Detailed characterization of cationic hydroxyethylcellulose derivatives using aqueous size-exclusion chromatography with on-line triple detection

Journal of Chromatography A, 1104 (2006) 145–153

Detailed characterization of cationic hydroxyethylcellulose derivatives using aqueous size-exclusion chromatography with on-line triple detection X. Michael Liu a,∗ , Wei Gao b,∗∗ , E. Peter Maziarz a , Joseph C. Salamone a , John Duex a , Erning Xia a b

a Bausch & Lomb, Inc., Global Research & Development, 1400 N. Goodman Street, Rochester, NY 14609, USA NSF-I/UCRC Center for Biocatalysis and Bioprocessing of Macromolecules, Othmer Department of Chemical and Biological Sciences and Engineering, Polytechnic University, Six Metrotech Center, Brooklyn, NY 11201, USA

Received 7 December 2004; received in revised form 23 November 2005; accepted 28 November 2005 Available online 19 December 2005

Abstract A more complete understanding of polymeric, cationic cellulose derivatives, including polyquaterium-10 (Polymer JR), has become increasingly important in the eye care industry as thorough characterization of raw materials helps promote product quality and process control. Often such detailed information requires utilization of a combination of analytical techniques. In this work three Polymer JR samples with different viscosities were characterized using aqueous size exclusion chromatography (SEC) with a light scattering detector, a differential viscometer, and a differential refractometer (triple detection). Detailed molecular information such as absolute molecular weights, molecular weight distributions, intrinsic viscosities, and molecular conformations were obtained. One major challenge of analyzing cationic polymers is abnormal size exclusion separation, which could be caused by the ionic interaction between sample molecules and the column packing material. A selection of mobile phases varying in pH, buffer, organic solvent content, and molar concentration of salts was employed to evaluate the correlation of obtained molecular weight values and mobile phase composition. Universal calibration concept was used to examine the abnormal size exclusion separation phenomenon of Polymer JR samples when using different mobile phases. It was observed that the abnormal size exclusion was dependent on both the separation conditions and molecular weights of the samples. Despite the changes in separation parameters and uncharacteristic polymeric structure compared to conventional SEC samples, the use of aqueous SEC with triple detection provided reproducible and valuable molecular information of Polymer JR samples with low to medium molecular weights. By using a combination of high buffer content and adding organic solvent, the abnormal exclusion separation of high molecular weigh Polymer JR could be considerably reduced. © 2005 Elsevier B.V. All rights reserved. Keywords: Polymer JR; Cationic polymers; Absolute molecular weight; Molecular weight distribution; Intrinsic viscosity; Aqueous size exclusion chromatography; Triple detection

1. Introduction Many cationic polymers have been used in cosmetics and hair-care solutions. They have superior abilities to restore damaged hair by improving wettability and enhancing the appearance and feel of dry hair. These polymers are normally mild themselves and are compatible with a broad spectrum of surfactant systems. They are also used to reduce the potential irritation of surfactants. Polymer Technology Corporation, a ∗

Corresponding author. Tel.: +1 585 338 6674; fax: +1 585 338 5875. Corresponding author. Tel.: +1 718 260 4042; fax: +1 718 260 3075. E-mail addresses: [email protected] (X.M. Liu), [email protected] (W. Gao). ∗∗

0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.11.094

Bausch & Lomb company, pioneered the utilization of one of these cationic polymers, UCARE Polymer JR (polyquaternium10), in contact lens solutions for rigid gas permeable lenses [1]. Recent studies have also shown that Polymer JR has significant capabilities of treating and conditioning soft lens materials inthe-eye and/or out-of-eye solutions [2–5]. In general, Polymer JR is a water-soluble, polymeric quaternary ammonium salt of hydroxyethylcellulose. The molecular structure of Polymer JR is represented in Fig. 1. The number of repeating units, n, determines the size and subsequent molecular weight of these materials and provides the unique solution viscosities by which Polymer JR 30M, JR 400, and JR 125 are characterized. Polymer JR has a rather low charge density and a relatively high density of polar groups. Charge density is the residue molecular weight

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Fig. 1. Molecular structure of Polymer JR.

