Anal. Chem. 2001, 73, 4852-4861
Ultrahigh Molar Mass Component Detected in Ethylhydroxyethyl Cellulose by Asymmetrical Flow Field-Flow Fractionation Coupled to Multiangle Light Scattering Mats Andersson,† Bengt Wittgren,‡ and Karl-Gustav Wahlund*,†
Department of Technical Analytical Chemistry, University of Lund, Box 124, SE-221 00 Lund, Sweden, and AstraZeneca R&D Mo¨lndal, SE-431 83 Mo¨lndal, Sweden
Asymmetrical flow field-flow fractionation (flow FFF) was connected to multiangle light scattering (MALS) and refractive index (RI) detectors for characterization of the molar mass distribution and molecular radius of a cellulose derivative, ethylhydroxyethyl cellulose (EHEC). Experimental conditions were optimized to allow study of a wide range of molar mass including even ultrahigh molar mass (UHM) components. The weight-average molar mass was 3.1 × 105 g‚mol-1 representing a very broad range (of molar mass) from 4.0 × 104 to 107 g‚mol-1, which corresponds to from 13.5min retention time). The reason is the high imprecision in the measurement of the molar mass (cf. eq 10) mainly caused by a very low RI signal-to-noise ratio. Polymer Conformation. From light scattering measurements combined with a separation method, information about polymer conformation can be obtained from the well-known relationship48,49
〈r2g〉1/2 ) kMγ
(11)
By plotting the logarithm of rms radius versus the logarithm of molar mass, the coefficient γ is obtained from the slope. Typical values are 0.33 for a spherical shape and 0.5-0.6 for a random coil. For EHEC, the slope changes over the molar mass range, which indicates a conformational change (Figure 7). For the material between 2 × 105 and 5 × 105 g‚mol-1 (3.1-7.0 min in B of Figure 2), corresponding to 30% of the sample mass according (48) Tanford, C. Physical Chemistry of Macromolecules; Wiley: New York, 1961. (49) Richards, E. G. An introduction to physical properties of large molecules in solution; Cambridge University Press: Cambridge, U.K., 1980.
to Figure 4, the slope is 0.57, which corresponds to the expanded structure of a random coil type polymer. However, for the high molar mass part (106-107 g‚mol-1; 9.0-12.4 min), corresponding to 5% of the sample mass in Figure 4, the slope is lower, 0.35. This corresponds to a more globular shape. No reliable conformation analysis was possible for the ultrahigh molar mass fraction due to the low precision in molar mass and radius data. Ultrahigh Molar Mass Component. The UHM component that was eluted at 7-min retention time in Figure 2, case A, should have an extremely high molar mass. This is evident from the low concentration together with the high light scattering response. The molar mass is difficult to assess accurately due to experimental uncertainty (see above) but seems to be ∼108 g‚mol-1 (see Figure 2). In addition, the large size of the component is indicated by the hydrodynamic diameter, ∼0.35 µm, roughly estimated from the retention time (eq 1). The UHM component can originate either from the EHEC itself or may have been introduced during the sample preparation and analysis procedure. To test this, a large number of experiments were done. They were designed to indicate whether the UHM component was caused by (1) water-soluble particulates in the sample solution that had not been dissolved due to a slow dissolution process of EHEC, (2) particulates released from pumps and valves, (3) particulates released from the accumulation membrane or other parts in the channel, or (4) aggregation induced in the separation channel. Case 1 was tested by making 0.1% solutions of EHEC in the carrier and storing the solutions up to one month. However, several analyses during this period did not show any change in the MALS signal, and it was concluded that the dissolution must have come to equilibrium. Cases 2-4 were tested by filtering the sample solution with a 0.45-µm pore size syringe filter prior to analysis using the 1.2-µm filter at F5. The result was a significant decrease in the MALS signal in the retention time range of the UHM component (very similar to when a 0.45-µm in-line filter was used after the detector), which shows that the UHM component was not produced in the separation system but was present already in the sample solution. Moreover, tests of cases 2 and 3 were obtained by running blanks (carrier solvent) and these showed no indication of a UHM component whatsoever. In summary, there are strong indications that the UHM component originates from the EHEC. The identity of the UHM component is unknown. It may represent aggregated EHEC but there could be other causes. The presence of small quantities of large particulate matter in EHEC has been indicated before by extensive experimentation.50 This aggregated material was presumed to be due to the biological (50) Manley, R. S. J. Arkiv Kemi 1956, 9, 519-581.
