A 30 Minute Introduction to Size Exclusion Chromatography/Gel Permeation Chromatography

In natural and synthetic materials, bulk physical properties like toughness and strength mainly rely on their molecular properties. The same concept applies to the efficacy and activity of biopharmaceuticals and proteins. In most industries, there is a need to regulate the properties of molecules such as molecular structure and weight to exploit and manage the material’s bulk properties in a better way. It is important to have a reliable and consistent method for making these types of measurements.

A subset of high performance liquid chromatography (HPLC), size exclusion chromatography (SEC) is a separation method, which is also known as gel filtration (GF) or gel permeation chromatography (GPC). In this method, polymer molecules are isolated depending on their hydrodynamic volume. To explain this clearly, an appropriate sample is taken to which sample molecules are mixed and are able to travel via a gel packing matrix fused inside a column, and then to finally disperse in and out of pores within the gel packing matrix. Molecules of smaller size diffuse deeper and more frequently within the packing matrix pores which impair their passage via the column, while molecules of larger size enter only a fewer number of the pores. In this context, mixed sample molecules such as a mixture of protein molecules or a polydisperse polymer are arranged as per their size. Once the separation process is over, the sample is characterized using one or more detectors. In this method, a concentration detector is always used but intrinsic viscosity and sophisticated detectors like light scattering may or may not be used. The aim behind this characterization is to determine the molecular weight as well as the molecular weight distribution, and may even cover the measurement of structure, size and other properties. The values determined can be used to interpret the bulk properties of the sample.

Introduction

The toughness and strength of natural and synthetic polymers depend on their molecular properties. In polymer chemistry, for instance, different bulk properties can be influenced by different molecular parameters. In the case of proteins, molecular weight and the oligomeric state directly correlates to their activity in an assay or as a biopharmaceutical. Sample activity will depend on the size and amount of aggregates present in a sample. In the context of biopharmaceuticals, the loss of sample activity can trigger an immune reaction which would eventually impact the drug’s safety and efficacy aspects.

The following areas are covered in this whitepaper:

  • Separation Mechanism
    • Prerequisites
    • The Separation Process
    • Columns
  • Column Performance
    • Theoretical Plate Number
    • Slope of the Calibration Curve
  • Apparatus
    • Concentration Detectors
    • Differential Refractive Index Detector (RI)
    • UV/Vis absorption Detector (UV)
  • Data processing – What information to expect?
    • Molecular Mass
    • Conventional Calibration
    • Universal Calibration
    • Light Scattering
  • Sample Composition
  • Structural Information

Summary

SEC/GPC is a well- established technique that can be used for determining the molecular weight as well as the molecular weight distribution of materials. When protein or natural/synthetic polymer molecules are isolated, they can be fully characterized to create critical data about the molecules present in the samples. Subsequently, this information can be correlated back to its bulk physical properties. This method can be further improved by using sophisticated detectors, which produce highly accurate data like absolute molecular weight or offer supplementary data on composition, structure, and branching.

This information has been sourced, reviewed and adapted from materials provided by Malvern Panalytical.

For more information on this source, please visit Malvern Panalytical.

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