Influence of Substitution Pattern on Solution Behavior of

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Biomacromolecules 2009, 10, 522–529

Influence of Substitution Pattern on Solution Behavior of Hydroxypropyl Methylcellulose Anna Virideń,*,† Bengt Wittgren,‡ Thomas Andersson,‡ Susanna Abrahmseń-Alami,‡ and Anette Larsson† Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96, Gothenburg, Sweden, and AstraZeneca R&D, SE-431 83, Mo¨lndal, Sweden Received October 9, 2008; Revised Manuscript Received December 17, 2008

Industrially produced hydroxypropyl methylcellulose (HPMC) is a chemically heterogeneous material, and it is thus difficult to predict parameters related to function on the basis of the polymer’s average chemical values. In this study, the solution behavior of seven HPMC batches was correlated to the molecular weight, degree of substitution, and substituent pattern. The initial onset of phase separation, so-called clouding, generally followed an increased average molecular weight and degree of substitution. However, the slope of the clouding curve was affected by the substitution pattern, where the heterogeneously substituted batches had very shallow slopes. Further investigations showed that the appearance of a shallow slope of the clouding curve was a result of the formation of reversible polymer structures, formed as a result of the heterogeneous substituent pattern. These structures grew in size with temperature and concentration and resulted in an increase in the viscosity of the solutions at higher temperatures.

1. Introduction Hydroxypropyl methylcellulose (HPMC) is a water-soluble cellulose derivative with a number of properties that appeal to different industries. For example, HPMC is physiologically harmless, tasteless, and odorless and is therefore used in different types of food and pharmaceutical applications.1,2 The solutions are also stable in a wide pH interval and provide good viscosity stability in long-term storage;2 thus, one application is as a rheological modifier. The solution properties of HPMC can be altered by changing the molar mass and the degree of substitution. In the case of pharmaceutically approved HPMC, a number of different substituent and viscosity grades are commercially available, where each grade has certain specification limits.3,4 As these specification limits are quite broad, batch-to-batch variations are frequently observed in different applications. Studies have therefore been conducted to evaluate the influence of the average degree of substitution on applications such as matrix formers in hydrophilic matrix tablets.5-11 It has not always been possible to explain the behavior of the polymers by the average degree of substitution, however, and attention has instead been given to the influence of the substituent pattern, which has been shown to affect the physiochemical properties of cellulose derivatives.12-16 The substituent pattern can vary strongly, and hence also the solution behavior of the polymer. However, the substituent pattern is normally not controlled in the production of HPMC on an industrial scale, and it can thus be difficult to predict the parameters related to function for the desired applications. In production of HPMC, cellulose fibers are heated with caustic solution to disrupt the intermolecular hydrogen bonding.17 The caustic treated cellulose is then usually mixed with methyl chloride and propylene oxide. The average degree of substitution can be altered by the molar ratio of the substituents. * To whom correspondence should be addressed. E-mail: annlov@ chalmers.se. † Chalmers University of Technology. ‡ AstraZeneca R&D.

However, because the three hydroxyl groups on the glucose unit have different acidity and, hence, different reactivity in an alkali solution, they do not have the same probability of being substituted;18 hence, a certain inhomogeneity in the substituents’ pattern may occur. An additional reason for the appearance of a heterogeneous substituent pattern could be that propylene oxide is used in addition to methyl chloride, and because the hydroxypropoxylic (HPO) group is available for further substitution, the end product is dependent on whether the two reagents are added consecutively or as a mixture. Furthermore, even though the crystalline structure is extensively lost, there may be regions that become more highly substituted than others. This results in a nonuniform substituent pattern that can either arise between different chains or result in a heterogeneous substitution pattern along the chain. As described, there are endless possibilities as to where the substituents can be placed and, hence, result in a heterogeneous substituent pattern both among the glucose units along the chain or within the batch. This overall quite random process of producing HPMC can generate chemical heterogeneity in many orders and, thus, give the batch a wide chemical distribution. It has consequently been a challenge to develop sensitive and selective analytical tools for characterizing the chemical structure of cellulose derivatives.18,19 Today, however, there are more analytical tools to provide more information about the polymers, and new functional related characteristics for different applications can be characterized.20 For certain hydrogen bonding polymers such as cellulose derivatives, a decreased solubility is observed upon temperature increase, which eventually leads to phase separation. The hydrophobic influence of the substituents on HPMC further decreases the temperature of phase separation. Phase separation can be explained by the well-known relationship of free energy of mixing, which is the difference in Gibbs free energy (G) of the solution and the pure substances. This term can be divided into two parts, one enthalpic (H) and one entropic (S), according to

10.1021/bm801140q CCC: $40.75  2009 American Chemical Society Published on Web 01/27/2009

Solution Behavior of Hydroxypropyl Methylcellulose

∆Gmix ) ∆Hmix - T∆Smix

Biomacromolecules, Vol. 10, No. 3, 2009

(1)

