Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, March 16, 2013

Introduction to Size Exclusion Chromatography (SEC HPLC)

Size Exclusion Chromatography. Often known as “SEC”.

Other names used to describe SEC:

  • Gel Filtration Chromatography or “GFC” is a commonly used phrase when you are separating biological molecules in aqueous (or sometimes organic containing mobile phases). It is often described as a gentle form of chromatography leaving the protein or sample intact (*Proteins are one of the most common molecules separated using this technique, but if needed intact, must be kept away from denaturing agents). 
  • Gel Permeation Chromatography or "GPC" usually refers to the separation of polymers using an organic solvent.

Basic Principle: Used to separate molecules based on their molecular size in solution. The pore size and interstitial volume of a packed column must be determined to find out which molecules it excludes. Small molecules which are smaller than the pore size will enter the particles and spend more time navigating the channels within than larger molecules which will be excluded from entering the particles. Manufacturer’s often report these exclusion limits via calibration tables for linear standards such as dextran or polystyrene though some provide data using globular standards which provides more accurate data when running many proteins or peptides.

Support Types: Available supports are most commonly based on either silica gel or polymeric materials.

Technique: Improved resolution often results from chaining columns together with the same pore size. A broader range of molecules can often be separated using multiple columns with differing pore volumes together (very common in GPC applications). Single "Mixed Pore" columns are also available from many manufacturers which allow a wide range of molecular weights to be screened, though often at reduced resolution. It is important to make sure that there is no interaction between the stationary phase used and the solute employed to transport the sample. This will insure that the only mechanism being used is size exclusion.

Misc. Notes: (1) As the mode of chromatography is based on "size", achieving acceptable K prime values for retention are not applicable. (2) For silica based supports, strong salt buffers are often employed. You must insure proper miscibility of the sample and mobile phase at all times. Be sure and flush the system of all buffers at the end of each day. This is critical and not an optional step if you want to maintain the chromatography hardware. Salt crystals can be corrosive to the steel used in these system and may result in damage to the pump, injector and other components if not flushed out. Use a flushing solution that is similar to your mobile phase, but without the buffer. If you see any salt crystals forming on the instrument, then you have not been flushing the system down properly, or often enough. Salt should never be visible on the outside of the instrument.