HPLC - Size Exclusion

1. What’s the difference between SW/SWXL and PW/PWXL SEC columns?

The “S” in SW and SWXL stands for silica so these columns are created using porous silica. The “P” in PW and PWXL 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 G2000SWXL 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/SWXL SEC column?

The TSKgel SW/SWXL 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 200 mM, e.g. 100 mM phosphate buffer and 150 mM NaCl, pH 6.8.

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

TSKgel SW/SWXL columns can tolerate up to 100% polar organics such as acetonitrile and methanol. TSKgel PW/PWXL 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/SWXL 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/SWXL column we recommend 4 successive injections of 100 µg BSA to condition the resin. The PW/PWXL sizing columns are far less hydrophilic and normally do not require conditioning.

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

You can load from 1-3 mg of total protein on a TSKgel SWXL-type column. Maximum injection volume is 750 µL.

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

MW and molecular configuration, e.g. globular: Do they match 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/SWXL 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 TSKgel PW/PWXL columns shed microparticles?

Microparticles do not interfere with most detection methods used in HPLC. However, these particles can increase the noise level in laser light scattering detection systems. To minimize this effect using the PW/PWXL columns run the column overnight in 10-20% methanol at one-half the normal flow rate to clean any microparticles off the resin.

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