfromGC-MS and C NMR analysis of acid-hydrolyzed EC monomers. While the general trend of lesser substitution at the 3-postion is correct, the relative amount of 3substitution for an EC with a DS value of 2.7 (as detennined by the STZ technique), as is the case with this EC, cannot drop below 26%. This is because if ethylation of C2 and C6 is complete, positional DS at C2 (PDS ) = 1, PDS = 1 and then PDS = 0.7. Since 0.7/2.7 = 26%, the minimum relative substitution at C3 is 26%. l 3

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Table 1. Relative Positional DS of intact ethylcellulose polymer. Position 3 2 6 Percentage 10% 50% 40% (29% ,31% ) (34%*,35% ) (36%*, 33% *) a. calculatedfromGC-MS data on an acid hydrolysate b. calculatedfromID carbon data on an acid hydrolysate a

b

b


332 l3

A review of the *H- C H M Q C (black) and H M B C (gray) spectra shown in Figure 3 shows the S3-CH H signal is split in the proton dimension into two peaks centered at 3.7 and 4.0 p p m The former peak overlaps the S2-CH peak in the lower resolution H O H A H A spectrum shown in Figure 4. As a result, relative positional DS values calculated from the H O H A H A spectrum would be low for C3 and high for C2. This work shows that 2-D N M R analysis of intact E C can provide structural information on polymer substitution, but not quantitative data on positional DS without substantially more effort to resolve peak overlaps spectroscopically or by deconvolution of overlapping 2-D peak volumes. !

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2-D N M R Analysis of Acid Hydrolysed Ethylcellulose Previous positional DS determination of ethylcelluloses were performed on samples subjected to exhaustive acidic methanolysis and analyzed by 1-D C N M R (17). This method required four days for the digestion of each sample, and assignments of the side chain methylene carbons bonded to C2, C3 and C6 were made by comparison with synthetically prepared reference materials. Recently, a faster digestion method was developed (as described in the Experimental section), and it was necessary to confirm that C assignments (Figure 4) used to determine positional DS after acidic methanolysis method were still valid for samples digested in T F A solutions. 1 3

1 3

Οόα,β

03α,β C2p

C2a

ppm

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Figure 4: C N M R Spectrum of TFA-Hydrolyzed Ethylcellulose Side Chain Methylene Carbons Assigned According to Reference Spectra from Acidic Methanolysis Method (a and β are anomers of ethylated glucose)


333 A combination of H M Q C and H M B C spectra, as described above, allowed for confirmation of the C chemical shifts without the need of re acquiring external reference standards. Figure 5 shows how scalar coupling interactions were tracked from βΟΙ to the sidechain methylene carbons substituted at the C2 position (Since the E C has been hydrolytically digested, the hydrolysate contains a mixture α and β anomers of glucose ethoxylated at C2,C3andC6). The same "spin-walk" analysis was also performed for the C3 and C6 side chain methylenes, on both α and β anomers. The C chemical shifts of the sidechain methylene carbons determined in this fashion confirmed the assignments used to determine positional DS by 1-D N M R , as shown in the assigned spectrum in Figure 5. Like die calculation of the relative DS made for the intact polymer, the relative positional DS of the TFA-hydrolyzed E C can also be calculated from the cross peaks on H O H A H A spectrum (Figure 6). The relative positional DS values are listed in Table 2. Because of the improved spectral resolution of the hydrolyzed E C relative to the intact EC, there are no signal overlaps for the sidechain methylene protons and the calculated values are almost the same as those determined by GC-mass spectrometry and ID C N M R analyses on the acid hydrolysate. However, since the C N M R method provides an absolute rather than relative positional DS value, it is still the method of choice for E C characterization.


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Table 2. Relative Positional DS of TFA-hydrolyzed ethylcellulose polymer 2 6 Position 3 34% Percentage 36% 30% (34% 35% ) (29% ,31% ) (36% \ 33% ) a. calculated from GC-mass data b. calculated from ID carbon data 1

b

b

b

Conclusions This study shows that 2-D N M R analysis of intact E C can provide structural information, such as confirming the presence of cellulose ethoxylated at the C3 position, but does not readily yield quantitative information such as positional DS. 2-D N M R analysis of TFA-hydrolyzed E C corifrrms formation of a complex mixture of ethoxylated α and β - glucose anomers. A combination of H M Q C and H M B C analyses was used to confirm the assignments used for quantitative 1-D C N M R analysis without reference to external standards. Because of improved spectral resolution, H O H A H A analysis of hydrolyzed E C afforded relative positional DS that agreed well with values determined by G C l 3



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Figure 5: H - C 2-D Short Range H M Q C (black) and Long Range H M B C (gray) Correlation Spectra of TFA-Hydrolyzed Ethylcellulose in d6-DMSO


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o

3.85

3.80

3.75

3.70

3.65 3.60 3.55 DI_ ippm)

3.50

3.45

3.40

3.35

Figure 6: A section of cross peaks in the H O H A H A spectrum of the ethyl cellulose hydrolysate l 3

M S and 1-D C N M R . In general, it was found that while 2-D N M R techniques were superior for absolute structure determination, 1-D N M R analyses were preferable for quantitative analysis.

References 1. "Aqualon Ethylcellulose (EC) Physical and Chemical Properties" Hercules Inc. 2. P B Deasy, MR Brophy, B . Ecanow, MM Joy J. Pharm Pharmacol 32, 1520(1980) 3. M . Ito, M Nakano, Chem. Pharm. Bull (Tokyo) 28, 2816-2819 (1980) 4. J. R. Nixon, M. R. Meleka J. Microencapsul 1, 53-64 (1984) 5. Abeygunawadana C., and C. A . Bush Adv. Biophys. Chem. 3, 199-249 (1993) 6. B . Molly, M o l Biotechnol 6, 241-265 (1996) 7. L . Lerner, Basic Life Sci. 56, 255-271 (1990) 8. T A Koerner, R K Y u , J N Scarsdale, P C Demou, J H Prestegard Adv. Exp. Med. Biol. 228, 759-784 (1988) 9. U . Piantini, O. W. S⇃renson, R. R. Ernst, J. Am. Chem. Soc. 104, 68006801 (1982) 10. M . Ranee, O. W . S⇃renson, G . Bodenhausen, G . Wagner, R. R. Ernst, K . Wuthrich Biochem Biophys. Res. Commun. 117, 479-485 (1983) 11. Α. Ε. Derome, M. P. Williams, J. Magn. Reson. 88, 177-185 (1990) 12. L . Braunschweiler, R. R. Ernst, J. Magn. Reson. 53, 521-528(1983) 13. A . Bax, D . G . Davis, J. Magn. Reson. 65, 355-360 (1985) 14. L. Muller, J. Am Chem. Soc. 101, 4481-4484 (1979) 15. A . Bax, R. H. Griffey, B . L . Hawkins, J. Magn. Reson. 55, 301-315 (1983) 16. A . Bax, M. F. Summers, J. A m . Chem. Soc. 108, 2093-2094 (1986) 17. J. Reuben and H.T. Conner, Hercules D D R 28-096-1, December 21, 1982


2D NMR Analysis of Ethylcellulose - American Chemical Society

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