While polymer dissolution is primarily an endothermic process, dissolution of the polymers still occurs due to the entropy gain through the increased number of possible conformations in solution. However, by increasing the temperature, more hydrophobic interactions between the substituents may take place, which induces an increased structure of the water and thus decreases the total entropy in solution.21,22 In addition, both the water structure and the hydrophobic interactions contribute to increased enthalpy, which in combination with the reduced entropy causes an increase in ∆Gmix and thus explains the phase separation upon heating. Phase separation of HPMC is connected to a drastic increase in the turbidity of the solution and can therefore be measured with light transmittance, also called clouding curves. The temperature required for HPMC to phase separate is obviously affected by the amount of the two substituents, where samples with an increased degree of total substitution and higher proportions of the more hydrophobic MeO group would phase separate at lower temperatures. However, HPMC is, as described, normally produced industrially under heterogeneous conditions, causing inhomogeneities in the substitution patterns. For both heterogeneously substituted MC and HPMC samples, the clouding curve has been found to decrease under a broader temperature interval, as compared to less heterogeneously substituted samples.23,24 It has, however, not been shown whether the broader temperature interval depends on a wider chemical distribution of chains being differently substituted or whether the broad temperature interval is actually a result of a different solution behavior caused by the heterogeneous substituent pattern. The substituent pattern may then either affect the temperature span needed for the heterogeneously substituted chains to phase separate or affect the phase separated polymer structures formed and thus the light transmittance. Therefore, to clarify the relationship between the chemical distribution and the phase behavior of HPMC, this study correlated the clouding curves of seven batches to the samples’ molecular weight, degree of substitution and substitution pattern. Two batches with different substitution patterns were further studied to discover the amount and chemistry of the fractions, the polymer-rich and the polymer-depleted phase, separated at different temperatures. The influence of the substituent pattern on the properties of the solution was elucidated by measuring both the rheological properties of the polymer solutions at various temperatures and the kinetics in the formation of the phase separated polymer structures.

2. Materials and Methods 2.1. Materials. Seven HPMC batches of the same substitution (USP 2208) and viscosity (100 cps) grade were supplied by Shin-Etsu (ShinEtsu Chemical Co., Ltd. Tokyo, Japan) and Dow (Dow Chemical Co., U.S.A.), under the trade names of 90SH100 and K100LV, respectively. 2.2. Molar Mass. The molar mass was analyzed using size exclusion chromatography with dual multiangle light scattering and refractive index detection (SEC-MALS/RI). The column was a TSK gel GMPWXL, 7.8 mm ID × 30.0 cm L, with a particle size of 13 µm (TOSOH Corp., Japan). The refractometer was an Optilab rEX, (Wyatt Technology, Santa Barbara, CA) and the MALS instrument was a DAWN EOS (Wyatt Technology, Santa Barbara, CA). This instrument setup makes it possible to determine the molar mass at each eluted fraction, which provides a distribution of the molar mass as well as different averages.25 The sample concentration was 0.4 mg/mL in 10 mM NaCl and the dry polymer was diluted in mobile phase for 48 h before analysis. The analyses were made at room temperature at a flow rate of 0.5 mL/min.