per unit of positive charge. Despite the low charge density, the cationic charges on Polymer JR molecules have significant effects on Polymer JR uptake onto human hair and interactions with various surfactants. The apparent cationic charge of the sample molecules becomes quite substantial as the percentage of modified amine content increases [6–8]. The cationic characteristics of this hydrophilic polymer in combination with high molecular weight and high viscosity give Polymer JR distinct properties of long lasting comfort in eye-care solutions. Polymer JR has a long history and wide range of applications from conditioners and moisturizers to deposition agents and film formers. It is produced and distributed with a number of physical properties in which performance is generally based on solution viscosity. It has become essential for Bausch and Lomb to fully characterize and understand Polymer JR for use in eye care products, requiring strict specification of raw materials; however, no reference was found for the determination of molecular weight, molecular weight distribution, molecular conformation, or molecular size of Polymer JR. The only information for Polymer JR that the manufacturer (Amerchol, Subsidiary of Dow Chemical, Greensburg, LA, USA) provides is the solution viscosity. According to Amerchol, Polymer JR 30M, Polymer JR 400, and Polymer JR 125 function as cationic, water-soluble, substantive conditioners for hair care and skin care products. They have same charge density, the percentage nitrogen contents of them are 1.8–2.2. The aqueous solution viscosities range from 1000 to 2500 cPs for 1% (%, w/w) Polymer JR 30M to 75–125 cPs for 2% Polymer JR 125. Amerchol Corporation provides no molecular weight or other physical and chemical data of these materials. Size exclusion chromatography (SEC), or gel permeation chromatography (GPC), is the most useful analytical method for molecular weight and molecular weigh distribution characterization. There are three different calibration methods by using different detection techniques: (1) conventional calibration: it can be utilized by SEC system with a single concentration detector. Multiple narrow standards or broad standard are need to generate the calibration curve, which is the plot of log M versus retention volume. Conventional SEC has been used successfully to measure relative molecular weight and molecular weight distribution with remarkable precision. However, conventional SEC is limited in that it only measures relative molecular weight and molecular weight distribution values and does not provide any information about molecular conformation or size. Furthermore, the analysis of cationic polymers by conventional SEC is constrained by potential ionic inter-

actions of the quaternary ammonium groups with the residual anionic end-groups of the column packing materials. This interaction results in abnormal size exclusion separation, such as poor chromatographic peak shape, low recovery and increased retention times, thus rendering erroneous determination of molecular weights and molecular weight distributions of the sample [9–11]. (2) Universal calibration: it can be utilized by SEC system with a single concentration detector when Mark-Houwink constants of standard and sample are known, or by SEC system with dual detectors (concentration and viscometry) when the Mark-Houwink constants are unknown. In the universal calibration concept, the separation of chemically different polymers is based on hydrodynamic volume, and it has been widely demonstrated. Based on Einstein viscosity law, the product of intrinsic viscosity ([η]) multiplied by molecular weight (M), [η]M, is proportional to hydrodynamic volume, so the plot [η]M versus retention volume is known as universal calibration curve, which need be generated by standards to calibrate the identical SEC condition. The absolute molecular weight averages of sample, conformation and size information could be obtained. However, when abnormal size exclusion exists, the universal calibration is invalid, thus this method will give erroneous results. (3) Absolute molecular weight calibration. When molecular weight sensitive detector (e.g. dynamic light scattering or mass detector) is added to SEC system with concentration detector, the molecular weight of each elution fraction is directly measured without using assumptions on size exclusion separation and without need to resort to external standards. The sensitivity and accuracy of mass sensitive detector crossing the whole elusion range is essential for molecular weigh averages determination [13], and good separation is still important to obtained accurate molecular weight distribution information. In this study, aqueous SEC with a triple detection system was employed that consisted of a right angle light scattering detector (LS), a differential refractometer (dRI), and a differential viscometer (VISC). A dRI was used to monitor the concentration change over the molecular weight distribution of the polymer. A VISC detector in combination with the dRI was used to determine the intrinsic viscosity [η] of the polymer samples in solution. In addition, the LS data combined with the dRI was used to directly determine absolute molecular weight values without using a calibration curve. Therefore, [η]–M-relationship, also known as Mark-Houwink plot, could be obtained from polydispersed sample, the molecular conformational information and size of the polymer could be obtained. The reduction of potential ionic interactions between the sample molecules and the column packing materials was essential and allowed for theoretical selectivity based solely on molecular size and conformation in solution [12–21]. In order to evaluate and validate the efficacy of reduction of ionic interaction, we employed four mobile phases with different types of salt concentrations, and pH. The size exclusion separations under different mobile phase conditions were evaluated by universal calibration concept. In this report, we used aqueous SEC with triple detection to obtain more complete molecular information of Polymer JR commercial materials.

