Division 5.5 Liquid-liquid partition

Transfer the eluate obtained in (5.4.2) into a 1 litre separating funnel. Rinse the conical flask with 5 to 10 ml methanol (3.2) and combine the washings with the contents of the separating funnel. Add 300 ml of hydrochloric acid solution (3.5) and 130 ml of dichloromethane (3.1). Shake for 1 minute and allow the phases to separate. Run off the lower (dichloromethane) layer into a 500 ml round bottomed flask. Repeat the extraction of the aqueous phase with two further 70 ml portions of dichloromethane and combine these extracts with the first in the round-bottomed flask.

Evaporate the dichloromethane extract down to dryness on the rotary evaporator (4.2) operating under reduced pressure at 40 ^oC. Dissolve the residue in the flask with approximately 5 ml of methanol (3.2) and transfer this solution quantitatively to a 10 ml graduated flask. Rinse the round-bottomed flask with a further two portions of 1 to 2 ml of methanol and transfer these to the graduated flask. Make up to the mark with methanol and mix. An aliquot portion is filtered through a membrane filter (4.6). Reserve this solution for HPLC-determination (5.6).