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The refractive index increment (dn/dc) used was 0.136 mL/g. The mobile phase was 10 mM NaCl with 0.02% NaN3, and the volume of the injected sample was 100 µL. The reported average molar weights are an average of three repeated analyses. The software used to process the data was Astra 4.90.07 (Wyatt Technology, Santa Barbara, CA). 2.3. Degree of Substitution. The degree of substitution was determined in acetylated samples with proton nuclear magnetic resonance (1H NMR), as described elsewhere.23,26 The samples were acetylized by dissolving 75 mg of each of the polymer samples in 2.25 mL acetic anhydride and 0.75 mL pyridine. The solutions were heated to 90 °C under stirring for 6 h and then dialyzed against deionized water in a Spectra/Por dialysis membrane (with a molar mass cutoff at 10 kDa) for 24 h. The samples were dried before being dissolved in deuterated chloroform (0.8 mg/mL). The 1H NMR measurements were carried out in a Varian 600 MHz Inova instrument operating at 14.09 T. The NMR instrument was equipped with a 5 mm Nalorac triple resonance probe. The 1H NMR measurement was carried out using a flip angle of 45° with an acquisition time of 4 s and a delay time of 10 s. The spectral width was at least between -3.5 and 10.4 ppm, with reference to the solvent peak of CDCl3 (7.26 ppm), and 16 transients on each sample were obtained. A line broadening factor of 0.3 Hz was used. The delay time between the pulse and the acquisition was optimized to give a zero first-order phase shift. The spectra were collected using oversampling with AnalogPlus as a digital filter. All spectra were recorded at 50 °C. 2.4. Cloud Point. The phase behavior of the polymer solutions was determined by light transmission on a Mettler Toledo FP90 central Processor, Mettler FB81C MBC, combined with IPClab software (Switzerland). The measurements were made in phosphate buffer (I ) 0.1, pH ) 6.5) in 1, 3, 5, and 10% (w/w) polymer solutions. If nothing else is specified, the temperature was raised by 1 °C/min, although both 0.1 and 10 °C/min were used. The light transmittance through the polymer solutions was normalized to 100% at the starting temperature. To characterize the clouding behavior of the polymers, two different temperatures (cloud point temperatures) were determined, CP50 and CP96, which refer to the temperatures at which the transmittance decreased to 50 and 96%, respectively. The average values reported are based on three measurements. 2.5. Enzymatic Hydrolysis. Enzymatic degradation was performed in the batches to compare the homogeneity of the substituent groups across the polymer backbone. The samples were dissolved in 5 mM of NaOAc (pH 5.0) to a concentration of 1 mg/mL. A total of 2 U of an endoglucanase enzyme (EGII from Trichoderma longibrachiatum) from Megazyme (Bray, Ireland) were added to 2 mL of HPMC solution, and the hydrolysis was carried out in a shaking water bath at 37 °C for 48 h. The released glucose was detected using high-performance anionexchange chromatography with pulsed amperometric detection (HPAECPAD) from Dionex (Sunnyvale, CA). The HPAEC-PAD system consisted of a GS50 gradient pump, a CarboPac PA-100 guard, an analytical column, and an ED50 electrochemical detector. The injection volume was 20 µL. Elution of the components in the hydrolysate mixture was carried out at a flow rate of 1 mL/min using a gradient program with 150 mM NaOH and H2O. 2.6. Centrifugation of Clouded HPMC Systems. Phase separation of the 1% (w/w) polymer solutions (phosphate buffer (I ) 0.1, pH ) 6.5)) was done in an oven (Termaks 8000) from Ninolab (Sweden), where the systems were left for equilibrium at each respective temperature for 2 h. The clouded systems were centrifuged (Wifuge, Laboratory Centrifuge, England) inside the oven for 10 min at 3000 rpm, and the upper and lower phases were then separated from each other. The phases were desalted by dialyzing them against deionized water in a Spectra/Por dialysis membrane (with a molar mass cutoff at 10 kDa) for 24 h. The samples were then dried in the oven at 50 °C for 24 h before measurements were made. 2.7. Rheology. Viscosities at a constant shear rate of 5 s-1 were recorded for the selected samples using an Anton Paar Physica MCR 301 rheometer (Germany) equipped with a double gap cylinder. HPMC

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Figure 1. Cloud point slopes of the seven batches.

solutions of 1, 3, 5, and 10% (w/w) were prepared in phosphate buffer (I ) 0.1, pH ) 6.5) and the viscosities were recorded at 40, 50, 60, 70, and 80 °C. A solvent trap was used to reduce solvent evaporation. The average values reported are based on two measurements.

3. Results and Discussion 3.1. Clouding Curve versus Chemical Heterogeneity. The phase behavior of seven HPMC batches (A-G) was investigated by measuring the transmittance of light through polymer solutions as a function of temperature, also called cloud point measurements. The transmittance was found to decrease as the samples passed the cloud point temperature and phase separated (Figure 1). Even though these batches were of the same substitution and viscosity grade, the lowest and highest cloud point temperatures measured at 96% transmittance (CP96) varied more than 7 °C (Table 1). Furthermore, the order of increasing cloud point temperature between the batches was G < B < F < E < D < A < C (Table 1). Phase separation for HPMC depends on the molecular weight27,28 and the chemical composition,29 where an increased molecular weight and a higher degree of total substitution should decrease the temperature at which phase separation takes place. The chemical differences between these batches were characterized, and variations were observed in both the average molecular weight and the degree of substitution. The deviation in average molecular weight was significant between the batches, where an increase of 50% was found in a comparison of the two batches with the lowest (batch D) and the highest (batch G) average values (Table 1). Furthermore, the variation in the degree of substitution was larger between the average amount of HPO groups than between the MeO groups, which can be exemplified by a 60% higher HPO content in batch G compared to batch A. There are indications that the average polymer characteristics could be correlated to the CP96 values, where for example batch G, having the highest average molecular weight and being one of the most highly substituted batches with both MeO and HPO groups, also had the lowest CP96 value (Table 1). However, there is no obvious correlation between the average polymer characteristics and the CP96 values in the seven batches. This may be explained by the width of the samples’ chemical distribution. HPMC is a rather polydisperse material and consists therefore of chemical distributions of both the molecular weight and the chemical substitution. From that point of view, the decrease in turbidity at CP96 may not reflect the phase separation of polymers having the average chemical properties but rather the phase separation of fractions in the sample with higher molecular weights and a higher total degree of substitution. To elucidate more than the incipient phase separation