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2. Experimental 2.1. Samples and materials Polymer JR samples were provided by Amerchol Corporation (Greensburg, LA). A narrow molecular weight poly(ethylene oxide) standard with Mp of 82,250 Da (Mw /Mn = 1.02) was purchased from Polymer Laboratories (Amherst, MA, USA). Sodium nitrate was obtained from Sigma-Aldrich (Milwaukee, WI, USA). Sodium acetate, acetic acid, and acetonitrile were obtained from Fisher Scientific (Fairlawn, NJ, USA). Triethylamine hydrochloride was purchased from Fluka Chemika (Buchs, Germany). Sodium nitrate, sodium acetate, acetic acid, acetonitrile, and triethylamine hydrochloride were HPLC grade quality. All chemicals and solvents were used as received. 2.2. SEC with triple detection The solvent delivery system used an Alliance 2690 Separation Module (Waters, Milford, MA, USA). The separation was performed using two Ultrahydrogel Linear columns (300 mm × 7.8 mm i.d.) purchased from Waters Corporation. The average particle size of the packing materials was 6 ␮m. Polymer JR samples were analyzed using four different mobile phases as follows: (a) 0.5 M sodium acetate/acetonitrile (80:20 by volume, pH 4.94); (b) 0.8 M sodium nitrate solution (pH 5.51); (c) 0.1 M triethylamine hydrochloride with 1% acetic acid (pH 3.18); and (d) 0.1 M triethylamine hydrochloride solution (pH 4.78). All mobile phases were filtered using 0.22 ␮m nylon membrane filters (Sigma-Aldrich) prior to use. Samples were prepared in the corresponding mobile phase for the analysis. Polymer JR samples were prepared at concentrations ranging from 0.5 to 2 mg/ml as molecular weights decreased. One hundred microliters of sample solutions were used for each injection. The flow rate was set at 0.5 ml/min. The triple detection system (Model 310) was purchased from Viscotek Corporation (Houston, TX, USA). The three detectors were connected in series such that the light scattering detector was placed directly downstream of the SEC column set, followed by the refractometer, and viscometer. One hundred microliters of a narrowly dispersed poly(ethylene oxide) standard (Mp = 82,250 Da) solution at a concentration of 1.0 mg/ml was first injected into the SEC with the triple detection system. Three goals were achieved by the injection of the narrow polymer standard solution. First, the inter-detector delay volumes among the three detectors were compared and corrected. Second, the calibration factors and constants for the three detectors were obtained. Third, the band broadening effect due to serial connections of three detectors was effectively rectified. No flow rate marker was used in the analysis. The dn/dc value of PEO in aqueous used for calibration is 0.142 ml/g [22]. All sample and standard solutions were filtered through 0.45 ␮m nylon Acrodisc syringe filters prior to injection. The temperature for the SEC column set and the detector chamber was 45 ◦ C to ensure high chromatographic efficiency, stable baselines, and consistent

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results. Data acquisition and calculations were performed using Viscotek OmniSEC software version 2.0). The dn/dc value of polymer JR in aqueous used for calculation is 0.144 ml/g [23]. The calculated data is exported from OmniSEC software, the value of log[η] and log M at each retention volume were used to generate the universal calibration curve by Origin 6.1 software (OriginLab, MA, USA), the value of log[η] and adjusted dRI at each retention volume were used to obtain molecular weight averages from universal calibration by Microsoft Excel software. The following simplified equations reflect the correlation between the detector signals and polymer molecular weight, size, and conformation determination: RdRI = KRI C

dn dc

Rvisc = Kvisc C[η] RLS (θ) = KLS

4π2 n20 (dn/dc)2 CP(θ)Mw λ40 NA

(1) (2) (3)

[η] = kM × Mva

(4)

Rh = 0.251([ηM])1/3

(5)

Rg = 0.328([η]M)1/3

(6)

where RdRI , Rvisc and RLS (θ) are signals of the refractive index detector, viscosity detector and light scattering detector, respectively; KRI , Kvisc and KLS are the instrument constants of refractive index, viscosity and light scattering detectors respectively. C is the concentration of sample solution. dn/dc is the specific refractive index increment. Mv and Mw are the viscosity and weight average molecular weight, respectively. For individual fraction from SEC, Mv is equal to Mw since they are treated as monodispersed sample. [η] is the intrinsic viscosity of the polymer. θ is the scattering angle, which is 90◦ for the particular instrument. P(θ) is the form factor, it relates the angular variation in scatting intensity to mean square radius of the particle. The excess light scattering intensity at zero angle is calculated from 90◦ excess scatting intensity by using the particle size information for P(θ) calculation under appropriate assumptions [24]. n0 is the refractive index of the solvent, λ0 is the wavelength of the light and NA is the Avogadro number. Eq. (4) is the Mark-Houwink equation. KM and exponent a are Mark-Houwink’s constants. The value of exponent a (or conformational coefficient) can also be used to predict the combined effect of polymeric chain conformations and the presence of branching. It is well known that values of a ≥ 1.0 indicate a rigid rod conformation, values of 0.5 and 0.8 indicate random coils in a theta and good solvent respectively, and a ≤ 0.5 indicates a spherical or branching conformation. [6]. Eqs. (5) and (6) are the formulas used in OmniSEC software for calculation of hydrodynamic radius and radius of gyration, which are resembles from Einstein viscosity law for particles in suspension under certain assumptions [24].

