GPC/SEC is a vital analytical technique for characterizing polymers.
Gel permeation/size exclusion chromatography (GPC/SEC) is a vital analytical technique used to characterize synthetic and natural polymers, including biologically important macromolecules such as proteins and DNA. Evolving challenges—such as the development of smart, novel polymers with exacting performance specifications; the replacement of conventional polymers with those that are more environmentally benign; and the formulation of biopharmaceuticals—tax the capabilities of traditional GPC/SEC and invite advances in the technology.
A critical tool for molecular characterization
A basic understanding of how GPC/SEC works provides an important basis from which to assess the boundaries of the technique and the value of recent innovations.
GPC/SEC is a two-step analytical technique in which samples are separated into fractions on the basis of hydrodynamic size, followed by characterization of the fractions. Separation is achieved by passing the sample through a packed chromatography column. Detecting the amount of sample in each sized fraction enables determination of the size distribution and, more importantly, molecular weight and molecular weight distribution data.
Traditional GPC/SEC systems use a single concentration detector; most commonly a refractive index (RI) detector. Column calibration, using appropriate standards, provides the correlation needed to estimate a molecular weight distribution from measurements of the amount of sample (concentration) in each sized fraction. However, the resulting molecular weight data are only accurate if the relationship between molecular size and weight is the same for the sample as for the standard. This is a crucial limitation, most especially for novel materials where there are often no appropriate standards, and where absolute rather than relative data may be critical.
Boosting informational productivity
In recent years, the limitations of a single detector set-up have been addressed by increasing the use of multiple detectors. For example, a light scattering detector, in combination with a concentration detector, enables direct measurement of the absolute molecular weight of the eluting molecules, eliminating the need for column calibration standards. Further complementary additions include a viscometer, to enable the measurement of structural features, such as branching or conformation, and a photodiode array (PDA) detector for detailed investigation of the distribution of chromophores as a function of molecular size/weight.
The two-step nature of GPC/SEC means the associated instrumentation is well-suited to progressive enhancement. In many laboratories, the evolution of the technique can be seen from the design of successively attached detectors. However, today’s busy laboratories typically demand levels of accuracy and productivity that can’t be delivered by such “user-developed” systems, often with multiple software packages and complex data integration. It’s increasingly important that all users, often a variety of researchers and analysts measuring very different sample types, can quickly and easily access high quality data.
Innovations in the latest GPC/SEC systems answer to these requirements with easier-to-use integrated systems that deliver greater sensitivity and accuracy, while requiring only a small amount of sample, often a critical requirement at early stages of development.
| Performance Improvement | The practical value | Features to look for |
|---|---|---|
| Better baseline stability and improved signal-to-noise ratio. | Improved sensitivity. | High-performance pumps for low pulsation. Efficient degasser performance. |
| Quicker set-up and reduced time to changeover from one sample type to another. Lower sample usage. |
Ease-of-use, especially when analyzing a range of different sample types and/or when samples are precious. |
Low-volume degassers. Injector designs with minimal or zero sample loss. |
| Precise temperature control. | Ease-of-use, especially for samples that are temperature sensitive, or when using high viscosity solvents. Better resolution, higher accuracy. |
Precise temperature control of the three key instrument regions: All detectors and the inter-detector tubing connecting them; The autosampler storage tray (4 C required for proteins); And the column oven that can be controlled at 20 C, and up to 65 C. |
Faster, more precise separation
Precisely resolved separation of the sample is a critical first step in any GPC/SEC experiment, and has a defining influence on the overall accuracy of the data. The performance of the separation module directly influences the stability of the analysis baseline and, consequently, the signal-to-noise ratio achieved during detection. Better separation performance translates directly into more accurate measurements (Table 1).