Virideń et al.

temperature and hence also achieve more information about the samples’ properties in solution, the cloud point is sometimes defined as the temperature at 50% transmittance (CP50). A comparison of the CP50 values showed a difference of more than 15 °C between the batches (Table 1). Furthermore, compared to the CP96 values, the relative order between the batches changed. This difference can be explained by different temperature dependencies of transmittance, which can be seen in the shallow clouding curves of batches A, C, and D compared to the other batches (Figure 1). A shallow clouding curve is here defined as (1) a curve with a slope decreasing less than 5% per °C at 50% transmittance and (2) a curve not approaching zero percent transmittance below 100 °C (Figure 1). Shallow clouding curves from cellulose derivatives have been observed before11,23,24,28 and it has long been possible only to speculate whether these shallow curves depend on the chemical distribution (i.e., the difference in degree of substitution between different chains) or on the distribution of the substituents along the chain. Now, when analytical tools have developed further, groups such as Fitzpatrick et al. have been able to more thoroughly characterize both the distributions and average values of cellulose derivatives.23 They found after hydrolysing fractions of methylcellulose with enzymes that the most homogeneous fractions had the steepest clouding curves, whereas fractions that had not been hydrolyzed to the same extent had more shallow curves. They implied that these shallow curves were the result of a nonuniform substitution level because a broader range of temperatures was required for the entire sample to phase separate. To clarify the connection between the slope of the clouding curve and the substituent pattern of HPMC, enzymatic hydrolysis was carried out in the seven batches. The heterogeneity of the substituents along the cellulose chain was investigated in these seven batches by enzymatic degradation, where the samples were hydrolyzed by an endoglucanase from Trichoderma longibrachiatum.20 To form the enzymesubstrate complex, the polymer needs to be unsubstituted over a few glucose units.30 This means that polymer samples with more frequently occurring unsubstituted regions would liberate more glucose after being hydrolyzed by the enzyme; hence a comparison can be made between the substitution patterns of the different batches.18,20,30 As can be seen in Table 1, the amount of glucose liberated between the batches varied quite a lot, where the most heterogeneously substituted batches liberated more than four times the amount of glucose than the most homogenously substituted batch. As could have been expected, there are some indications that the least substituted samples liberated more glucose than the most substituted samples. However, there is no absolute correlation between the amount of liberated glucose and the total degree of substitution of the batches. This can be exemplified by comparing two batches with the same total degree of substitution (B and G), where batch B liberated three times more glucose than batch G, which indicates that the substituents in batch B were distributed in a more nonuniform manner than the substituents in batch G. To elucidate the effect of the substituent heterogeneity on the phase behavior, the slope of the clouding curves at 50% transmittance was plotted as a function of the amount of liberated glucose. As can be seen in Figure 2, there is a linear correlation between the amount of glucose liberated and the slope of the clouding curve, where the most heterogeneously substituted batches were found to give the shallowest curves (Figure 2). Even though the results obtained in the present study demonstrate the great influence the substitution pattern seems to have on the solution properties of HPMC, and thus the phase



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Table 1. Polymer Characteristics of Seven HPMC Batches of the 2208 and 100 cps Grade a w

4

a

10.4(0.2) 12.4(0.1) 10.2(0.2) 9.1(0.0) 12.9(0.3) 13.9(0.3) 14.1(0.3)

2.2(0.3) 2.8(0.5) 1.9(0.2) 1.9(0.2) 3.0(0.6) 2.9(0.6) 2.8(0.6)

sample M /10 g/mol Mw/Mn g/mol MS A B C D E F G

a Standard deviations in parentheses. between two repeated analyses.

b,c

(HPO) MS

0.18 0.29 0.19 0.17 0.26 0.23 0.29 b

b,c

% (mole) dglucose units cloud pointa at 96% cloud pointa at 50% liberated transmittance (CP96) transmittance (CP50) MeO)

1.54 1.50 1.50 1.50 1.45 1.54 1.51

1.4(0.1) 0.9(0.1) 1.4(0.1) 1.2(0.1) 0.8(0.0) 0.7(0.1) 0.3(0.1)

Average amount of mole per glucose unit.