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3. Results and discussion 3.1. Mobile phase selection In the SEC analysis of cationic polymers, mobile phases with high salt concentrations, or competitive cations, are often required to mask the anionic sites at the column packing materials. In the study high salt solutions (0.8 M NaNO3 or 0.5 M NaOAc) were used to suppress non-size exclusion effects such as adsorption and ionic interactions including ion exclusion, ion inclusion, as well as ion exchange between Polymer JR molecules and the column packing materials. Insufficient salt concentrations in the mobile phase would cause severe peak tailing, longer retention problems, and thus erroneous determination of molecular weights. These phenomenons are also found in other cationic polymer studies. For example, Wu and Senak reported a much higher salt content (0.5 M LiNO3 ) is needed in the SEC mobile phase for the cationic copolymer, quaternized copolymer of vinyl pyrrolidone and dimethylamineoethyl methacrylate (PVP/DMAEMA), than the salt contents for nonionic and anionic copolymers (0.1 and 0.2 M LiNO3 ) to improve the separation and recovery of polymer [25]. Under certain circumstance, it is necessary to add organic solvent into the mobile phase to eliminate the interactions between polymer sample and the column packing material. Herman and Field [26] demonstrated the 100% recovery of poly(vinyl pyrrolione) could be achieved by using 40% acetonitrile in 0.01 M KH2 PO4 , pH 2.1, while the recovery could be as low as 0–25% when using water as elute. Therefore, in this study high salt concentration (0.5 M NaOAc) and organic solvent are combined as mobile phase to achieve size exclusion separation. The Ultrohydrogel column used in SEC is packed with semirigid polymeric gel, they are hydroxylated PMMA in nature. They can be expected to have a small amount of free carboxyl groups as a result of hydrolysis, which can interact adversely with the cationic polymers [27]. In this case competitive cations could considerably reduced the ionic interactions between columns and analyze polymer. When 0.1 M triethylamine hydrochloride was used, with or without 1% acetic acid, as a mobile phase, partially ionized triethylamine effectively capped the anionic sites on the column packing materials. Meanwhile, Polymer JR was also partially ionized during the chromatographic separation process. As a result of the concentration of ionized triethylamine being much higher than that of Polymer JR in the SEC separation process, minimal or no ionic interaction between Polymer JR and the column packing materials could be achieved.

Fig. 2. VISC (—), LS (- - -), and dRI ( ) chromatograms of Polymer JR 30 M analyzed by SEC with triple detection (SEC-TD) using 0.1 M triethylamine hydrochloride as the mobile phase.

than would the low molecular weight sample (JR 125). When comparing the peak intensities and signal-to-noise ratio (S/N) from the three detectors, it was found that the viscometer gave the most intense signal and lowest S/N, while the differential detector gave lowest signal intensity and highest S/N. Due to the broad polydispersity of the samples, the front shifting of the VISC and LS traces compared to the dRI trace can be explained by the fact that both the VISC and LS detectors are more sensitive to high molecular weight components. The dRI detector has no differentiation across the molecular weight distributions (see Eqs. (1)–(3)). Figs. 4 and 5 illustrate absolute molecular weights, hydrodynamic size, and intrinsic viscosities of Polymer JR 30M and 125 across the SEC-TD separation, respectively. As expected, the absolute molecular weights, hydrodynamic sizes, and intrinsic viscosities decrease with increasing elution times. The linear trends for all three components suggested that these polymers were well behaved without abrupt change in molecular composition and/or conformation.

3.2. SEC-TD chromatograms of Polymer JR Figs. 2 and 3 show typical SEC-TD chromatograms of high (JR 30M) and low molecular weight (JR 125) Polymer JR obtained when using 0.1 M triethylamine hydrochloride as the mobile phase. They illustrated the well-behaved peak shapes under test conditions. It was expected that the high molecular weight sample (JR 30M) would elute earlier from the column set

Fig. 3. VISC (—), LS (- - -), and dRI ( ) chromatograms of Polymer JR 125 analyzed by SEC-TD using 0.1 M triethylamine hydrochloride as the mobile phase.

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Fig. 4. VISC chromatogram of Polymer JR 30M analyzed by SEC with triple detection. The absolute molecular weight [log(Mw )], intrinsic viscosity [log(η)] and radius of gyration (Rg ) are plotted as a function of retention volume.

