advantages and disadvantages of gel filtration chromatography 5 | Page 2

3. For size estimation
Free information;
Gives an estimate of molecular size in any practically any solution;
Precision is not so good.
Troubleshooting:
1. Poor resolution
Reason: Poor packed bed
Recommended solution: Check the efficiency of the column and re-pack if
necessary.
Always check the efficiency of a new column, especially one that has been
packed in the lab, before using it for critical applications.
Reason: Contamination of the bed with lipids, denatured protein etc.
Recommended solution: Thoroughly clean the column following the
recommended protocols given in the instructions provided by the column
manufacturer.
2. Poor yields
Reason: Contamination of the bed with lipids, denatured protein etc.
Recommended solution: Thoroughly clean the column following the
recommended protocols.
Tip: Running eluent slowly through the column can prevent microbial growth.
Cleaning the column thoroughly and keep it under cold conditions in case that the
column will not be used for a long time.
3. Elution earlier than expected
Reason: Ionic aggregates and complexes due to carbohydrate chains
(glycoproteins).
Recommended solution: Include 150mM NaCl or up to 10% betaine in buffer.
Reason: Complexes associated via SH-groups.
Recommended solution: Add reducing agent (e.g. DTT) to buffer.
Reason: Hydrophobic aggregates.
Recommended solution: Add 10-20% ethanol or other organic solvents to buffer.
Reason: Gel filtration of micelles/protein aggregates.
Recommended solution: Decrease buffer detergent concentration until below
CMC or use a different detergent (stay below its CMC).
Remember to check that your sample and your column are stable in the buffer
before using special additives to aid solubilisation.
4. Elution later than expected
Reason: Digestion by proteases.
Recommended Solution: Add protease inhibitors to buffer.
Reason: Hydrophobic adsorption.
Recommended Solution: Add 10-20% ethanol or other organic solvents to buffer
or decrease salt concentration in high ionic strength buffer.
Reason: Ionic adsorption due to negatively charged groups on the support
(Carboxyl-, Sulphate-) and positively charged roups on sample molecules.
Recommended Solution: Include 150mM NaCl (or other salt) in buffer.