GFC, using primarily aqueous solvents, is used primarily for the analysis of biological polymers, such as proteins and peptides, polysaccharides and polynucleotides.

The Support material can be silica or a polymer. Silica has the advantage that it has a larger pore volume and narrower pore size distribution. hence it generally gives sharper peaks and can give better resolution in some cases. However, large pore size silica particles are brittle and quite fragile, and if the pressure rises too high the particles collapse. The other problem as always is the pH limitation. Because silica dissolves at an unacceptable rate ove pH 7-8, it is necessary not to exceed pH 7.5 in eluents used with silica columns. In general silica has been found to give the best results for small proteins, from 10,000-1,000,000MWt, but for other samples, we recommend using a polymeric support.

pH control in GFC. The need to control the pH is really only to keep samples in solution, since the extent of ionisation does not affect retention time. However do see the note below about the addition of salt to GFC eluents. If Cl ions are present in the eluent, the eluent pH should be maintained in excess of 6.0, and if silica columns are used, the eluent should not exceed pH7.5.

Separation occurs on the basis of decreasing molecular size, which to a first approximation equates to decreasing molecular weight. No chemical interaction must occur between the sample and the packing material for a pure GFC separation to occur. However there are columns available which do offer a measure of interaction with the column in addition to the GPC separation.

Mixed mode columns. Sometimes it is hard to achieve resolution by GFC alone. In this situation, Shodex offer a range of Mixed mode columns, which allow some elements of traditional HPLC interactions to cause delayed retention of certain species on the column. In this case, the ability to derive molecular weight information from the retention time is sacrificed in order to achieve resolution.

A column is defined by its Exclusion Limit. Molecules bigger than this cannot enter the pores and pass unretained through the column. However columns with high exclusion limits offer very limited resolution of species with low molecular weight, so it is normal to require more than one column.

The total volume of the pores is known as the "Inner Volume" or "Pore Volume" of the system, Vi. The largest molecular weight components (at or above the Exclusion Limit) all elute in the Void volume of the system (V0). All components smaller than the exclusion limit of the column will elute between V0 and V0+Vi. So it follows that the bigger the pore volume of the system, the better resolution can be achieved, and this is why GFC columns are usually wider and longer than normal HPLC Columns.

Use of NaCl or NaNO3 in the eluent. GFC samples which are ionic will adopt positive or negative charges throughout the molecule. This causes parts of the molecule containing the charges to repel each other and the molecule expands significantly. Hence the apparent size of the molecule in GFC (and hence its retention time) is pH dependent. A form of ion-suppression can be used to minimise this effect, and this is achieved using approx 0.3M NaCl in the eluent. Should it be necessary to work at lower than pH6, then NaCl should not be used, and a salt such as NaNO3 used instead.

Use of organic solvents in the eluent. To avoid any hydrophobic interaction with the column, it is sometimes desirable to add polar organic solvents to the eluent, such as methanol or acetonitrile. This is generally no problem, although for some columns it is recommended not to go as far as 100% organic solvent.

Temperature control. Changes in temperature affect the viscosity of the eluent, and the kinetic energy of the samples. As a consequence, retention times are temperature dependent. For this reason it is recommended to select a temperature a few degrees above the highest ambient likely to occur in the lab, and control to this temperature accurately. However, sometimes back pressure becomes an issue, either because of the viscosity of a solvent used in the eluent (eg DMSO), or because higher salt concentrations are being used. In this situation, the use of elevated temperature (over 50oC) are recommended. However take care to note the maximum temperature which is compatible with the column chosen.

To separate a wide range of molecular sizes, there are two standard approaches:

Shodex have a range of columns to suit both of these approaches. Mixed bed columns are designated with an M.

Column format in GFC.

Max. Temp. (C)