Fig. 6. Plots of log[η]M vs. retention volume of Polymer JR 125 in different mobile phases. (A) 0.5 M NaOAc/ACN (80:20), (B) 0.8 M NaNO3 , (C) 0.1 M TEA/1% HOAc and (D) 0.1 M TEA.

3.3. Size exclusion separation examination by universal calibration concept

ever, the curve for mobile phase containing 20% acetonitrile is shifted from the others and the polymer fraction has smaller hydrodynamic volume than the others at the same retention volume. These phenomena could be caused by the column packing material swollen and therefore the pore size shrinking when acetonitrile is added to aqueous mobile phase. In the universal calibration concept, different samples under identical SEC condition follow the same calibration curve. This phenomena was also demonstrated by Fig. 7, which was the log[η]M versus retention volume plots and dRI chromatograms of three polymer JR samples using 0.5 M NaOAC/ACN (80:20) as mobile phase. The curves of three different polymer JR samples are almost overlapped according to the main peak ranges, therefore, it is demonstrated under this condition the chromatographic separation based mainly on the hydrodynamic volume of the sample molecules. Similarly, the universal calibration

In the universal calibration concept, the separation of chemically different polymers by SEC is believed based on hydrodynamic volume (VH ). As addressed in introduction section, [η]M is proportional to VH and the plot of [η]M versus retention volume is known as universal calibration curve. When the [η] and M of each eluent fraction are tested from SEC-TD, the measured universal calibration curve becomes an efficient tool to exam the size exclusion behavior under the identical SEC conditions. Fig. 6 is the plot of log[η]M versus retention volume of polymer JR 125 in different mobile phases. It is very interesting to know that the universal calibration curves are perfect linear and the curves of three aqueous mobile phases (0.8 M NaNO3 , 0.1 M TEA/1% HOAc and 0.1 M TEA) are overlapped. Since the same column set was used for all tests, this demonstrates that under these conditions the column had same separation ability. How-

Fig. 5. VISC chromatogram of Polymer JR 125 analyzed by SEC with triple detection. The absolute molecular weight [log(Mw )], intrinsic viscosity [log(η)] and radius of gyration (Rg ) are plotted as a function of retention volume.

Fig. 7. Plots of log[η]M vs. retention volume (left Y-axis) and dRI chromatograms (right Y-axis) of three Polymer JR samples using 0.5 M NaOAc/ACN (80:20) as mobile phase. (black,
) Polymer JR 125, (red, ) Polymer JR 400, (green, ) Polymer JR 30M.

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very different mobile phase compositions. The numbers listed in the tables were averages from multiple injections. From Table 1, the calculated sample mass recovery percentages of Polymer JR 125 were near 100% under all four mobile phase conditions, the slight difference of recoveries could also be caused by small variation of the dn/dc values in different solutions. Therefore, the obtained molecular weights and other physical properties can represent the real sample since the high mass recovery under all conditions. The weight average molecular weight (Mw ) values obtained from the different mobile phase compositions were amazingly comparable with an average of 299 × 103 Da. The RSD was determined to be 1%. This could be explained by the fact that Mw was determined by using the direct method from the light scattering detector and no column calibration was used (Eq. (3)). In contrast, the number average molecular weight (Mn ) values obtained from the four different mobile phases differed considerably with a RSD of 13%. The error could be caused by the abnormal size exclusion effect and/or the extreme sensitivity of this average to the presence of low molecular weight components [28–30]. As shown in Fig. 4, the log M at peak edges are noisy. This high RSD in Mn value could also explain the relatively high RSD value for polydispersity (PD = Mw /Mn ). The values for intrinsic viscosity [η], the Mark Houwink exponent, a, and hydrodynamic radius, Rh , of Polymer 125 were determined to be very close in four different mobile phases, had the values in the range of 2.89–3.16 dl/g, 0.80–0.87, and 22.2–23.0 nm, respectively. The [η] and Rh of Polymer JR 125 in 0.8 M NaNO3 is slightly lower than those in the other three mobile phases, this could be explained by salt concentration effect on polyelectrolytes, the higher salt concentration could course a decreasing coil expansion, therefore, the [η] and Rh values are lowered. It is important to note that the Mark-Houwink exponent a is close to 0.8, suggesting that all four different mobile phases acted as good solvents for Polymer JR 125 and Polymer JR 125 existed as random coil conformations in all four different solvents [11,18]. Comparing to Polymer JR 125, Polymer JR 400 and JR 30M were determined to have higher molecular weight averages,

Fig. 8. Plots of log[η]M vs. retention volume of three Polymer JR samples using 0.1 M TEA/1% HOAc as mobile phase. (black,
) Polymer JR 125, (red, ) Polymer JR 400, (green, ) Polymer JR 30M.

