Purification of Coomassie Brilliant Blue G-250 by multiple dual mode countercurrent chromatography

Commercial samples of Coomassie Brilliant Blue G-250 (CBB) were not pure enough to give reliable results when used as indicator of amine content in biological material. The polar and apolar impurities produce unacceptable biases in the results. Counter current chromatography (CCC) was used to purify significant amounts of CBB. The liquid system heptane/1-butanol/water 2:3:4 (v/v) was appropriate to separate crude CBB in three groups of components: polar, partitioning in the aqueous lower phase, intermediate, partitioning well between the aqueous and organic phases, and apolar, preferring greatly the organic phase. The dual-mode way of using a CCC chromatograph was found appropriate for the separation injecting the crude CBB in the middle of a two coil CCC instrument. A multi dual-mode purification was performed allowing to eliminate the polar impurities in the aqueous phase at the column tail and the apolar ones in the organic phase at the column head, trapping the purified dye inside the CCC column. 200mg of purified CBB were obtained from 1g of crude CBB in 3h using as little as 150 mL of butanol and 70 mL of heptane with 200 mL of water. The purified CBB gave total satisfaction in testing amine content in polyclonal antibody containing monolith pipettes.