Overcoming bottlenecks in the laboratory, and thus saving valuable time and money, is an important goal for scientists. This article discusses a seventh-generation gel permeation chromatography (GPC) instrument that features improvements in column oven and pump fluidics, enhancements in temperature control functions, and high sample throughput. The all-in-one system rapidly characterizes polymers with high efficiency, reliability, and reproducibility. The system’s performance when used with the manufacturer’s proprietary multipore GPC columns is also presented.
One factor that affects GPC analysis is system reproducibility, as well as systemt-to-system variation. Often these variations are the result of one or more factors, including flow rate reproducibility, baseline drift, and injection reproducibility. For the most part, measurements can be reproduced using the same equipment, but may differ when another system is used.
Another important variable is the influence of temperature fluctuations on solvent delivery and baseline stability. In the EcoSEC® GPC system (Tosoh Bioscience LLC, Montgomeryville, PA), temperature control for the pumps, columns, and tubing has been added. A built-in column oven, which also houses pump tubing and columns, ensures that the mobile phase remains at constant temperature from injection to the waste vial. This constancy of temperature is unaffected by changes in ambient temperature.
Results and discussion
Figure 1 - Elution time reproducibility of polystyrene standards.
A study was performed to assess elution time and molecular weight determination reproducibility using different polymer samples. The results (see Figures 1–3) show that the engineering design concepts of the system produce highly reproducible elution times and molecular mass determinations. The data from Figure 1 show that the intra- and interday elution time reproducibility is <0.03% for the two nearly monodisperse polystyrene samples. For poly(vinyl chloride–vinyl acetate) and poly(ε-caprolactone), as shown in Figure 2, intra- and interday Mw reproducibility, calculated from a linear polystyrene calibration curve, were <0.2 and <0.3% CV, respectively. Reproducibility studies were also conducted using a terpolymer, poly(vinyl chloride–vinyl acetate–vinyl alcohol) (Figure 3). Intra- and interday data also show reproducibility values of less than 0.2% in terms of molecular weight. (The tenfold increase when going from elution time to molecular weight is caused by the logarithmic function of the calibration curve.) These data exceed results typically obtained from other size exclusion chromatography (SEC) systems.
Figure 2 - Molecular weight reproducibility of a copolymer and polyester.
The GPC system also incorporates an extra cooling fan to remove heat from the pump heads, a unique check valve design, and better pressure monitoring to control pump speed. To assess the environmental influence on solvent flow, a test was done in which the EcoSEC GPC instrument and a conventional GPC system were placed in a chamber, where the temperature was cycled. A series of injections of polystyrene were made over time. The elution volume at peak maximum was measured, and the resulting data are shown in Figure 4. The elution volume drift of the EcoSEC GPC system was approx. 20% lower than that of the conventional GPC system. Since the SEC mechanism is temperature independent, this elution volume drift was ostensibly caused by expansion and contraction of the mobile phase, important considerations especially when using on-line viscometric detection.
Figure 3 - Molecular weight reproducibility of a terpolymer.
With a stagnant, referenced-flow refractive index (RI) detector, the RI of tetrahydrofuran (THF) mobile phase in the reference cell slowly alters over time, presumably caused by the buildup of peroxide-related compounds. To reduce RI baseline drift, the GPC system features a second, thermostatically controlled pump used to pump mobile phase at a controlled rate through the reference side of the RI detector. Thus, any minor changes in mobile phase composition are “zeroed” out, giving negligible baseline drift. This baseline stability is demonstrated in Figure 5 using RI detection and a low-MW test probe, dicyclohexyl phthalate. With the system, superposition of five consecutive chromatograms demonstrates negligible baseline drift (Figure 5a), compared to the same experiment repeated with a stagnant reference cell (Figure 5b) from a conventional GPC unit.
Figure 4 - Mobile phase delivery reproducibility with ambient temperature changes.
To test instrument reliability, a round-robin study was undertaken in which the same polydisperse poly(vinyl chloride–vinyl acetate–vinyl alcohol) sample was run on EcoSEC GPC systems located at four different sites. Each site used the same types of columns and similar calibration protocol (see Table 1). The sample was run using a TSK-GEL SuperMultiporeHZ-M column (Tosoh Bioscience LLC) (4.6 mm i.d. × 15 cm, 4 μm) × 2 at a flow rate of 0.35 mL/min at 40 °C using THF as the mobile phase. As shown in the table, reproducibility from system to system and from location to location was excellent, as would be expected from the intralaboratory studies given in Figures 2 and 3. These results demonstrate the stable SEC flow rate and baseline found with the GPC system.