Figure 2. Cloud point slopes at 50% transmittance versus the amount of glucose after enzymatic hydrolysis.

behavior, the results do not reveal whether the shallow curves are caused by the difference in the degree of substitution between different chains or by the heterogeneous distribution of the substituents along the chain. Schnider et al. titrated mixtures of two polyvinyl alcohol samples of the same viscosity but different degrees of hydrolysis.31 They found that the shape and the position of the transmission curve were dependent on the percentage of the two samples in the solution. In a polydisperse HPMC solution, each chemical fraction has its own phase diagram where the largest and most hydrophobic fractions are expected to phase separate at lower temperatures. It can thus be expected, as in the study of Schnider et al., that the shallow batch, which has a more heterogeneous substitution pattern, would also have a broader chemical distribution and that different fractions would have phase separated over a wider temperature interval, causing the shallower curve. This line of argument furthermore suggests that the shape of the curve and thus the turbidity of the polymer solutions are related to the amount of phase separated material. Therefore, the incomplete turbidity (20% transmittance) even at 100 °C in the heterogeneously substituted samples should depend on high fractions of nonphase separated material. If this were accurate, the shallow clouding curve would not be a result of a heterogeneous substitution pattern along the chains but rather to a nonuniform sample having fractions of chains with different degrees of substitution. Another explanation for the shallow clouding curves obtained would be the nature of the light scattering structures formed during phase separation. If the samples that liberated more glucose after enzymatic hydrolysis formed other kinds of polymer clusters than the more homogeneous samples, these would then also scatter light differently and hence influence the shape of the clouding curve. Further measurements were made to investigate the different hypotheses. 3.2. Fractionation of Two Batches with Different Slopes of their Clouding Curves. Two samples were further characterized to gain a better understanding of the cause of the

c

66(0.1) 61(0.2) 67(0.3) 64(0.8) 62(0.4) 62(0.1) 60(0.1)

80(0.7) 65(0.2) 79(0.2) 78(0.1) 68(0.2) 65(0.1) 63(0.1)

RSD of 0.02 according to an in-house validation.

d

Difference

different clouding curve shapes, one with a shallow clouding curve (batch A) and one with a steep clouding curve (batch B). These two batches are referred to in the following as the shallow batch and the steep batch. Polymer solutions (1% (w/w)) of the steep and shallow batches were equilibrated at temperatures related to CP96 and an end temperature at which the transmittance had decreased to 20% (CP20). An additional temperature related to CP50 was chosen for the shallow batch. The phases were macroscopically separated by centrifugation and further analyzed (Table 2). 3.2.1. Amount of Separated Material. The amount of material in the polymer-rich phases in the steep batch was about 30 and 50% (w/w) at temperatures corresponding to CP96 and CP20, respectively. In contrast, the shallow batch had a considerably higher amount of material in the polymer-rich phases (Table 2), where approximately 60% (w/w) had already phase separated at CP96 and as much as 80% (w/w) was found in the polymerrich phase at CP20. A comparison between the results in the steep and shallow batch clearly showed that, at the different cloud point temperatures, more material was found in the polymer-rich phases of the shallow batch than was found in the steep batch. At CP96 the shallow batch had as much as double the amount of material in the polymer-rich phase as compared to the steep batch. A possible explanation for the relative decrease in transmittance is the amount of material phase separated at the particular temperature in question. However, according to these results, a smaller amount of material from the polymer-rich phase of the shallow batch was needed to decrease the transmittance as compared to the polymer-rich phase of the steep batch. This can be exemplified by comparing 60% (w/w) phase separated material of the shallow batch at CP96 to 50% (w/w) phase separated material of the steep batch at CP20. The conclusion that can be drawn from these results is that the transmittance seemed to be highly affected by the phase separated polymer clusters of the steep batch, where smaller amounts of these structures cause the transmittance to drop dramatically. This implies that either the slope of the clouding curve is not solely related to the total fraction of material precipitated during the centrifugation process or that the mechanism by which these two batches form light scattering structures is different. If the solution behavior varies, the kinetics in forming the structures might not be the same in the two batches, and hence the amount of material collected after 2 h of equilibration cannot be related to the clouding curve, where heating was increased by 1 °C/min. The phases were further characterized to understand the underlying mechanism in the separation at the temperatures measured. 3.2.2. Chemical Composition of the Polymer Fractions. The degree of substitution affects the solubility of the polymer chain27 and, thus, the phase behavior (eq 1). Of the two substituents in HPMC, the MeO group is the most hydrophobic28 and, hence, is expected to have a greater impact on the phase

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Virideń et al.

Table 2. Polymer Characteristics of the Fractionated Phases phasesa (temperatures in parentheses) shallow batch PR (66) PD (66) PR (80) PD (80) PR (95) PD (95) steep batch PR (60) PD (60) PR (70) PD (70)

% (w/w)

b

60 40 60 40 80 20 30 70 50 50

material

cloud pointc at 96% transmittance (CP96)

cloud pointc at 50% transmittance (CP50)

M /10 g/mol

66 (0.1) 65.2 (0.4) 75.9 (0.2) 66.2 (0.7) 79.6 (0.1) 66.1 (0.3) 83.1 (0.3) 61 (0.2) 58.1 (0.2) 70.0 (0.1) 59.2 (0.1) 67.1 (0.2)

80 (0.7) 79.7 (0.1) 80.0 (0.1) 80.0 (0.4) 86.5 (0.1) 79.8 (0.1) 93.4 (0.1) 65 (0.2) 62.0 (0.1) 71.6 (0.1) 63.3 (0.1) 72.0 (0.1)

