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, but water soluble polymers are also applicable too.

Basic Principle: Used to separate molecules based on their molecular size in solution (as the primary mode of separation). 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 and exit quickly. It is extremely important to measure this so you know what the actual column volume is AND what the exclusion limit is. 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. Please keep in mind that the actual confirmation (hydrodynamic volume) of the compound in the mobile phase may be different than what any of these standards are so the best column to choose may be one with a different pore size than suggested (this is why it is so important to test your compound on actual columns). Determine the actual exclusion volume running actual samples. They should elute at the Tzero point (column void volume).

Support Types: Available supports are most commonly based on either silica gel or polymeric materials (e.g. DVB). Their properties and chemical compatibility may vary so be sure to document which back-pressure ranges, pH, flow rates, temperature and/or solvents are safe to use with them.

Technique: Improved resolution often results from chaining columns together, in-series, with the same pore size. Additionally, a broader range (size) of molecules can often be separated using multiple columns with differing pore volumes together, in-series (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. Method Development Notes: 

(1) As the primary mode of chromatography is based on "size", achieving acceptable K prime values for retention are not applicable in this mode. K prime is NOT applicable to ion exchange or SEC modes. You must achieve retention past the initial pore exclusion point to demonstrate that the compound(s) are interacting with the pores of the phase. Measure the actual column volume to determine Tzero (this is very important). Inject an unretained compound to confirm and record the pore exclusion limit with a suitable high Mw standard.

(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. 

(3) Method development using buffered mobile phase solutions may employ several key variables to achieve good results. After selecting the correct column(s) use a linear flow rate and systematically adjust: (a) the molarity of the buffer salt used (e.g. 10 mM, 50 mM, 100 mM, 0.5M ...); (b) the pH of the solution (acidic, neutral, basic); (c) the temperature of the column to achieve satisfactory resolution. Note: Selecting the best column is the single most important aspect of success. If you select a column that is poorly suited to the separation, a great deal of time and money will be spent on the method development with poor results. Start with the most suitable column(s).