Size Exclusion Chromatography Variations And Innovations

Interview conducted by G.P. ThomasAug 23 2013

Stephen Ball, Product Marketing Manager for Nanoparticle and Molecular Characterization at Malvern Panalytical, tells AZoM how the latest addition to the company’s range of detectors for size exclusion chromatography is all about delivering choice.

Could you provide a brief description of Size Exclusion Chromatography and its major areas of use?

As an analytical technique Size Exclusion Chromatography (SEC), also known as Gel Permeation Chromatography (GPC), is widely used in characterizing natural and synthetic polymers, biopolymers, proteins and nanoparticles. It separates dissolved molecules on the basis of their size by passing them through special chromatography columns that contain microporous packing materials. As the separated sample elutes from the column, it is characterized using a single concentration detector (Conventional Calibration) or a series of detectors (Multiple Detection).

Most macromolecular samples typically consist of molecules of varying sizes; for example a polysaccharide or a protein with oligomers. Separating by size enables the generation of distributive information for these molecules, most especially molecular weight distributions.

The number of detectors and detection techniques applied to the eluting, size fractionated sample dictates what and how much information is gathered from each experiment. For example, when SEC separation is followed by analysis using light scattering, viscometer and concentration detectors, it provides distributions of absolute molecular weight, molecular size, and intrinsic viscosity, as well as information on macromolecular structure, conformation aggregation and branching.

This information is extremely useful in the development and use of polymers, proteins and a wide variety of other macromolecules and is an important factor driving the trend away from single detector measurement to the growing use of multi-detector set ups.

Molecular structure of an Immunoglobulin. Image Credit: Malvern Panalytical.

Malvern Panalytical talks a great deal about multi-detector systems and the right detectors are clearly crucial to the success of SEC applications. Can you tell us something about the different types of detector and why people use light scattering detectors in particular?

Once the sample is separated, the success and productivity of subsequent analyses depend on choosing a detector array appropriate for the application. The traditional configuration is a single refractive index (RI) detector which measures concentration, but this approach requires calibration of the column with a series of standards similar in size and structure to the unknown sample. For new polymers or macromolecules it’s very difficult to assess the best calibration standard to use and there may be nothing suitable so single detector analysis can be limiting. That said, it offers a good solution for certain well-established polymers. Analysts working in development areas, however, increasingly want multi-detector arrays to deliver absolute data without column calibration and to glean as much detail as possible from each SEC experiment.

To get the best multi-detector set up for a given application it’s important to know a little about the different types of detector available and how they can be combined for maximum productivity.

A viscometer detector measures the intrinsic viscosity of the molecules, which can be correlated with the structure. Used in tandem with a refractive index (RI) or other concentration detector, a viscometer enables universal calibration, removing the need to calibrate the system with a standard similar to the test material. So, simply adding in a viscometer takes away relative calibration and enables more accurate measurement of any sample. On the other hand, if molecular weight is being measured using a light scattering detector, viscosity measurements can be used in a different way - to help quantify structural information such as polymer branching.

A schematic of a GPC/SEC system. The degasser, columns, injection loop and pump are standard, with optional autosampler. Here, the RI detector is positioned after the light scattering detector. Image Credit: Malvern Panalytical.

Static light scattering detectors directly measure absolute molecular weight and come in different types: LALS, RALS, and MALS. All determine molecular weight by measuring the intensity of scattered light, but in each case the method of obtaining molecular weight from the raw detector signals is different. In terms of the hardware, low angle light scattering (LALS) is detection at 7^o to the incident beam, right angle light scattering (RALS) is detection at 90^o to the incident beam and multi-angle light scattering (MALS) is detection at many angles.

In addition, for larger molecules (greater than about 10 – 15 nm) the intensity of the scattered light varies as a function of angle to the incident beam and the molecular size can be extracted from the data. The different technologies have evolved in response to needing the best solution for every type of sample.

The mathematics of molecular weight determination depends on knowing scattering intensity at 0^o to the incident beam, where it can’t be measured. For small molecules this isn’t a problem as detection at any angle produces the same result. Measurement at 90^o gives the best signal to noise ratio and is therefore usually preferable.

For larger molecules there are two alternative solutions. LALS detectors measure at an angle so close to the incident beam that extrapolation to 0° is unnecessary. MALS detectors measure at multiple angles and use the resulting data to extrapolate a value for intensity at 0^o. Overlaying this technical discussion are industry trends and preferences so, for example, SEC-MALS has become very common in protein characterization where, on the basis of molecular size RALS could be argued as the better choice.

