Packing a Column and Performing a Separation with Sephadex® LH-20

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

Packing a Column

Sephadex® LH-20 should be packed in a solvent-resistant (SR) column selected from Table 6.3 according to the column volume required for the separation.

Table 6.3. Sephadex® LH-20 packing volumes and bed heights in SR columns

Column Volume (ml) Bed height (cm)
SR 25/454 73 to 220 15 to 45
SR 25/100 343 to 490 70 to 100

Simple steps to clarify a sample before applying it to a column will avoid the risk of blockage, reduce the need for stringent washing procedures and extend the life of the chromatography medium. Filter or centrifuge all solvents and samples before use.

  1. Refer to Table 6.4 to calculate the amount of dry medium required as the extent of swelling depends upon the solvent system. Swell Sephadex® LH-20 for at least 3 h at room temperature in the solvent to be used for the separation
  2. Prepare a slurry 75:25 settled medium:solvent and decant any fine particles of medium.
  3. Equilibrate all materials to room temperature.
  4. Resuspend and pour the slurry into the column in one continuous step (using a glass rod will help to eliminate air bubbles).
  5. Fill the column reservoir to the top with solvent. Seal, attach to a pump, and open the column outlet.
  6. Pack at 300 cm/h until the bed has reached a constant height. Stop the flow, empty, and remove the packing reservoir.
  7. Carefully fill the column with solvent and insert a wetted adapter into the column. Ensure no air bubbles are trapped under the net and adjust the adapter O-ring to give a sliding seal against the column wall.
  8. Connect all tubings, ensuring that there are no air bubbles in the flow path.
  9. Slowly slide down the adapter so that any air in the tubings is displaced by solvent and lock the adapter into position on the surface of the medium.
  10. Open the column outlet and continue packing until the packed bed is stable and a final adjustment of the top adapter can be made.

In solvents such as chloroform, Sephadex® LH-20 is less dense than the solvent and the medium will float. Pour the medium into the column and drain until the second adapter can be inserted. Lock the adapter in position at the surface of the medium and direct the flow of chloroform upwards. The bed will be packed against the top adapter and the lower adapter can be pushed slowly upwards towards the lower surface of the medium.

Close the column outlet when moving the adapter to avoid compressing the bed.

Performing a Separation

Start at a linear flow of 1 cm/h to check resolution. Low flow rate improves the resolution.

  1. Equilibrate the column with at least 2 column volumes of the solvent until a stable baseline is achieved.
  2. Apply a sample volume equivalent to 1% to 2% of the total column volume.
  3. Elute in 1 column volume. Re-equilibration is not needed between runs with the same solvent.

Wash the column with 2 to 3 column volumes of the solvent, followed by re-equilibration in a new solvent if changing the separation conditions.

Table 6.4. Approximate values for packed bed volumes of Sephadex® LH-20 swollen in different solvents

Solvent Approx. bed volume (ml/g dry Sephadex® LH-20)
Dimethyl sulfoxide 4.4 to 4.6
Pyridine 4.2 to 4.4
Water 4.0 to 4.4
Dimethylformamide 4.0 to 4.4
Methanol 3.9 to 4.3
Saline 3.8 to 4.2
Ethylene dichloride 3.8 to 4.1
Chloroform* 3.8 to 4.1
Propanol 3.7 to 4.0
Ethanol† 3.6 to 3.9
Isobutanol 3.6 to 3.9
Formamide 3.6 to 3.9
Methylene dichloride 3.6 to 3.9
Butanol 3.5 to 3.8
Isopropanol 3.3 to 3.6
Tetrahydrofuran 3.3 to 3.6
Dioxane 3.2 to 3.5
Acetone 2.4 to 2.6
Acetonitrile‡ 2.2 to 2.4
Carbon tetrachloride‡ 1.8 to 2.2
Benzene‡ 1.6 to 2.0
Ethyl acetate‡ 1.6 to 1.8
Toluene‡ 1.5 to 1.6

* Containing 1% ethanol.
† Containing 1% benzene.
‡ Solvents that give a bed volume of less than 2.5 mg/ml dry Sephadex® LH-20 are not generally useful.


Chemical stability
Sephadex® LH-20 is stable in most aqueous and organic solvent systems. The medium is not stable below pH 2.0 or in strong oxidizing agents.

Store dry at 4°C to 30°C. Store packed columns and used medium at 4°C to 8°C in the presence of a bacteriostatic agent.

Transferring Sephadex® LH-20 from aqueous solution to organic solvents
Transfer Sephadex® LH-20 from an aqueous solution to the organic solvent by moving through a graded series of solvent mixtures. This will ensure efficient replacement of the water by the required solvent.

To transfer from aqueous solution or organic solvent (100% A) to a new organic solvent (100% B), proceed as follows: transfer to 70% A:30% B then to 30% A:70% B and finally to 100% B. If A and B are not mutually miscible, make the transfer via an intermediate solvent, for example from water to chloroform via acetone, as shown in Figure 6.3.

Suggested routes for changing to organic solvents

Figure 6.3. Suggested routes for changing to organic solvents.


  1. Transfer the required amount of medium to a sintered glass Buchner funnel and remove the excess aqueous solution by gentle suction.
  2. Add the next solvent and resuspend the medium by stirring gently.
  3. Suck out the excess solvent and resuspend in the same solvent.
  4. Repeat the process with the next solvent in the series. Perform at least two resuspensions for each change of solvent conditions until the final solvent composition is reached.
  5. Pack the medium into solvent resistant SR 25/45 or SR 25/100 columns.


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