10.4 (0.2) 10.4 (0.2) 6.2 (0.0) 11.0 (0.4) 3.9 (0.1) 11.0 (0.2) 3.0 (0.1) 12.4 (0.1) 20.4 (0.4) 6.4 (0.1) 18.8 (0.5) 5.5 (0.1)

a Polymer-rich (PR) and polymer-depleted (PD). b Separated from 1% (w/w) solutions. mole per glucose unit. e RSD of 0.02 according to an in-house validation.

separation temperature compared to the HPO group. This hydrophobic difference between the substituents was observed in the phases where the polymer-rich phases from both batches contained on average more of the MeO groups than did the polymer-depleted phases (Table 2). The average amount of HPO groups in the different phases from both batches showed hardly any difference. This might depend on a minor variation of HPO within the batch; hence, the effect was excluded by the distribution of the MeO groups, which has a greater affect on phase separation (Table 2). It is known that phase separation of cellulose derivatives is also dependent on molecular weight, where higher molecular weight samples phase separate at lower temperatures. This can be explained by a decreasing contribution of entropy with increasing molecular weight.32,33 It can therefore be expected that higher molecular weight fractions (with the same degree of substitution) would phase separate at lower temperatures. This dependency on molecular weight was evident in the batch with the steep clouding curve, where the average molecular weight of the polymer-rich phase (30% (w/w)) at CP96 was almost twice as high as that of the original batch. At the end temperature (70 °C), the amount of material in the polymer-rich phase increased to 50% (w/w), and hence, the molecular weight distribution more closely resembled the initial distribution. Regardless of the increasing amount of material, the average molecular weight was 50% higher than the original sample and thus emphasized the effect of the molecular weight on phase separation (Table 2). The material in the polymer-depleted phase and the average molecular weights decreased compared to the polymer-rich phase and decreased with increasing temperature, which is in agreement with theory (Table 2). The average molecular weight of the polymer-depleted phases of the shallow batch was also lower than the original samples’ average molecular weight. At CP96, the average molecular weight was 40% lower and decreased with increasing temperature (Table 2). In contrast to the steep batch, however, the polymer-rich phases from the shallow batch did not have any significantly different average molecular weights than the original batch. This partly depends on the higher amount of material (60 and 80%) in these phases, which made them resemble the original sample’s distribution. However, the increasing amount of material in the polymer-rich phase cannot solely explain the loss of an increased molecular weight of these phases, especially since there was only a small difference in the degree of substitution between the polymer-rich and the polymer-depleted phases. Instead, the absence of an effect of molecular weight on phase separation might be a result of the

c

c w

4

c n

Mw/M

2.2 (0.3) 1.8 (0.2) 1.6 (0.1) 1.7 (0.2) 1.7 (0.1) 1.7 (0.1) 1.6 (0.1) 2.8 (0.5) 1.5 (0.0) 2.5 (0.0) 1.5 (0.0) 2.3 (0.0)

Standard deviations in parentheses.

d

MSd,e (HPO)

MSd,e (MeO)

0.18 0.19 0.17 0.18 0.17 0.18 0.17 0.29 0.32 0.29 0.31 0.28

1.54 1.53 1.44 1.52 1.39 1.52 1.38 1.50 1.61 1.40 1.56 1.38

Average amount of

mechanism by which heterogeneously substituted samples phase separate as compared to more homogeneously substituted samples. It would have been expected that a variation in the degree of substitution should have affected the phase separation at the different temperatures and that the polymer-rich phases should have been more substituted compared to the polymer-depleted phases. While the polymer-rich phases were slightly more substituted compared to the polymer-depleted phases, the differences were quite small. The minor fractionation regarding the average substitution therefore indicates that the degree of substitution did not vary substantially between the chains in the two batches. As concerns the steep batch, this minor variation might explain the fractionation clearly obtained owing to the molecular weight. A possible explanation for the shallow slopes of the clouding curves is a wider chemical distribution and that fractions with different chemical compositions and molecular weights would have phase separated over a larger temperature interval, thus causing the shallower slope. This study has shown the opposite, however, where the shallow batch was separated only to some extent according to the average molecular weight and the degree of substitution. The shallow slope can therefore not be explained by a wider distribution of either the molecular weight or the degree of substitution. All of the above results imply that the process by which a heterogeneously substituted sample phase separates is different as compared to a sample with a more homogeneous substituent pattern. To gain a better understanding of the process by which these two batches phase separate, the solution behavior was further investigated at increased temperatures. 3.3. Properties of Phase-Separated Solutions. 3.3.1 Kinetics and Concentration Dependency. The amount of material found in the polymer-rich phases of the shallow batch was greater than the amount found in the polymer-rich phases of the steep batch at the different cloud point temperatures. This led us to question whether the actual transmittance decrease could be related to the amount of polymers in the polymer-rich phase or whether the kinetics in the formation of the light scattering structures was different. The latter would then suggest that HPMC with a heterogeneous substituent pattern forms other kinds of structures in solution than are formed in more homogenously substituted batches. To investigate the kinetics in the formation of these light scattering structures in relation to the clouding curves, the heating rate in the polymer solutions was raised by two additional speeds, 0.1 °C/min and 10 °C/ min, besides the 1 °C/min otherwise used. The three different clouding curves of the steep batch showed almost the same



