Journal of Chromatography A, 1232 (2012) 134–141 Contents lists available at SciVerse ScienceDirect Journal of Chromatography A j our na l ho me p ag e: www.elsevier.com/locate/chroma Purification of Coomassie Brilliant Blue G-250 by multiple dual mode countercurrent chromatography Nazim Mekaoui, Joseph Chamieh, Vincent Dugas, Claire Demesmay, Alain Berthod ∗ Laboratoire des Sciences Analytiques, Université de Lyon, CNRS, Bat. CPE, 69622 Villeurbanne, France a r t i c l e i n f o Article history: Available online 19 November 2011 Keywords: Countercurrent chromatography Multi dual-mode Coomassie Brilliant Blue Amine detection a b s t r a c t 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. 200 mg of purified CBB were obtained from 1 g of crude CBB in 3 h 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. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Biomolecules and amines immobilized on solid supports were developed for micro-volume reactions and/or biocatalysis [1–3]. They found important uses in enzymatic conversion [4], molecu- lar recognition [5] or biosensors engineering [6]. These supports need to be fully characterized. Spectroscopic techniques were used for that purpose but they are limited to accessible sur- faces [7–11]. Other techniques including molecular labeling or protein total analysis have been described but these applications remained destructive analytical methods, lacked in terms of speci- ficity or sensitivity or required the use of expansive reagents for the assays [12,13]. Recently, a simple and non-destructive colori- metric analytical method to quantify protonated amino groups in proteins, peptides or any amino containing structure adsorbed on solid supports has recently been proposed [14,15]. It was called the ADECA method for “Amino Density Estimation by Colori- metric Assay”. This non-destructive method is based on specific and reversible Coomassie dye-amino group binding to positively charged amino groups. The surface containing protonated amino groups is stained by the dye. Next the excess dye is washed out without disrupting the dye-amino bonded part. Finally, a special sodium carbonate methanol buffer is used to release the attached ∗ Corresponding author. Tel.: +33 472448296; fax: +33 472431078. E-mail address: berthod@univ-lyon1.fr (A. Berthod). dye whose colorimetric measure is easily related to the amino group concentration [15]. The ADECA method can be applied to a wide variety of supports of very differing hydrophobicity. Being reversible, the method is non-destructive. It means that the very same surface that was characterized by the ADECA method can be readily used for further coupling application or protein reaction [14,15]. However, the method requires the use of an extremely pure Coomassie Blue as the staining agent to avoid biases mea- suring the desorbed dye amount. Regular and commercial grade Coomassie Blue will produce biased results due to non-specific bindings [16]. Coomassie Brilliant Blue (CBB) is a triphenyl methane dye exten- sively used to stain proteins in electrophoretic profiles and in solution quantification. There are however two types of CBB dyes: CBB R-250 where R refers to the reddish tint of the product and CBB G-250 where G is related to the greenish tint of the dye [17]. CBB G differs from CBB R-250 with only two additional methyl groups (Fig. 1). The methylated dye is the preferred dye used in protein quantification assays by the Bradford method [18]. Coomassie is a trademark name still detained by Imperial Chemical Industries (ICI, later acquired by Akzo Nobel Inc.), and the 250 code is related to the dye content. In fact, both varieties of CBB commercially available very often contain up to 50% impurities accompanying the main staining agent [19–21] making them unusable with modified solid supports. It is therefore critical to purify the staining agent from high binding impurities in order to achieve better analytical per- formances and reproducible assays. A low pressure LC purification 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.11.022