curves of different polymer JR samples in other mobile phase are compared. When using 0.8 M NaNO3 as mobile phase, overlapped curves also obtained. However, when 0.1 M triethylamine hydrochloride was used, with or without 1% acetic acid, as a mobile phase, the curve of JR 30M slightly difference for those of Polymer JR 125 and JR 400 (Fig. 8) This phenomena indicates that the interaction between cationic polymer and the column packing materials could also be effected by the molecular weight. For polymers with higher molecular weight, the interaction is stronger and more difficult to be reduced. 3.4. Analysis results from SEC-TD Tables 1–3 summarize average molecular weights and other physical properties obtained for the three Polymer JR samples using the aqueous SEC with triple detection (SEC-TD) and four

Table 1 Average molecular weight values and other physical properties of Polymer JR 125 determined by SEC-TD using four different mobile phase compositions Mobile phase

Mn (×103 g/mol)

Mw (×103 g/mol)

[η] (dl/g)

Rh (nm)

a

Mass recovery (%)

(a) 0.5 M NaOAc/ACN (80:20) (b) 0.8 M NaNO3 (c) 0.1 M TEA/1% HOAc (d) 0.1 M TEA

105 121 142 135

304 299 296 297

3.09 2.89 3.16 3.11

22.6 22.2 23.0 23.0

0.81 0.80 0.87 0.86

104 102 93 95

2.90 2.46 2.08 2.21

Average RSD (%)

126 13

299 1

98 6

2.41 15

PD

Table 2 Average molecular weight values and other physical properties of Polymer JR 400 determined by SEC-TD using four different mobile phase compositions Mobile phase

Mn (×103 g/mol)

Mw (×103 g/mol)

[η] (dl/g)

Rh (nm)

a

Mass recovery(%)

PD

Relative mass recovery (%)

(a) 0.5 M NaOAc/ACN (80:20) (b) 0.8 M NaNO3 (c) 0.1 M TEA/1% HOAc (d) 0.1 M TEA

136 183 193 183

467 461 437 444

3.91 3.76 3.98 3.96

28.0 27.9 28.1 28.3

0.83 0.92 0.85 0.84

104 101 93 93

3.43 2.53 2.26 2.43

100 100 101 98

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Table 3 Average molecular weight values and other physical properties of Polymer JR 30 M determined by SEC-TD using four different mobile phase compositions Mobile phase

Mn (×103 g/mol)

Mw (×103 g/mol)

[η] (dl/g)

Rh (nm)

a

Mass recovery (%)

PD

Relative mass recovery (%)

(a) 0.5 M NaOAc/ACN (80:20) (b) 0.8 M NaNO3 (c) 0.1 M TEA/1% HOAc (d) 0.1 M TEA

549 538 556 525

1012 918 829 771

9.31 7.83 9.00 8.56

50.5 46.2 47.4 45.4

0.78 0.79 0.80 0.80

96 82 83 85

1.84 1.71 1.49 1.47

95 88 89 87

[η], and Rh values (Tables 2 and 3). In order to better compare the mass recoveries among different Polymer JR in same mobile phase, the relative recovery percentages are also listed in Tables 2 and 3. The relative recovery percentage is defined as the mass recovery of Polymer JR 400 or Polymer JR 30M relative to the mass recovery of Polymer JR 125 when using the same mobile phase. Therefore, the slight vibrations of dn/dc of Polymer JR samples in different mobile phase could be cancelled out, and it better reflected the mass recovery differences among different samples. The relative recovery percentages of Polymer JR 400 in all four different mobile phases were in the range of 98–100%, these results agreed with the [η]M versus retention volume curves in Figs. 7 and 8, i.e. the interactions between Polymer JR 400 and column packing materials were eliminated when using all four mobile phase conditions. However, the relative recovery percentages of Polymer JR 30M were in the range of 87–95%, which was significantly lower. The lower relative recovery percentage could be caused by the higher impurity of the sample as while as the stronger absorption of sample with column packing material. It was interesting to find that Polymer JR 30M had the highest relative recovery percentage (95%) when using 0.5 M NaOAC/ACN (80:20) as mobile phase. This result indicated that the interaction/absorption between Polymer JR 30M and the column packing material could be considerably reduced when using this mobile phase. Therefore the abnormal size exclusion separation could be considerably reduced under this condition, just as it was demonstrated by universal calibration concept (Fig. 7). When using this mobile phase, Polymer JR 30M followed same curve as other samples. It is worth mentioning that different polydispersity values (PD) were obtained for the three different Polymer JR samples. Polymer JR 125 and 400 samples gave comparable PD values

