Performing a Separation with Superdex

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 ionic interactions with the matrix. At very low ionic strength, the presence of a small number of negatively charged groups on the medium can cause retardation of basic 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.

First-time use or after long-term storage

  1. Equilibrate the column with 2 column volumes of water to remove the storage solution. Use a low flow rate (Table 2.3) 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 containing 150 mM sodium chloride at recommended flow rate during run (Table 2.2).
  5. Apply a sample volume equivalent to 0.5% to 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, up to 2.4 ml for HiLoad 16/600, and 6.4 ml for HiLoad 26/600). Note that small sample volume improves the resolution.
  6. Elute with 1 column volume of buffer.
  7. Before applying a new sample, re-equilibrate column with buffer 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.

See Appendix 1 for how to check column efficiency.

See Chapter 1 for advice on optimizing the separation.

Make sure to not exceed the pressure limits of the column and consider flow limitations (Table 1.3). 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.

Table 2.3. Recommended flow rates at different stages using prepacked columns containing Superdex Increase, Superdex, or HiLoad columns containing Superdex prep grade media

Flow rates at room temperature

  3.2/300 GL 5/150 GL 10/300 GL HiLoad 16/600 HiLoad 26/600
First-time use or after long-term storage 0.05 ml/min 0.3 ml/min 0.5 ml/min 1 ml/min*
(30 cm/h)
2.6 ml/min*
(30 cm/h)
First-time use or after long-term storage (Superdex 200 Increase) 0.075 ml/min 0.3 ml/min 0.75 ml/min N/A N/A
Cleaning-in-place, CIP 0.02 ml/min 0.3 ml/min† 0.5 ml/min 0.8 ml/min
(25 cm/h)
2.2 ml/min
(25 cm/h)

*HiLoad: To save time, higher flow rate can be used for the equilibration with buffer: 1.6 ml/min and 4.3 ml/min for HiLoad 16/600 and HiLoad 26/600, respectively. Reduce the flow rate to the recommended flow rate during run before sample application.

† 0.13 ml/min for Superdex 200 Increase 5/150 GL.


  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 (see Table 2.3).
  2. 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.

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.

Removing severe contamination

  1. Reverse the flow.
  2. Wash with 4 column volumes of 1M NaOH (to remove hydrophobic proteins or lipoproteins) followed by 4 column volumes of distilled water.
  3. Wash with 0.5 column volume of 30% isopropanol to remove lipids and very hydrophobic proteins, followed by 2 column volumes of distilled water.
  4. Equilibrate the column with at least 5 column volumes of buffer, or until the baseline monitored at A280 and the pH of the eluent are stable, before beginning a new separation.

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