Performing a Separation with Superose

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

Buffer: 10 to 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.0 to 7.4, 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.

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 (Table 1.3).

Table 1.3. Example of flow rate limits at different viscosity and temperature, Superose 6 Increase 10/300 GL

Temperature Flow rate (ml/min)  
20ºC to 25ºC Maximum flow rate, water 1.5
  Maximum flow rate, 20% ethanol 0.75
4ºC to 8ºC Maximum flow rate, water 0.75
  Maximum flow rate, 20% ethanol 0.35

First-time use or after long-term storage

  1. Equilibrate the column with 2 column volumes of water at room temperature to remove the storage solution. Use a low flow rate (Table 3.2) to avoid any gap formation.
  2. For first-time use: Determine the column specific pressure limit according to section Setting column pressure limits (Chapter 1).
  3. Perform a column efficiency test (see Appendix 1).
  4. Equilibrate with 2 column volumes of buffer at recommended flow rate during run (Table 3.1).
  5. Apply a sample volume equivalent to between 0.5% and 4% of the column volume.
    For most applications the sample volume should not exceed 2% to achieve maximum resolution (up to 50 μl for 3.2/300 GL and 5/150 GL, up to 500 μl for 10/300 GL). Note that smaller sample volumes generally lead to improved resolution.
  6. Elute with 1 column volume of buffer.

Before applying a new sample, re-equilibrate the column with buffer until the baseline monitored at A280 is stable.

Table 3.2. Recommended flow rates at different stages using prepacked columns containing Superose 6 Increase, Superose 6, and Superose 12 media

Flow rates (ml/min) at room temperature

  3.2/300 GL 5/150 GL 10/300 GL
First-time use or after long-term storage 0.04 0.25 0.5*
Cleaning-in-place, CIP 0.02 0.1 0.5

*Start with 0.2 ml/min for 0.5 column volumes.

Column performance should be checked at regular intervals by determining the theoretical plate number m-1 and peak a symmetry factor, As. See Appendix 1 for how to check column efficiency.

Ensure that the pressure limit of the column is not exceeded. This is particularly important when working at low temperatures, such as in a cold room, or when the column is used with 20% ethanol or other viscous solutions.

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 1 column volume of 0.5 M sodium hydroxide, alternatively 0.5 M acetic acid.Use a low flow rate during the entire CIP procedure. Use 0.2 M sodium hydroxide for Superose prep grade media.
  1. Immediately wash with 1 column volume of distilled water followed by at least 2 column volumes of buffer until the baseline monitored at A280 and the pH of the eluent are stable.

For extreme cases of contamination, check the instructions supplied with the product. In special cases, it might be necessary to change the bottom filter or to remove and discard the top 2 to 3 mm of the gel. These operations must be done with extreme care to avoid serious loss of resolution. Note that Precision Columns (3.2/300) should not be opened. 

Superose prep grade 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 Precision Columns (3.2/300) cannot be repacked. 

Degas the ethanol/water mixture thoroughly and use a low flow rate. Connect the transport tool to the capillary tubing at the column outlet to prevent air from entering the column. Fill the tool up to approximately 50% of the total volume.

Avoid changes in temperature, which can cause air bubbles in the packing.


Related Links