around 2.5, Polymer JR 30M had the narrowest polydispersity with an average of 1.6. When using 0.5 M NaOAc/ACN (80:20) as mobile phase, the obtained PD values of Polymer JR 125, 400 and 30M were determined to be 2.90, 3.43 and 1.84, respectively. In the case of SEC combined with triple detection system, the abnormal size exclusion behavior has less effect on the obtained weight-average molecular weight as long as the mass recovery is acceptable, because the weight-average molecular weight values of each fraction are directly measured by the light scattering detector without theoretical assumption on size exclusion separation. It was reported that poor resolution and noise baseline could lead to the overestimation of Mn determined by multiangle light scattering detection [31–32]. Therefore, they lead to the underestimation of polydispersity values. In order to better understand the PD values in this study, the comparison of Mn , Mw and PD from universal calibration with those from SEC-TD measurement were calculated and listed in Table 4. Four injections of two Polymer JR samples with two mobile phase conditions were used as examples. The universal calibration curve for each condition was generated by linear fitting of log[η]M ∼ Rt of Polymer JR125 in the mobile phase, and the obtained constants were used for the calculations of Mw , Mn and PD of JR 400 and JR 30M by universal calibration method (SEC-UC). When using 0.5 M NaOAc/ACN as mobile phase, the Mw values from the SEC-TD and the SEC-UC are very similar. The Mn obtained from SEC-UC is significantly lower than that from SEC-TD, and therefore PD is higher when using SECUC method. When changing the peak limits (more specifically, eliminating the peak tail) the Mn and PD values had a much more significant decrease when using SEC-UC method than those using the SEC-TD method. For example, the PD value of JR 30M using the SEC-UC methods decreased from 3.98 to

Table 4 Average molecular weights and polydispersity of Polymer JR 125 and 30M determined by SEC-UC and SEC-TD using two different mobile phase compositions Mobile phase

(a) 0.5 M NaOAc/ACN (80:20)

(c) 0.1 M TEA/1% HOAc

a

Sample

Peak range

Mass (%)a

Mw (×103 g/mol)

Mn (×103 g/mol)

PD

SEC-TD

SEC-UC

SEC-TD

SEC-UC

SEC-TD

SEC-UC

JR125

10.92–18.16 10.92–16.00

100 93.3

298 316

288 307

126 161

89 164

2.36 1.96

3.23 1.87

JR 30M

10.08–16.30 10.08–14.60

100 93.7

1020 1080

746 795

547 659

187 387

1.87 1.64

3.98 2.05

JR125

11.11–19.35 11.11–16.50

100 94.0

294 311

287 304

132 165

91 161

2.23 1.88

3.14 1.89

JR 30M

10.29–17.96 10.29–15.50

100 94.3

879 919

570 604

560 616

57 212

1.57 1.49

9.99 2.85

The relative percentage of sample mass used for calculation.

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2.05, when the peak tail was not counted and the 93.7% of the mass was considered for the calculation. In the case of SEC-TD methods the PD value is not as sensitive as the SEC-UC method, the PD value decreased from 1.87 to 1.64. These comparisons demonstrated that the Mn and PD values calculated by the SECUC method are extremely sensitive to peak tail comparing with SEC-TD. When the resolution is poor or the abnormal size exclusion exists, the fractions at peak tail could have higher molecular weight component. Therefore, the measured molecular weight M and intrinsic viscosity [η] are higher then expected ideal values. In the case of using the SEC-TD method no calibration curve is applied, and the weight average molecular weight of each fraction is measured by light scattering detector, so the Mn could be overestimated by using the molecular weight from LS. In the case of the SEC-UC method, the molecular weight of the fraction derivates from calibration curve ([η]M versus retention volume) and measured [η], so the molecular weights of the fractions at peak tail are underestimated and lower Mn and higher PD will be erroneously calculated. Based on the mathematic difference between these two methods and the statistic definition of Mn . The extent of the underestimation of Mn by the SECUC is more serious than that of the overestimation of Mn by the SEC-TD. As demonstrated by the universal calibration curves in Fig. 8, when using 0.1 M TEA/1% HOAc, the curve of polymer JR 30M shows slightly difference from JR 125. These differences are translated into a significant difference of Mn and PD values by SEC-UC method. As listed in Table 4, the obtained PD of JR 30M under this condition could be as high as 9.99, which is obviously greatly overestimated. To summarize, the SEC-TD method could underestimate the PD and SEC-UC could overestimate the value, the variation of PD from SEC-UC is much larger than that from the SEC-TD methods. Based on the obtained PD values when using 0.5 M NaOAc/ACN as mobile phase and the relative mass recovery of the samples, the polydispersities of three polymer JR sample are believed to be in the similar range. Based on the viscosity data provided by Amerchol, the use of aqueous SEC with triple detection confirmed the trend toward lower molecular weights for Polymer JR 30M, JR 400, and JR 125. Polymer JR 30M was reported to have the highest molecular weights as well as the greatest solution viscosity. Polymer JR 30M gave the largest values in all four mobile phase, and Polymer JR 125 gave the smallest values under the corresponding mobile phase conditions. 4. Conclusions In this report, three Polymer JR samples with different solution viscosities were successfully characterized using aqueous SEC with triple detection and four different solvent systems. For polymer JR 125 and JR 400, all four mobile phases, with either high salt concentrations (ionic strength) or addition of competitive ions, effectively minimized or eliminated the undesired ionic interactions between the polymer molecules and the column packing materials. Universal calibration concept was used to evaluate the abnormal size exclusion separation of Polymer JR samples when using different mobile phases. It was discovered that the abnormal size exclusion was dependent on not only the

