Gel permeation or size exclusion chromatography (GPC/SEC) is a highly valuable tool for characterizing natural and synthetic polymers and proteins. However, basic GPC/SEC systems that have only a UV or refractive index (RI) detector provide only relative molecular weight measurement. This limits their use in research. Low angle light scattering (LALS) detectors, on the other hand, directly measure the absolute molecular weight of the sample as it elutes from the column.
A GPC/SEC column separates a sample on the basis of hydrodynamic size, rather than molecular weight. Larger molecules pass through the column more quickly than smaller ones and thus have shorter retention volumes.
With a system that has only an RI detector, the concentration of macromolecule or polymer in the eluting solution is determined from the change in RI brought about by its presence. The detector provides no information about molecular weight, which is instead inferred from calibration data. Calibration with a standard generates a molecular weight versus retention volume plot. In combination with the RI concentration data, this produces a relative molecular weight distribution.
This approach is limited in that the molecular weight reported is only correct if the hydrodynamic size-to-molecular-weight relationship of the sample is identical to that of the standard. For many systems, standards are not readily available, so reporting relative (rather than absolute) molecular weight is usual. In some instances, this creates massive errors.
Absolute molecular weightLight scattering is a well-established analytical technique for absolute molecular weight determination. The excess scattering caused by a known concentration of macromolecules in a solution, relative to the scattering from the pure solvent, is related to the molecular weight of the material present.
Therefore, measuring the intensity of scattered light produced when a laser beam passes through the sample allows the direct determination of molecular weight. The true scattering intensity, however, which gives the correct molecular weight, can only be measured at or very close to an angle of 0 degrees from the incident beam (see Figure 1).
Any large macromolecules that are present will scatter light asymmetrically. Destructive interference then reduces intensity at all angles greater than 0 degrees. Figure 1 illustrates the shape of this asymmetry, showing how the measurement error introduced depends on the angle at which scattering intensity is detected. This angular dependence is not constant and increases with molecular size, making it more important to measure close to 0 degrees for larger molecules.
Developing LALS detectors
In recent decades, the development of new polymers for industrial, pharmaceutical, biomedical and food applications has stimulated the commercialization of light scattering detectors. The first ones appeared in the 1970s, but technical limitations hampered both their design and construction. Early development then became driven by the need to overcome these limitations rather than focusing on adopting the best scientific approach.
It has long been clear that measuring close to 0 degrees minimizes errors in molecular weight. However, it presents a technical challenge because it demands accurate discrimination between the incident and scattered light.
This problem was not properly solved in the design of early instruments and, as a result, they were arduous to use. Any particulates present in the eluent showed up as spikes in the scattered light signal, so preparing both eluent and sample demanded exemplary cleanliness. Also, at the time, columns often contained manufacturing particulates, which exacerbated the problem.
These difficulties encouraged the adoption of multiple angle light scattering (MALS), which was promoted as an alternative method for molecular weight measurement. In theory, MALS avoids the issue of measuring at 0 degrees by measuring at a range of much higher angles and then using the data to extrapolate back to 0 degrees.
Unfortunately, this approach does not solve the underlying problem because the accuracy of measured molecular weights is highly dependent on the low angle signals. Furthermore, selection of the correct data fitting algorithm for the extrapolation process depends on prior knowledge of the sample. So, although MALS provides a method for measuring molecular weight, the reliance on an extrapolation places it far from most people’s understanding of an “absolute” measurement.
More recently LALS technology has been revisited, and in 2001 the modern LALS detector was introduced. Combining new advanced optics with clever instrument design, it accurately captures the scattering signal at an angle of 7 degrees, effectively discriminating it from the incident beam. This angle is so close to 0 degrees that measurement errors are very small, even for the largest molecules (see Figure 2).
With this system, a mirrored prism removes the incident laser beam from the optics after it has passed through the solution, allowing collection of the 7-degree signal by the LALS photodiode detector. This avoids any need for extensive solvent or sample preparation and permits the use of normal instrumentation and columns. The efficient optical design produces optimal sensitivity at lower laser powers, and innovative software processes results without data fitting, giving excellent molecular weight accuracy.
Multiple detection benefits
The modern LALS detector is ideal for GPC/SEC systems incorporating multiple detectors. It has a cell volume of just 18 uL, many times smaller than other light scattering detectors, and the compact detector measures only 20 cm long. Within a multiple detection system, it provides absolute molecular weight measurement and eliminates the need for calibration. The molecular weight is calculated at each retention volume slice, 5x per second.
The LALS is used together with RI or UV detectors to provide concentration data and with the viscometer detector to provide molecular size and information relating to molecular structure and branching. The LALS detector, therefore, complements the other detectors, maximizing information flow from the GPC/SEC system.
Conclusion
Modern LALS detectors provide polymer and biopolymer chromatographers with a powerful and valuable tool for direct, absolute molecular weight measurement. The accuracy of results is excellent and use of the detector straightforward. No assumptions are required about molecular size or shape, and there is no need for data fitting and/or extrapolation. When used in combination with other detectors (RI, UV and viscometer) in multiple detector systems, it provides comprehensive macromolecule characterization: absolute molecular weight, molecular size and information about branching and structure.
For more information, contact Paul Clarke, Viscotek, a Malvern company, at paul.clarke@viscotek.com or visit www.viscotek.com.