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Figure 6. Root-mean-square radius obtained across a fractogram. Flow conditions as in Figure 2, case B. Above 13 min the recorded rms radii were too imprecise to be reported.
Figure 7. Conformation plot. The lines represent linear regression to the data points. Their slopes are 0.57 in the molar mass range 2 × 105-5 × 105 g‚mol-1 (A) and 0.35 in the molar mass range 1 × 106-1 × 107 g‚mol-1 (B). Flow conditions as in Figure 2, case B.
structure elements of the original cellulosic material, i.e., underivatized regions of cellulose perhaps retaining its crystalline character. More recently, several studies of cellulose and partially derivatized cellulose in solution have been performed and discussed, for example, for hydroxypropylmethyl cellulose and hydroxyethylmethyl cellulose in water.51 On the basis of static and dynamic light scattering as well as small-angle neutron scattering the presence of supramolecular structures was suggested, “particles”, having rms radii in the range 150-600 nm and an unrealistically high degree of polymerization (corresponding to extremely high apparent molar mass) as compared to what is expected from a single cellulose chain.51 The size is much larger (51) Schulz, L.; Seger, B.; Burchard, W. Macromol. Chem. Phys. 2000, 201, 20082022.
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than obtained for cellulose in the form of cotton linters (∼2 × 106 g‚mol-1) dissolved in aqueous metal-complexing solvents,52 which shows that the particles are composed of many polymer chains.51,53 Calculations from light scattering data caused the suggestion that the particles were aggregates of many chains forming so-called fringed micelles having a rigid stem and semiflexible corona chains.51,53 As to the origin of the particles, it has been supposed that they could be remnants of the semicrystalline fiber structure of the parental cellulose.51 Indeed, it has been shown54 that in carboxymethylation of cellulose the noncrystalline regions can be preferentially reacted when the reaction is performed at low alkalinity. Thus, the crystalline regions are excluded from alkalization and will therefore be nonderivatized. Then the formation of a blocklike substituent distribution in cellulose ethers was obtained. However, in conventional derivatization reactions, a high alkalinity is used which is expected to cause breakup of the crystalline regions so that they become fully reacted. One may, however, speculate that the industrial alkalization process may be incomplete leading to incomplete derivatization so that the product contains the abovementioned particles. It is possible that the UHM component observed in the present study is of such origin. Actually, smallangle X-ray scattering was suggested to indicate a crystalline component in EHEC solutions.55 The ability to find very large components of low concentration in industrial polymers is dependent on the analytical technique used. It is necessary both to isolate the component and to detect it. The latter is possible by the high-concentration sensitivity of light scattering for ultrahigh molar mass components. With only an RI detector, the presence of this component would not have been detected. The isolation must be made by a separation method that is applicable to such ultralarge polymers/particles. Among (52) Saalwaechter, K.; Burchard, W.; Kluefers, P.; Kettenbach, G.; Mayer, P.; Klemm, D.; Dugarmaa, S. Macromolecules 2000 33, 4094-4107. (53) Schulz, L.; Burchard, W.; Donges, R. ACS Symp. Ser. 1998, No. 688, 218238. (54) Mann, G.; Kunze, J.; Loth, F.; Fink, H. P. Polymer 1998, 39, 3155-3165. (55) Thuresson, K.; Lindman, B. Colloids Surf., A- 1999, 159, 219-226.
