As the properties of a polymer can depend greatly on its size, there has always been a need to be able to measure it, both in terms of molar mass and physical size. The earliest polymers studied were materials such as elastomers and, later, synthetic polymers like polyethylenes or polypropylenes. The range of samples studied has since increased enormously with the realisation that many biological functions are also controlled by molar mass, size and shape.
The initial techniques used were based on the principle of measuring physical characteristics and then relating these properties to molar mass. The most useful of these were: u Membrane osmometry transfer across a membrane. u Sedimentation settling experiments, later vastly improved with the use of ultracentrifuges.
Another instrumental approach was also possible with the development of light scattering by Einstein, Raman, Debye, Zimm and others.
All of these techniques, including light scattering, were absolute measures of molar mass (ie they did not rely on a calibration or other assumption in the measurement, and all measurements were made from first, physical principles). However, the main disadvantage with measurements made using membrane osmometry and sedimentation was that they were only able to characterise the bulk properties of the polymer sample as a whole rather than the individual components. These measurements also proved excessively time consuming and subject to operational error.
Size exclusion chromatography
In order to gain information about a polymer made up of many different molar masses and sizes, a method was needed for separating the molecules present. This was achieved through the introduction of high performance size exclusion chromatography (HPSEC) later known as SEC or gel permeation chromatography (GPC).
SEC and GPC separate molecules in the column on the basis of size. A solvent containing the molecules is passed through a porous bed of polymeric gel stored in the column. As there is more interaction between smaller molecules and the stationary phase than the larger ones, the large molecules move more rapidly through the column, separating the mixture into its components. Thus the sample is separated according to the hydrodynamic volume (Vh) of the individual molecules or, put more simply, the larger molecules come out first (see below).
Before the advent of light scattering, polymer standards were used to convert the time at which the samples eluted into a measurement of molar mass. If the molar mass of the standard was known, then the time of elution would equate to the appropriate molar mass. Using multiple standards, a calibration curve of time versus molar mass could be drawn up. This was a major advance in polymer analysis because a single polymer could be shown to be made up of many different sized components, the complexity and distribution of which would also affect the physical properties.
However, because the system was calibrated according to the size characteristics of the standards (not the molar mass), the ratio between the molar mass and size of the standard had to be the same to ensure the calibration was valid. To be accurate, the standards not only had to be of the same polymer as the sample to be studied, but also in the same conformation (the shape of the molecule had to be the same) and in the same eluent. Moreover, there had to be no chemical or electrostatic interactions between the polymer and the column packing material (column interactions).
This process was improved somewhat by introducing an on-line viscometer which made relative viscosity measurements on the eluted polymer solution. This was better than straightforward calibration as it offered a way of detecting changes in the size density of the molecule. Nevertheless, it still depended on the accuracy and validity of the standards and on a uniform shape of the molecule.
The need to obtain accurate information on the molar mass and size of the polymers became more urgent as demands on polymer properties increased. This was especially important in pharmaceutical applications where changes in molar mass through aggregation or shape changes could actually have a deleterious effect instead of a beneficial one (for example prions, association of aggregated proteins with degenerative disease).
The obvious answer was to connect a light scattering detector to the separation system.
Traditional light scattering instruments worked by taking readings from multiple angles, each being measured in series. A low angle light scattering system was developed that allowed a single measurement to be used to calculate the molar mass (but this instrument sacrificed the ability to measure size and, by implication, conformation).
Although measurements at low angles are problematic for fundamental physical reasons molecules tend to scatter more light in lower angle directions than in higher angles, and these low angle scattering events caused by dust and contamination of the mobile phase easily overwhelm the scattering from the molecules of interest low angle laser light scattering or LALLS became popular in the late 1970s and early 1980s. The mid 1980s saw the development of multi-angle light scattering (MALS) instruments that were able to make measurements at the different angles simultaneously, and that included lasers which improved the intensity and consistency of the source.
The connection between MALS detectors and SEC systems was the link that made routine absolute molecular weight and size measurements a reality. The combined system (MALS+SEC) enabled molar mass and size to be determined from each slice of the polymer fraction, meaning that MALS instrumentation had a significant role in a wide range of R&D applications, including molecular biology, nanotechnology and environmental analysis.