Figure 5 - RI baseline drift comparisons.
Because of the high instrument-to-instrument reproducibility of the system, methods developed at one location, e.g., an R&D laboratory, can be transferred to a second site, e.g., a QC laboratory at a manufacturing site. The SEC methods can therefore be reliably duplicated from one site to another site, which is needed for compliance with Six Sigma and ISO 9000 certification.
Additional instrument features
The low dead volume of the system and the low-volume flow cell of the RI result in an instrument that provides reduced analysis time and reduced solvent use. The goal of decreasing solvent consumption and reducing solvent disposal costs is an operational demand of all laboratories. By using semimicro technology, less solvent is used and run times are shortened. The small stroke volume of the pump allows for reduced flow rate (10–2000 μL/min) suitable for ESI-MS of low-MW polymers.
Table 1 - Reproducibility at four different sites
To take advantage of the semimicro design, a line of GPC columns, the SuperMultiporeHZ series, was developed. Previously, when characterizing polymer samples with a wide molecular weight range, scientists were left with two options: either use columns containing a mixture of resins with varying pore sizes or use individual columns packed with given pore sizes. Here, a different approach was taken: Through the use of proprietary multipore technology, particles with widely varying pore sizes were synthesized.
There are currently three different column selections in the SuperMultiporeHZ series: SuperMultiporeHZ-M, -N, and -H, each with a different particle size, exclusion limit, and linear MW range. These columns have a smaller i.d. (4.6 mm) and shorter column length (15 cm), as compared to most other commercially available GPC columns. Table 2 details the physical properties of the columns.
Table 2 - Characteristics of the SuperMultiporeHZ series of semimicro columns for use with the EcoSEC GPC system
Another parameter that affects molecular mass analysis is the linearity of the SEC calibration curve. Even columns with the same separation range can have differently shaped calibration curves. The SuperMultiporeHZ columns produce very good linearity with a shallow slope, indicative of superior resolution across the molecular weight range (see Figure 6).
Figure 6 - Linearity of TSK-GEL SuperMultiporeHZ-M columns.
An advantage of using semimicro technology is that analysis time is 50% lower compared to conventional columns and systems, while solvent consumption is lowered by approximately one-sixth the volume for a given separation.
The versatility of the EcoSEC GPC system is realized with the addition of an optional column-switching valve. The user is able to conveniently switch from one column to another of different pore size and/or column length for optimal molecular mass separation. Furthermore, an empty capillary tube can be inserted in place of the second column for sample recovery studies. This approach can be used to determine whether or not complete elution of the sample has occurred without irreversible adsorption, a potential source of error occasionally encountered.
The system is controlled by EcoSEC GPC Work Station software, which is capable of running two instruments. This powerful, versatile software is intended to facilitate and simplify system operation and data analysis. Setting authentication, passwords, and user access privileges is effortless. The software manages the instrument, allows programming start and shutdown times, displays operating conditions in real time, audits the data, and prints custom reports.
To aid in SEC calibration, PStQuick calibration standard kits (Tosoh Bioscience) are available in a wide range of molecular weights. The kits are premixed polymer standards to facilitate column calibration. The calibration standards help give highly reproducible calibration curves, which are essential for reliable molecular mass analysis. In addition, calibration of a TSK-GEL SuperMultiporeHZ column with PStQuick standards can be completed in a few hours rather than the day or more that is required with conventional columns and standards.
Using TSK-GEL SuperMultiporeHZ series semimicro columns and PStQuick standards, the EcoSEC GPC system reduces solvent cost, increases throughput, and minimizes errors in setting up calibration standards. The instrument has been specifically designed for routine or standardized SEC analysis, where a premium is placed on high-precision molecular mass measurements.
Dr. Villarreal is GPC Marketing Manager, Tosoh Bioscience LLC, 156 Keystone Dr., Montgomeryville, PA 18936, U.S.A.; tel.: 215-283-5028; e-mail: firstname.lastname@example.org. Dr. Barth is a consultant specializing in the application of size-exclusion chromatography (e-mail: email@example.com).