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February 19, 2018

Master analytical SEC running conditions

By Jon Lundqvist, Scientist at GE Healthcare Life Sciences

Need to find out how to calibrate your size exclusion chromatography (SEC) column? Or how different additives affect your results? Read these useful insights.


A: The first step prior analysis is to equilibrate the column with the buffer you are going to use. Biomolecules with similar conformation to the target molecules should be used as standards for determination of molecular weight. For example, for soluble globular protein you could use the HMW or LMW Standard protein that GE provides.

For oligo- and polysaccharides you need to use standards with similar conformation. For a more accurate molecular weight, online detectors such as multi-angle light scattering (MALS) or mass spectrometry (MS) could be used.

Download the Size exclusion chromatography handbook for in-depth reading.

A: Removal of particles in the sample, buffers, and cleaning solutions is extremely important for SEC. Clarifying a sample before applying it to a column will minimize the risk of blockage, reduce the need for stringent washing procedures, and extend the lifetime of the column.

Use high-quality water and chemicals. Solutions should be filtered through 0.45 µm or 0.22 µm filters before use with, for example, Whatman filters.

A: For analytical separations, we recommend a sample volume from 0.3% to 1% of the total column volume. The focus is to get the highest possible resolution with a small sample volume. Here are sample volume examples for three different column sizes:

10/300 GL column: 72 to 240 µL
5/150 GL column: 9 to 30 µL
3.2/300 column: 7 to 24 µL

To learn more about SEC, download the Size exclusion chromatography handbook.

A: We recommend you inject not more than 0.3% and 1% of the total column volume dependent on how close the molecules you want to separate are from each other. If proteins are close in size you need to load not more than 0.3% of the column volume.

Download the Size exclusion chromatography handbook to learn more.

A: A running buffer of 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.0 works well for many separations. Alternatively, select the buffer best suited for the target molecule. Degas buffers before any SEC separation as air bubbles can reduce performance. Buffers are automatically degassed if filtered under vacuum.

Buffers, resins, or prepacked columns must have the same temperature before use. To prevent air bubble formation that can result in poorer separation, avoid rapid changes in temperature. For example, avoid removing packed columns from a cold room and immediately applying buffer at room temperature.

A: See recommended and max. operating flow rates for the different Superdex Increase and Superose Increase 10/300 GL columns and under different conditions in Table 1. For the other column sizes, see the Instructions for each respective column.

Table 1. Recommended and max. operating flow rates for Superdex Increase and Superose Increase 10/300 GL columns

Temperature Recommended and max. operating flow rates Flow rate (mL/min)
Superose 6 Increase Superdex 200 Increase Superdex 75 Increase Superdex 30 Increase
20°C to 25°C Recommended flow rate, water 0.5 0.75 0.80 0.80
Maximum flow rate, water 1.50 1.80 1.60 1.20
Maximum flow rate, 20% ethanol 0.75 0.90 0.80 0.60
Maximum flow rate, 10% glycerol 0.75* 0.90 0.80 0.60
Maximum flow rate, 30% acetonitrile 1.5* 1.80* 1.60* 1.20
Maximum flow rate, 40% methanol 0.75* 0.90* 0.80* 0.60
4°C to 8°C Maximum flow rate, water 0.75 0.90 0.80 0.60
Maximum flow rate, 20% ethanol 0.35 0.45 0.40 0.30
Maximum flow rate, 10% glycerol 0.35* 0.45 0.40 0.30
Maximum flow rate, 30% acetonitrile 0.75* 0.90* 0.80* 0.60
Maximum flow rate, 40% methanol 0.35* 0.45* 0.40* 0.30

* Not verified experimentally

Learn more about Superdex 30 Increase
Learn more about Superdex 75 Increase
Learn more about Superdex 200 Increase
Learn more about Superose 6 Increase

A: Below is a list of chemical compatibility for Superdex 200 Increase resin. You will find a similar list in the Instructions for each of the other Superdex Increase and Superose resins.

Chemical stability: long-term use

Refers to use where the resin is stable over a long period of time without adverse side-effects on its chromatographic performance.

  • Commonly used aqueous buffers, pH 3 to 12
  • Urea, up to 8 M
  • Guanidine hydrochloride, up to 6 M
  • Ionic and nonionic detergents, for example, 1% SDS
  • Guanidine hydrochloride, up to 6 M

Chemical stability: short-term use

Refers to the use during regeneration, cleaning-in-place, and sanitization procedures.

  • Acetonitrile, up to 30%
  • Sodium hydroxide, up to 1 M
  • Ethanol, up to 70%
  • Methanol, up to 100%
  • Acetic acid, up to 1 M
  • Isopropanol, up to 30%
  • Hydrochloric acid, up to 0.1 M
  • Trifluoracetic acid, up to 10%
  • Formic acid, up to 70%

Avoid:

  • Oxidizing agents
  • Unfiltered samples and eluents

Download instructions for the different SEC columns:

A: Up to 10% dimethyl sulfoxide (DMSO) can be used during regeneration, cleaning-in place, and sanitization procedures.

A: Proteins tend to interact with the charged or active surface sites of the SEC resin. Two types of nonbinding interactions predominate, electrostatic interactions and hydrophobic interactions. The first involve charged surface residues of the resin and the proteins. The latter can occur between hydrophobic sites of the resin beads and the proteins.

Electrostatic interactions

Electrostatic interactions can result in shifts in protein elution time, adsorption, and peak tailing. Two types of electrostatic interactions can be obtained that might affect the retention of the proteins in SEC, ionic adsorption and ion exclusion.

When ionic adsorption is obtained, low sample recoveries and peak tailing might occur. With ion exclusion, the analyte with the same charge as the resin will effectively be excluded from the pores due to the ensuing repulsive forces and will be eluted earlier than predicted by its hydrodynamic radius.

Adjustments to the ionic strength and pH, and increasing the salt and/or buffer concentration of the mobile phase are the primary means of reducing electrostatic interactions between the analyte and the SEC column.

Hydrophobic interactions

For decreasing hydrophobic interactions, using a more chaotropic anion, such as perchlorate, can be advantageous. Another approach is adding an organic modifier, such as acetonitrile, detergent, or glycerol.

Another mobile-phase modifier that has been widely used to improve SEC protein and peptide separations is the basic amino acid arginine. Arginine both stabilizes protein structure and prevents interactions between the protein and the column.

A: IgG from different sources might retain differently on a SEC column. The reason is that IgG can have different characteristics in form of hydrophobic, aromatic, and ionic groups on the surface. Such interactions can lead to an analyte eluting later than expected and could give the appearance of a lower molecular weight.

To overcome undesirable secondary interactions, it might be necessary to perform method optimization to change the conditions of the SEC runs. Changing the salt concentration or adding organic modifiers such as glycerol or detergents in the elution buffer are two examples of appropriate method optimization.

A: Our general recommendation is to clean the column with 0.5 M NaOH after 10 to 20 analysis cycles. Always clean the column if you are going to change sample type to avoid unwanted contamination.

Download Maintenance and cleaning of size exclusion chromatography columns for further reading.