MALS
MALS can be applied to synthetic polymers, proteins, pharmaceuticals and particles, such as liposomes, micelles and encapsulated proteins. Measurements can be made in one of two modes: un-fractionated (batch mode) or in continuous flow modes (with SEC, HPLC or any other flow fractionation method).
Batch mode experiments can be performed either by injecting a sample into a flow cell with a syringe, or with the use of discrete vials such as scintillation vials or microcuvettes where sample availability is low.
The batch measurements are most often used to measure timed events like antibody-antigen reactions or protein assembly, but can also be used to determine the second virial coefficient, a value that gives a measure of the likelihood of crystallisation or aggregation in a given solvent. This is useful in protein crystallography, for example.
Continuous flow experiments can be used to study the material eluting from virtually any source. Wyatt Technology was able to study the on-going process in a reaction vessel by coupling a reactor bleed to a MALS instrument. The detectors have also been coupled to a variety of different chromatographic separation systems. By combining these separation systems, Wyatt Technology achieved an absolute measurement of the mass and size of the materials as they eluted.
MALS measurements work by calculating the amount of light scattered at each angle detected. This process both overcomes the problems associated with low angle detectors where typically there is around ten times the non-sample noise at an angle of fifteen degrees or below compared to observing at ninety degrees, and it allows a reliable and accurate measure of the light scattered; the greater the number of detectors, the better the accuracy of the experiment. Statistically, the greater the number of measurements, the greater the precision of the measurement.
Why use light scattering?
The utility of SEC or a similar separation combined with the advantages of an absolute detection method is achieved by adding a MALS detector after a chromatographic separation. The light scattering data is purely dependant on the light scattering signal and the concentration; the elution time is irrelevant and the system can be changed for different samples and solvents without recalibration. In addition, a non-size based separation method such as HPLC or IC can also be used (see below).
Fig.2 shows the SEC separation of a sample of RNase. Under the conditions used, the native RNase elutes at around 24.5minutes. If the same sample is then reduced, this makes the molecule unfold and become much less compact (larger in size) meaning the RNase will elute earlier (in this case 19minutes). However the light scattering data shows a molar mass of 13700g/mol regardless of when the sample elutes.
As the light scattering detector is mass dependant you get more scattering at higher molar masses or greater concentrations it becomes more sensitive as the molar mass increases. Thus it is an excellent tool for detecting aggregation.
An example of how powerful light scattering can be in aiding the identification of components in an unknown mix can be demonstrated by reference to a real life example. A large UK pharmaceutical company was studying the behaviour of a recombinant protein in the presence of Rotamase.
The sample was initially examined by SDS-PAGE which showed that the protein and the Rotamase were present, along with some additional components (below).
Aqueous SEC was then carried out on the sample which confirmed there was in fact a complex mix. Adding Wyatt's DAWN multi-angle light scattering detector to the SEC experiment allowed the scientists to measure the molar mass in each fraction of the chromatogram (next diagram).
It is clear from the resulting molar mass versus volume plot that there is an interaction between the protein and the Rotamase, which subsequently affects the behaviour of the protein.
The poor resolution of the peaks is due to the fact that this is a dynamic equilibrium. Thus, there is no single species that can be resolved.
Summary
Wyatt's MALS instruments coupled to SEC or any other liquid fractionation techniques can provide an absolute means for measuring the molar mass, size and distribution of polymers of all sorts.
MALS also enables the elucidation of additional data such as branching, conformation and eluent behaviour.
The same instrumentation also allows observation of molecular interactions in a real-time environment.
As multi-angle light scattering is an absolute mass and size measuring technique, the separation mechanism itself is irrelevant and as such, MALS is suitable for both highly variable research applications as well as routine QA of previously characterised samples.
Kevin Jackson is Technical Director with Wyatt Technology. Wyatt Technology Corporation (WTC) is involved in the research, development, and commercialisation of absolute macromolecular characterisation techniques and their instrumentation.
Founded in March 1982, the company was formed around certain of his patents, ideas and inventions in industrial, military and medical domains. To date, WTC has developed instruments capable of measuring the multi-angle light scattering characteristics of macromolecules (and particles) in solution, refractometers, as well as instruments for airborne aerosol samples.
The DAWN detectors by some of the largest corporations, including DuPont, Dow, IBM, Glaxo, Unilever, Wyeth, Bayer and Merck.
For more information about absolute macromolecular characterisation and light scattering instrumentation, please visit www.wyatt.com or e-mail info@wyatt.com