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Figure 3. Cloud point slopes conducted at three different temperature rates: light grey, 0.1 °C/min; dark grey, 1 °C/min; and black, 10 °C/min.

Figure 4. Cloud points conducted at different concentrations. (A) Cloud point at 96% transmittance; (B) cloud point at 50% transmittance; 9, shallow batch; 4, steep batch.

temperature dependency, and only a small deviation between the curves could be seen in the initial decrease of the light transmittance, CP96 (Figure 3A). In contrast, there was a larger deviation between the clouding curves of the shallow batch, displaying not only a difference in the initial decrease in light transmittance but also in the curvatures obtained. The CP96 values decreased and the light transmittance declined with a lower temperature dependency as the speed decreased, which caused the CP50 values to increase (Figure 3B). These results indicate that the light scattering objects that were formed upon heating were dependent on the rate of the temperature increase. The somewhat higher CP96 at 10 °C/min for both batches might be explained by cluster growth being time dependent; hence, equilibrium was not reached at faster heating at the actual phase separation temperature, and a higher cloud point was detected. The light scattering polymer clusters formed in the shallow batch were affected much more by the rate of the temperature increase. This behavior might be compared to crystallization from supersaturated solutions. When the temperature is decreased in a supersaturated solution, the size and numbers of the crystals formed are dependent on the rate of the temperature change, where a faster temperature decrease results in a larger number of smaller crystals.34,35 In parallel, it could be suggested that, at faster heating, there is less time for the polymers to diffuse and hydrophobically interact with each other; therefore, as the phase separation temperature is reached, the polymers collapse and form a larger amount of smaller clusters. In contrast, at the slower temperature increase, there is more time for diffusion before a total polymer collapse occurs and hence the growth of clusters starts at lower temperatures where the number of clusters is smaller and scatter the light less extensively, thus making the cloud point curve shallower (Figure 3B). This argument suggests that the more heterogeneously substituted polymers in the shallow batch are more associative than the polymers in the steep batch. This is further strengthened by the amount of material (60%) in the polymer-rich phase that already separated at CP96, compared to only 30% at the same transmittance in the steep batch. If more

heterogeneously substituted HPMC batches are more associative, one would expect their phase behavior also to be affected more by an increased concentration. To elucidate the concentration dependency of the two batches, clouding curves were obtained on additional concentrations: 3, 5, and 10% (w/w) solutions. The results can be seen in Figure 4A,B, where the CP96 and CP50 values are plotted as a function of concentration. The concentration dependency of the steep batch is minor, where the decrease in CP96 and CP50 is not even 5 °C between 1 and 10% (w/w) solutions. These results are in accordance with earlier findings for HPMC, where the concentrations at these intervals affected the cloud point only a few degrees.28,36 In contrast, the shallow batch showed a larger concentration dependency, where cloud points CP96 and CP50 decreased more than 15 °C between 1 and 10% (w/w) solutions. Although the cloud points decreased, the temperature dependency remained approximately the same at all concentrations (Figure 4). The large concentration dependency in the shallow batch supports the indications that the polymers in this batch are more associative as compared to the polymers in the steep batch. In 1987, Takahashi et al. proposed that the MeO groups in a heterogeneously distributed sample of methylcellulose could act as cross-linking loci upon heating.16 The same behavior might explain the associative behavior of the shallow batch, where the heterogeneous substituent pattern facilitates regions that are highly substituted and, hence, may interact hydrophobically at an increased temperature. Enhanced viscosity was obtained in the solutions of the heterogeneously substituted samples of methylcellulose, and thus, the rheological properties of the shallow and steep batch were investigated. 3.3.2. Rheology. The rheological properties of the turbid solutions were investigated to understand more about the structural differences between the two systems. The samples were exposed to a constant shear rate at 40, 50, 60, 70, and 80 °C at concentrations of 1, 3, 5, and 10% (w/w; Figure 5). When HPMC starts to phase separate, the polymers lose their water of hydration, which is normally connected with an initial decrease in viscosity.28,29 This

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Figure 5. Viscosities (left, y-axis) and cloud point slopes (right, y-axis) conducted at different temperatures and concentrations: 1% (w/w), 3% (w/w), 5% (w/w), and 10% (w/w); black, shallow batch; gray, steep batch.