In novel separation modules—such as Malvern Instruments’ OMNISEC RESOLVE—each element of the system—degasser, pump, autosampler and column oven—has been refined and optimized to deliver superior performance specifically for GPC/SEC. For example, the latest degasser design combines low volume, which makes it easier to switch between different solvent systems, with the degassing performance needed to secure high signal-to-noise ratios on all detectors. New low pulsation pumps deliver highly stable flow rates for smooth experimental baselines, and also have features such as integrated seal back flushing that reduces seal wear and allows use of high salt buffers without increasing servicing requirements.
The latest autosamplers have zero overhead injection, which means they can inject the sample with no sample waste. They also have the precise temperature control needed to ensure all samples are maintained in an optimal state. For example, proteins, which have a tendency to aggregate, can be cooled to protect them from degradation.
Successful analysis relies on maintaining the sample at a closely controlled temperature during both separation and detection. For example, certain polymers will dissolve only in relatively high viscosity solvents. Here integrated column ovens make it possible to run at elevated temperature, to reduce the solvent viscosity and the pressure in the separation columns, thereby helping to improve system performance.
| Detector type | Features to look for | Practical benefit |
|---|---|---|
| Refractive index | Robust flow cell design. Temperature stability. |
Enables first-in-series connection for improved stability and sensitivity. Better baseline stability; improved sensitivity. |
| UV | PDA designs that offer detection over the full UV/Vis wavelength range. |
Accurate concentration measurements for proteins and other samples containing chromophores; detection and quantification of different components in blends, copolymers or protein conjugates. |
| Light scattering | Inclusion of RALS. Inclusion of LALS. Fiber delivered laser. |
Accurate measurement for low molecular weight samples. Accurate and direct measurement for high molecular weight samples. Greater sensitivity for all measurements—greater accuracy with less sample. |
| Viscometer | Automated mechanical bridge balancing. User-exchangeable capillary module and delay volumes. Robust, all stainless steel pressure transducers with a wide operating range. |
Optimized accuracy and sensitivity for each analysis. Easy user set-up and reduced downtime. Ease-of-use—system is suitable for a very wide range of solvent and buffer types. |
High-sensitivity detection
As multi-detector arrays become more widely used, there’s more focus on ensuring these detectors work together effectively. Although detectors can be sourced individually, integrated arrays offer a number of distinct advantages (Table 2). These include:
- Detectors connected in a sequence that optimizes the performance of each one.
- Reduced inter-detector tubing lengths, to minimize band broadening.
- Precise temperature control over the full detector array and connecting tubing.
As a result, an integrated detector array—such as in the OMNISEC REVEAL—delivers more accurate results and has higher sensitivity than modular detector set-ups. As well as the shift to integration, the performance of individual detectors is also being substantially enhanced to meet evolving analytical needs.
For example, as a result of more efficient optics and advanced fiber connectivity, the latest light scattering detectors are many times more sensitive than older designs. This greater sensitivity delivers more accurate measurements for low molecular weight materials that produce relatively weak light scattering signals, and provides more precise analysis with smaller sample injection volumes. As a result, the newest light scattering systems deliver high performance across a wide range of molecular weights and concentrations.
Increased sensitivity in the measurement of molecular weight and concentration calls for higher accuracy and productivity in measurements made for structural analysis. This requirement is addressed in new viscometer designs, where the use of a self-balancing mechanism ensures viscosity measurements are made at optimum sensitivity. Productivity is also improved by the use of user-exchangeable capillary modules and user-selectable delay volumes, which support the optimization of viscometer performance.
New levels of analytical productivity and accuracy
GPC/SEC is an essential technique for those working with macromolecules, whether polymers, proteins or polysaccharides. Advances in GPC/SEC technology therefore have widespread impact in polymer, food, pharmaceutical and biopharmaceutical industries, as well as in academic research. Recent innovations have produced fully integrated separation and detection modules that break new ground in critical areas such as the accuracy and sensitivity of measurement, instrument productivity, ease-of-use and the management of the analytical workload associated with achieving high-quality data. Such improvements widen the field of applications GPC/SEC can be successfully used for, and have the potential to positively impact the efficiency of analysis in all application areas.
