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Interaction-free GPC/SEC (Theoretical background) 

Gel Filtration, Gel Permeation, Size Exclusion Chromatography


The key to successful GPC/SEC separation is the correct choice of columns and solvent. Interaction-free GPC/SEC requires a balanced system of sample, mobile phase (solvent), and stationary phase (GPC/SEC column material). 

The sample is the determinant factor: the polarity of the sample defines the polarity of the solvent and that of the stationary phase. Since the polarity of the sample can be from very polar (e.g. Poly (acrylic acid) and other polyelectrolytes) to very unpolar (e.g. Poly(styrene)) it is inescapable to have polar (e.g. SUPREMA) and unpolar (e.g. SDV) stationary phases.  

For some applications interaction-free chromatography can be achived by adding a salt or co-solvent to the mobile phase. E.g. in aqueous GPC/SEC often phosphate buffer solutions are used to overcome interactions between the column material surface and the sample. Therefore a careful method development is essential for GPC/SEC.

  
Why is it important that GPC/SEC is interaction-free? 

GPC (Gel permeation chromatograpy), or SEC (Size exclusion chromatography), is a chromatographic method that relies on the separation according to the size of the polymer in solution. The size determines the elution volume; larger molecules elute first, since they can not penetrate the pores in the column (stationary phase) and therefore spend less time on the separation column.

GPC/SEC allows to determine molar mass averages (e.g. Mn, Mw, PDI) and the molar mass distribution if

  •  a calibration curve, that describes the relation between the elution volume and the molar mass, is available (e.g. established using a calibration kit or PSS ReadyCals)
  •   molar mass sensitive detectors (e.g. an on-line MALLS detetor or an on-line viscometer), that measures the molar mass of the eluting fractions on-line is available

For reliable and precise results both methods require a perfect separation according to size only. If any kind or interaction with the column material appears (leading to a shift in the elution volume), both methods fail. In some cases bulk values, like the Mw for light scattering or the intrinsic viscosity for the viscometer, can still be measured. But distribution information and PDI will be wrong. 

For reliable and precise results both methods require a perfect separation according to size only. If any kind or interaction with the column material appears (leading to a shift in the elution volume), both methods fail. In some cases bulk values, like the Mw for light scattering or the intrinsic viscosity for the viscometer, can still be measured. But distribution information and PDI will be wrong. 

So the first goal in GPC/SEC, no matter what detection method is used, is to achieve interaction-free chromatography without any kind of interactions between column material (stationary phase) and sample.

Click picture for close up view   

GPC vs.HPLC interaction
  Chromatogram of the same protein measured under 2 different conditions:
green: Protein on PROTEEMA in water with 0.3M NaCl. Perfect size separation, expected result.
red: Protein on PROTEEMA in water with 0.03M NaCl. Distorted chromatogram, interactions lead to adsorption, wrong peak form, and wrong elution volumes
The results on a column material with the wrong polarity could even be worse, leading e.g. to irreparable column damage.

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