Performing a Separation with Sephacryl®

Extracted from Size Exclusion Chromatography Principles and Methods, (PDF)
GE Healthcare, 2014

Buffer: 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.2 or select the buffer in which the sample should be stored or solubilized for the next step.

Use 150 mM sodium chloride, or a buffer with equivalent ionic strength, to avoid pH dependent nonionic interactions with the matrix. At very low ionic strength, the presence of a small number of negatively charged groups can cause retardation of basic proteins and exclusion of acidic proteins.

The sample should be fully dissolved. Centrifuge or filter to remove particulate material (see Appendix 3). Always use degassed buffers and maintain a constant temperature during the run to avoid introducing air into the column (see Table 1.3)

Set an appropriate pressure limit on the chromatography system to avoid damage to the column packing. Use lower flow rates for high viscosity solutions and low temperature

First-time use or after long-term storage

  1. Equilibrate the column with at least 0.5 column volumes of distilled water at 15 cm/h (0.5 ml/min for 16/60 column or 1.3 ml/min for 26/60).
  2. Equilibrate with 2 column volumes of buffer at 30 cm/h (1.0 ml/min for 16/60 column or 2.6 ml/min for 26/60).
  3. Reduce flow to 15 cm/h and, for optimal resolution, apply a sample volume equivalent to 1% of the column volume (1.2 ml for 16/60 column or 3.2 ml for 26/60). Sample volumes between 0.5% and 4% can be applied.
  4. Elute with 1 column volume of buffer.
  5. Before applying a new sample-re-equilibrate column with 1 column volume of buffer at 30 cm/h until the baseline monitored at A280 is stable.

Column performance should be checked at regular intervals by determining the theoretical plate number per meter and peak symmetry. Prepacked columns are supplied with recommended values. See Appendix 1 on how to check column efficiency.

See Chapter 1 for advice on optimizing the separation.

Exposure to temperatures outside the range 4°C to 40°C will negatively affect the efficiency of a packed bed and the column will need to be repacked.


  1. Wash with 0.5 column volumes of 0.2 M sodium hydroxide at a flow of 15 cm/h (0.5 ml/min for column 16/60 or 1.3 ml/min for 26/60) to remove most nonspecifically adsorbed proteins.
  2. Re-equilibrate immediately with 2 column volumes of buffer or until the baseline monitored at A280 and the pH of the eluent are stable.

Further equilibration might be necessary if the buffer contains detergent.

Routine cleaning after every 10 to 20 separations is recommended, but the frequency of cleaning will also depend on the nature of the samples being applied.

If required, Sephacryl HR may be autoclaved repeatedly at 121°C, pH 7, for 30 min without significantly affecting its chromatographic properties. The medium must be removed from the column as autoclaving can damage column components. Note that HiPrep columns cannot be repacked.

To remove severe contamination

Reverse the flow and wash at a flow rate of 10 cm/h (0.3 ml/min for column 16/60 or 0.8 ml/min for 26/60) at room temperature using the following solutions:

  1. Wash with 0.25 column volumes of 0.5 M sodium hydroxide (to remove hydrophobic proteins or lipoproteins) followed by 4 column volumes of distilled water.
  2. Wash with 0.5 column volumes of 30% isopropanol (to remove lipids and very hydrophobic proteins), followed by 2 column volumes of distilled water.

For extreme cases of contamination, check the instructions supplied with the product.

Reversing flow through a column packed with Sephacryl® media should only be considered under cases of severe contamination. Reversing the flow can cause channeling through the packed bed leading to poor resolution, reduced efficiency, and the need to repack the column. Professionally packed columns are less likely to be affected, but extreme care must be taken.


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