Figure 6. Schematic illustration of polymer structures formed upon phase separation. To the left is an illustration of the steep batch and to the right is an illustration of the shallow batch.

behavior was recognized in the solutions of the steep batch, except for the 1% (w/w) solution, which showed a steady increase in viscosity. However, for 3, 5, and 10% (w/w) solutions, the viscosity decreased as the sample started to phase separate, and only a slight increase could be seen at 80 °C in the 3 and 5% (w/w) solutions (Figure 5). In contrast to the steep batch, the viscosity of the shallow batch increased with an increased temperature at all concentrations. A viscosity peak was found in the 5 and 10% (w/w) solutions, where at temperatures 20-30 °C higher than the CP96, a decreased viscosity could be observed (Figure 5). These results clearly show that the rheological properties of the two solutions were different upon temperature increase, which strengthens the indication that the two batches formed different kinds of structures during phase separation. Figure 6 schematically illustrates the possible structures that might explain the differences observed in both the clouding curve and rheological behavior. At low temperatures, short-lived polymer interactions may take place between the polymer chains in both batches at all concentrations. However, as the temperature increases, the polymer starts to phase separate and the hydrophobic interactions become more long-lived. In the case of the steep batch, the higher fraction of chains having more homogeneously distributed substituents cause the chains to phase separate as discrete structures and thus prevent the formation of more long-lived hydrophobic interactions between different chains. The kinetics of the phase separation of these chains is thus fast, since the neighboring chains have only limited influence on the phase separation. Consequently, the polymer clusters that are formed are smaller and more discrete,

as illustrated in Figure 6. The limited influence that the different chains have on each other is also reflected in how the turbidity is affected by the concentration (Figure 4). Because the onset of turbidity, in the case of the steep batch, is affected very little by the concentration at the measured interval, it will force the concentration in the polymer-rich and the polymer-depleted phases to differ significantly, hence, the phase separated polymer clusters become more discrete. These small and compact polymer clusters scatter light intensively and thus reduce the transmittance as soon as they form. In contrast to the steep batch, the shallow batch had a more heterogeneous substitution pattern, which seems to facilitate an amphiphilic behavior. More long-lived hydrophobic interactions between the chains can therefore take place without a total precipitation of the polymer chains. On the basis of the results presented, we suggest, in parallel to heterogeneously substituted methylcellulose samples,16 that these interactions result in the formation of a gel-like structure (Figure 6) where blocks, which are less substituted, stay soluble at higher temperatures and function as links between more hydrophobic domains. As the size of these structures grows with time and temperature, the transmittance decreases, although not to the same extent as in the steep batch because the phase separated polymer clusters in the shallow batch have a less dense structure. However, at higher concentrations, the gel structure becomes larger and more compact, which reduces the light transmittance to a larger extent (see the 10% (w/w) sample in Figure 5). Moreover, the observed increase in viscosity with temperature and concentration may be a result of the formation of a more continuous and compact gel structure. The decrease in viscosity at the higher temperatures in the 5 and 10% (w/w) solutions might be explained by total phase separation of the polymer chains, causing the continuous gel layer to collapse and transform into smaller phase separated clusters, hence, reducing the viscosity to the same level as measured in the steep batch.

4. Conclusion We have shown a linear correlation between the slope of the clouding curves of seven HPMC batches and their substituent pattern, where the most heterogeneous batches showed a very


weak temperature dependency in their clouding curves. To investigate this correlation, two batches with differences in substituent patterns and thus in the slopes of their clouding curve were fractionated into one polymer-rich phase and one polymerdepleted phase at various temperatures. The phase separation in both batches was related to the molecular weight and the degree of substitution. The polymer-depleted phases had a lower average molecular weight and were less substituted than the polymer-rich phases. However, neither the amount of material in the different phases nor the chemical differences between the two phases could prove that the shallowness and the incomplete turbidity of the clouding curve was a reflection of the width of the samples’ chemical distribution. By analyzing the kinetics and the concentration dependency of the two different clouding curves we could conclude that the two batches formed light scattering structures by different mechanisms. The more homogenously substituted batch with the steep slope was affected only to a minor extent by the temperature rate and the alteration of the concentration in contrast to the more heterogeneously substituted batch. Rheological measurements supported the indication that the shallow curves were caused by the formation of different structures. These structures consisted of clusters of reversible gels that increased the viscosity of the solution as the temperature was raised. We conclude that the possibility of forming these structures is enhanced by HPMC polymers’ heterogeneous substituent pattern. This study has shown that different properties in solution can be achieved by altering the substituent pattern of two HPMC batches of the same commercial grade. Consequently, both users and producers must be aware of the polymers’ complex structure, which generates a wide variety of solution properties that change within the same material and of the same commercial grade. We thus emphasize the importance of carefully characterizing the parameters related to the functionality of cellulose derivatives in order to understand their behavior in different applications.


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