Table 2. Weight-Average Molar Mass, z-Average rms Radius, and Recovery of EHEC after Different Heating Timesa heating time (h)
Mw (g‚mol-1)
rg,z (nm)
recovery (%)
0 6 14
3.2 × 1.0 × 105(5) 0.21 × 105(18)
70(3) 62(17)
99(6) 77(7) 38(5)
105(2)
a Values in parentheses represent the percentage relative standard deviations.
Figure 8. MALS 90° and RI fractograms after heating for three different time periods: (A) no heating; (B) heating for 6 h; (C) heating for 14 h; Fc ) 0.39 mL‚min-1, Fin ) 0.8 mL‚min-1, Fout ) 0.39 mL‚min-1, Fc/Fout ) 1.0, and t0 ) 0.61 min.
chromatography-like techniques, the only alternatives would be hydrodynamic chromatography and size exclusion chromatography (SEC) but SEC is rarely applicable for separation of macromolecules of hydrodynamic size of 300-400 nm as the largest available average pore size of commercial column SEC packings (e.g., LiChrospher DIOL 4000) does not exceed 400 nm. In most cases, the UHM component would then be eluted close to the exclusion limit and be difficult to resolve and discern from various noise. There is also a high risk that the UHM component would be trapped in the SEC column filter frits and never be eluted from the column. Moreover, it is a common standard procedure in SEC to purify the sample solutions by filtration through a 0.45-µm microfilter in order to prevent the column from being clogged by particulate matter. Such sample filtration will of course remove a part of the information about the UHM component as follows from Figure 3. Those precautions are not necessary in FFF. Hence, the combination FFF-MALS is ideal for the task of detecting the presence of UHM components. Experiments at Elevated Temperature. The presumption that the UHM component may contain remaining crystalline parts of cellulose suggests that these aggregates may be dissociated and dissolved at elevated temperatures. For this purpose, a 0.1% EHEC solution was heated to 120 °C under stirring for 6 and 14 h. After reaching room temperature, the solutions were analyzed
with FFF-MALS-RI. The 6-h sample showed a significant shift of the two light scattering maximums to lower retention times, i.e., lower hydrodynamic radii (see Figure 8). Obviously the polymer had been partly depolymerized, as indicated by the shift of the main first peak. This resulted in a decrease of the weight-average molar mass (Table 2). The shift of the second UHM peak indicates a decrease of the hydrodynamic diameter to ∼0.3 µm. In the 14-h sample, the UHM peak had totally disappeared and the major peak was further shifted to lower retention times, indicating further depolymerization so that the weight-average molar mass was drastically decreased (Table 2). Obviously the UHM component has disappeared at the elevated temperature and had not been regenerated during the 1-h time that it was kept at room temperature before analysis. As the main fraction of EHEC was also broken down, it is not possible to say whether the UHM was degraded by only decrystallization or also by depolymerization. The recovery of the 14-h sample was low, 38%, most probably because a large part is smaller than the pore size of the ultrafiltration membrane (cutoff 10 kDa) and thereby was lost through the membrane. It is obvious that thermal treatment can degrade EHEC. As the heating was performed in the presence of oxygen, an oxidative degradation mechanism seems to apply. ACKNOWLEDGMENT This study was financed by the Centre for Amphiphilic Polymers from Renewable Resources at Lund University, supported by the Swedish National Board for Technical and Industrial Development, and the Swedish Research Council for Engineering Sciences. Dr. Bedrich Porsch is acknowledged for advice in light scattering experimentation. This work was presented as a poster at the 13th Bratislava International Conference on Polymers “Separation and Characterization of Macromolecules”, July 4-9, 1999, Bratislava, the Eighth International Symposium on FieldFlow Fractionation, September 6-8, 1999, Paris, International Symposium on Analysis of Carbohydrates, September 27-29, 1999, Stockholm, and the 23rd International Symposium on Chromatography, October 1-5, 2000, London. Received for review April 27, 2001. Accepted July 25, 2001. AC0104734
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