1. What’s the difference between SW/SW_XL and PW/PW_XL SEC columns?

The “S” in SW and SW_XL stands for silica so these columns are created using porous silica. The “P” in PW and PW_XL refers to a polymeric support of porous methacrylate. The  subscripted XL is used to denote smaller particle, higher efficiency columns. However, the pore size is the same for a given type of column, e.g. G2000SW and G2000SW_XL have the same fractionation range.

2. How does SEC work?

Molecules in solution assume 1 of 3 possible configurations: linear, e.g. PEG; branched, e.g. dextran; and globular, e,g, protein. As such a given molecule will have a unique hydrodynamic radius in solution. It is the differences in molecular dimensions that enables separation of compounds in a mixture by means of size exclusion liquid chromatography. Whereas large molecules will have limited or no access to the pores in an SEC column, smaller species will fit in the pores and be retained. Thus, the molecules will elute from the column in reverse order of molecular weight. Unretained compounds will come out in the column’s void volume (the space between particles in the resin bed). A fully retained molecule will elute at the column’s included (or mobile phase) volume which is the sum of the void volume and pore volume.

3. What causes peak tailing on a SW/SW_XL SEC column?

The TSK-GEL SW/SW_XL columns contain porous silica that has been chemically bonded with a proprietary hyprophilic material. This coating stabilizes the silica in aqueous solvents. When charged, strongly polar or hydrophobic molecules interact with the silica support, it is possible to have secondary electrostatic, hydrophilic or hydrophobic interaction between the solutes and the silica matrix. Thus, the separation involves two mechanisms, size exclusion and electrostatic, hydrophilic or hydrophobic interaction. When this occurs the chromatogram may exhibit peak tailing, as the number of adsorption sites that cause the secondary interaction is limited. The best way to avoid secondary interaction is to use a buffer with an ionic strength in excess of 200mM, e.g. 100mM phosphate buffer and 150mM NaCl, pH 6.8.

4. Can I use organic solvents with SEC columns?  What about surfactants like Triton or SDS?

TSK-GEL SW/SW_XL columns can tolerate up to 100% polar organics such as acetonitrile and methanol. TSK-GEL PW/PW_XL columns can handle up to 20% polar organics in the mobile phase, and as high as 50% in special cases.  Please contact the technical service group for these special cases.  Using a surfactant in the mobile phase, e.g. 0.1% SDS will require that the column be dedicated to the application as the surfactant tends to irreversibly bind to the column and thereby alter the stationary phase.

5. When I use a new SW/SW_XL column I get low recoveries of my protein.  How can I avoid this?

There are active sites on the resin that require conditioning with either your sample protein or another material such as bovine serum albumin (BSA).  For the SW/SW_XL column we recommend 4 successive injections of 100mg BSA to condition the resin. The PW/PW_XL sizing columns are far less hydrophilic and normally do not require conditioning.

6. How much sample can I load on a 7.8mm ID x 30cm size exclusion column?

You can load from 1-3 mg of total protein on a TSK-GEL SW_XL-type column.  Maximum injection volume is 750 mL.

7. What factors should I consider when selecting the correct SEC column for my application?

• MW and molecular configuration, e.g. globular: match this to the fractionation range of the column.
• Solvent solubility: what mobile phase is required and is the column compatible with this solvent, e.g. SW/SW_XL column operate in the pH range of 2.5-7.5.
• Hydrophilic vs. hydrophobic: this will dictate the ionic strength of the eluent used in the mobile phase. Some proteins have hydrophobic sites that require a small amount of organic solvent in the mobile phase such as 5% methanol. This inhibits secondary hydrophobic interaction.
• Other factors: temperature,  sample characteristics,  HPLC system requirements, fittings required.

8. Do the TSK_GEL PW/PW_XL columns shed microparticles?