What is gel filtration and when is it used?
Gel filtration is a liquid chromatography technique that separates molecules according to their sizes. It is sometimes called size exclusion or gel permeation chromatography.
Gel filtration can be used for group separations, fractionation or size analysis.
Group separations such as desalting, buffer exchange or removing reagents are quickly accomplished (much faster than dialysis!).
Applications include adjusting pH, buffer type or salt concentration during sample preparation, either before an assay or for a further chromatography step. It can not be used when the protein will precipitate at the new conditions. Group separations are also useful for removing small molecules such as EDTA, fluorescent labels or radioactive markers.
Fractionation by gel filtration is excellent during the polishing stage of purification of proteins and peptides. It may be used to remove multimers and other aggregates. It is not suitable if the sample is very crude or the volume is large.
Gel filtration is a non-destructive means of size estimation & size homogeneity analysis. Although not very precise, it can give an estimate of molecular size in practically any solution.
What is the ??void volume???
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The void volume or "Vo" is the elution volume of molecules which are too large to enter even the largest pores of the gel. For non-rigid gels like Superose, Sephacryl and other APBiotech Gel Filtration gels, we estimate the void volume of a column to be approximately 30% of the total bed volume. It can be quite a bit higher in rigid gels like silica (up to 40% of the bed volume).
What is an exclusion limit?
The exclusion limit is the molecular weight of the smallest molecule which cannot enter the pores of the matrix. All molecules bigger than the exclusion limit elute in the void volume. The exclusion limit means that the sample is no longer fractionated and will be excluded from entering the pores. This limit will be just above the upper limit of the fractionating range.The exclusion limit is an extrapolated value defined by convention.
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The suggested flow rates and volumes in the following procedure are appropriate in most cases but may need to be adjusted, depending on the condition of the column.
Always keep the backpressure below 2.4 MPa for the Superdex 75.
Always keep the backpressure below 1.5 MPa for the Superdex 200.
The following observations may indicate a dirty column:
- Increased back-pressure.
- Color change in the gel bed.
- Loss of resolution.
- Decreased sample recoveries.
Consider possible contaminants when choosing cleaning conditions.
General cleaning method:
- Inject 2 ml 1 M NaOH through a 2 ml loop on to the column (flow rate 0.02 ml/min). Repeat the injection with another 2 ml if necessary.
- Rinse the column with 5 - 10 ml of water at 0.04 ml/min. Ensure the base-line is stable before proceeding to the next step.
- Equilibrate with 5 - 10 ml buffer at a flow rate of 0.04 ml/min.
For very hydrophobic proteins and lipids:
- 5 ml of 70% ethanol or 30% acetonitrile, flow rate 0.02 ml/min.
- Rinse the column with 5 - 10 ml of water at 0.04 ml/min. Ensure the base-line is stable before proceeding to the next step.
- Equilibrate with 5 - 10 ml buffer at 0.04 ml/min.
If the column is still not restored, inject solution of 1 mg/ml pepsin in 0.1 M acetic acid and 0.5 M NaCl and then leave it overnight at room temperature or 1 hour at 37??C.
Note: After enzymatic digestion, careful rinsing is required.
Other enzymes can be used, e.g. DNAse. After enzymatic treatment, repeat the cleaning process.