Dynamic light scattering (DLS) is a technique used in nanoscale particle sizing, protein analysis being a prime example, which accurately measures hydrodynamic molecular radius. DLS detectors also have static light scattering capability and may therefore supply molecular weight measurement.

Last but not least, there are detectors based on UV (ultra violet) technology. A UV detector may be a more precise alternative to RI concentration measurement for samples containing a chromaphore, but in combination with an RI can determine the proportion of a specific component, each monomer in a copolymer for example, within each eluting size fraction.

Figure A shows an isotropic scatterer is small relative to the wavelength of the light and scatters light evenly in all directions. Figure B shows an anisotropic scatterer has significant size compared with the wavelength of the incident light and scatters light in different directions with different intensities. Image Credit: Malvern Panalytical.

Could you briefly summarise the new detector that Malvern Panalytical has introduced and why it is needed?

Because light scattering detectors can directly measure molecular weight without needing a closely similar standard their use is increasing, particularly in R&D where there may be no relevant calibration standards and where there is a need for information-rich analysis. By adding a MALS detector to its existing range, which already includes RALS and LALS detectors, Malvern Panalytical is ensuring that every application can be addressed with the most appropriate technology choice.

The Viscotek SEC-MALS 20 offers versatility as it can be added to any existing SEC system, or purchased as part of a complete system package, but also superior accuracy of data due to its high number of measurement angles and optimised performance at lower angles.

What makes the Viscotek SEC-MALS 20 unique, what are its particular applications and can it be used on any SEC set up?

The Viscotek SEC-MALS 20 detector runs with established OmniSEC software and is compatible with any commercially available SEC instrument. As mentioned, MALS detectors are widely used within biopharmaceutical research. In some instances this is because they provide the most accurate data for measuring the radius of gyration, and in others because they have become an accepted technique.

The new Malvern Panalytical product has more detectors than any other commercially available MALS system for SEC and the incorporation of a vertical flow cell with radial optics, rather than the more conventional lateral flow cell, means increased sensitivity and accuracy for the lower angles. It also allows the use of a variety of different mobile phases without the need to alter the optical properties of the detector. This increased level of detection, particularly at lower angles, gives the best possible data fit for the extrapolation of molecular weight and size. Consequently there are significant gains in accuracy, even at high molecular weights.

The Viscotek SEC-MALS 20 from Malvern Panalytical. Image Credit: Malvern Panalytical.

What will be the long term benefits for users?

The primary benefit for the market is that users now have a wider choice of supplier and, more importantly, because we now offer a full range of light scattering detector types, we can offer unbiased guidance as to the most appropriate choice of technology for a given application. Our portfolio allows people to buy individual detectors or to specify a fully integrated system.

What other detectors does it sit alongside in the Malvern Panalytical range?

Full details of all our detectors are available at http://www.malvern.com and we offer everything from viscometer, RI, light scattering and UV detectors to dual, triple and tetra detector arrays. The most widely known and used is our 305TDA instrument which is available as a Triple-Detector array (RI, viscometer and light scattering) or a Tetra-Detector array (RI, UV, viscometer and light scattering). The TDA is equally at home in both protein and polymer applications and benefits from a close integration of all the detector modules.

How do you see the field of Size Exclusion Chromatography expanding in the next decade and will you be adding to the range in the future?

The SEC/GPC field continues to grow faster than many other chromatography areas, particularly as the benefits of multi-detection become apparent to more users. Malvern Panalytical will continue to research in this area and add to our range of products in the future. In parallel, we will also address the challenge of making multi-detection in general, and light scattering specifically, more accessible to a greater number users. We can use our experience in the DLS (Zetasizer) and laser diffraction (Mastersizer) fields to simplify the use of the technique without ‘dumbing’ it down.

Not all users have the time or background to be a light scattering expert but most require and will benefit from what light scattering can bring to their research or analytical problems. This means that our focus will not just be on adding technology, but on adding technology which is readily accessible and useful. We will also continue our tradition of education via our free to use website resources and strong technical specialist and applications teams.

About Stephen Ball

Stephen Ball is Product Marketing Manager, Nanoparticle and Molecular Characterization, at Malvern Panalytical.

He holds a degree in Computer Aided Chemistry from the University of Surrey, UK, which included a year in industry working as a research chemist for the Dow Chemical Company in Horgen, Switzerland.

Before joining Malvern Panalytical, he worked for Polymer Laboratories as an applications chemist, then took on a marketing position as a product manager for light scattering instrumentation at Agilent Technologies.