separation conditions, but also the molecular weights of the samples analyzed. By using a combination of high buffer content and organic solvent, the abnormal exclusion separation phenomenon of high molecular weigh Polymer JR (JR 30M) could be considerably reduced. Therefore, the absolute molecular weights, molecular weight distributions, and hydrodynamic sizes of all three Polymer JR samples were obtained. The reliability of PD values obtained form SEC-TD was discussed in comparison with that from SEC-UC. The absolute molecular weight and intrinsic viscosity values of the Polymer JR samples agreed reasonably well with the solution viscosity data provided by the manufacture. All three Polymer JR samples were determined to have a random coil conformations in the four different solvent systems evaluated. In summary, aqueous SEC with triple detection was a very powerful technique for determining absolute molecular weight, molecular weight distribution, molecular size, and conformation of cationic hydroxyethylcellulose derivatives, as well as a powerful tool to examine the abnormal size exclusion separation phenomenon under the testing conditions. The knowledge of these detailed molecular information of hydroxyethylcellulose derivatives is extremely valuable in predicting product performance and process control in the personal and health care industry. References [1] E.J. Ellis, J.C. Salamone, Contact lens with a hydrophilic, polyelectrolyte complex coating and method for forming same, US Patent 4,168,112 (1979). [2] E.J. Ellis, J.Y. Ellis, Method for wetting contact lenses, US Patent 5,711,823 (1998). [3] E.J. Ellis, J.Y. Ellis, Composition for wetting contact lenses, US Patent 5,872,086 (1999). [4] Z. Hu, C.E. Soltys, Method for treating extended-wear contact lenses in the eyes, US Patent 6,274,133 (2001). [5] D.J. Heiler, S.E. Maier, S.P. Spooner, Composition and method for inhibiting of protein on contact lenses, US Patent 6,323,165 (2001). [6] C.R. Robbins, Chemical Physical Behavior of Human Hair, Van Nostrand Reinhold Company, New York, 1979. [7] E.D. Goddard, J.V. Gruber, in: E.D. Goddard, J.V. Gruber (Eds.), Principles of Polymer Science and Technology in Cosmetics and Personal Care, Marcel Dekker, New York, 1999. [8] P. Hossel, R. Dieing, R. Norenberg, A. Pfau, R. Sander, Int. J. Cosmetic Sci. 22 (2000) 1. [9] W.W. Yau, J.J. Kirkland, D.D. Bly, Modern Size-Exclusion Liquid Chromatography, Wiley-Interscience, New York, 1979. [10] S.T. Balke, in: H.G. Barth, J.W. Mays (Eds.), Modern Methods of Polymer Characterization, Wiley, New York, 1991. [11] L.H. Sperling, Introduction to Physical Polymer Science, WileyInterscience, New York, 2001. [12] D. Nagy, Am. Lab. 2 (1995) 47J. [13] X.M. Liu, E.P. Maziarz, D.J. Heiler, J. Chromatogr. A 896 (2004) 19. [14] J. Brandup, E.H. Immergut, in: J. Brandup, E.H. Immergut (Eds.), Polymer Handbook, fourth ed., Wiley, New York, 1999, p. 738. [15] D.J. Nagy, J. Liq. Chromatogr. 16 (1993) 2041. [16] I. Hall, D. Gillespie, K. Hammons, J. Li, Adv. Chitin. Sci. 1 (1996) 361. [17] S.V. Greene, in: J. Cazes (Ed.), Encyclopedia of Chromatograph, Marcel Dekker, New York, 2002, p. 738. [18] M.U. Beer, P.J. Wood, J. Weisz, Carbohydr. Polym. 39 (1999) 377. [19] M.L. Fishman, D.T. Gillespie, S.M. Sondey, Y.S. El-Atawy, Carbohydr. Res. 215 (1991) 91. [20] D.J. Nagy, J. Liq. Chromatogr. 13 (1990) 677.

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