Wide Column fractionation Procedure - Outline with Videos

Irene Mueller-Harvey, University of Reading

Normal lab safety procedures should be followed at all times. Please make sure you have read, understood and signed the relevant risk assessments.

The sample we use for fractionation is prepared by extracting a dried and ground plant sample in 70% acetone/30% water, then adding dichloromethane, separating the layers and removing the acetone from the upper (aqueous) layer on a rotary evaporator. This material (referred to as A0 extract) is then stored frozen prior to fractionation. The column is packed with Sephadex LH-20; the sample is loaded in water and the column isrinsed with copious amounts of water. Tannins are eluted with 30% acetone/70% water to give the F1 fraction, then with 50% acetone/50% water to give the F2 fraction.

1 – Prepare the column. (The column is stored in 80% acetone/20% Water and this needs to be washed out with a litre of water before the sample is loaded). Drain the column so that the liquid level is just above the Sephadex LH-20 bed. Add 1 litre of water to the top reservoir and open the tap to let the water flow very slowly onto the bed. Once a few cm of water has collected above the bed the flow can be increased. Once 20-30 cm of water has collected above the bed the lower tap can be opened and the water allowed to flow very rapidly through the bed. Stop when the water level is just above the top of the bed. (These steps are identical to those used to load the sample – step 4 below).

2 – Defrost the sample. The easiest way to do this is in a bowl of warm water, you may need to use several changes of water.

3 – Filter the sample. (In this example the sample has been diluted to 1 litre before filtration, alternative procedures are to filter before dilution or to centrifuge before dilution).


4 – Load the sample. If the sample is loaded too quickly (as in this example) the surface of the bed will be disturbed. In this case the bed can be reformed by twisting the column. Once a suitable level of sample has collected above the column the lower tap can be opened and the sample allowed to flow through the bed.


5 – Wash the column with 1-2 litres of water.

6 – Elute the F1 fraction with 1 litre of 30% acetone/70% Water. The procedure is exactly the same as for “loading the sample” and “Washing the column” steps above. Start collecting the eluant after approximately 200 ml has eluted, the signal to start collecting is when refractive index ripples are seen in the liquid under the column. These are shown in clip 1 and clip 2, you may need to adjust the angle of view to see these ripples.


7 – Rotary evaporate the F1 sample until there is no smell of solvent, then freeze it in a thick walled beaker.

8 – Elute the F2 fraction with 1 litre of 50% acetone/50% Water. The procedure is exactly the same as for “loading the sample” and “Washing the column” steps above. Actually just let approximately 500 ml flow through the column to elute the F2 material. The remaining acetone water mixture can be left to prevent the bed from drying out.

9 – Rotary evaporate the F2 sample until there is no smell of solvent, then freeze it in a thick walled beaker.

10 – The following day place the frozen samples on the freeze drier, they will take several days to dry down. The fractions can then be weighed into sample tubes and stored in the freezer.

All contents copyright © Legume Plus 2017


Sample Extraction Protocol

(Preparation of sainfoin [A0] crude extract)


  1. Weigh 20 g of dry grounded plant material (ball-milled) into a conical flask.
  2. Stir with 250 ml of 70% of acetoneaq for 60 min.
  3. Filter the mixture into a Buchner flask with filter paper using low vacuum.
  4. Pour the filtrate into a separating funnel.
  5. Add 125 ml of dichloromethane (CH2Cl2).
  6. Shake and collect the upper layer.
  7. The organic solvent is evaporated and the remaining aqueous solution is centrifuged for 6 min at 4500 rpm.
  8. Freeze the solution overnight.
  9. Freeze dry the solution to give A0 sample
  10. Weigh the A0 sample.

To determine unextractable tannins…

  1. Collect and freeze dry the solid residue.
  2. Weigh it.
  3. HCl butanol reaction on it and on original plant material, and on A0 extract.