Detection of bovine whey proteins by on-column derivatization capillary electrophoresis with laser-induced fluorescence monitoring

Abstract

1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS), 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) and 2-( p-toluidino)naphthalene-6-sulfonic acid (2,6-TNS) were evaluated as additives in different buffers for the detection of bovine whey proteins using laser-induced fluorescence (LIF) monitoring in capillary electrophoresis (CE). These N-arylaminonaphthalene sulfonates furnish a large fluorescence emission when associated to some proteins whereas their emission in aqueous buffers, such as those used in CE separations, is very small. To select the best detection conditions, the fluorescence of these probes was first compared using experiments carried out in a fluorescence spectrophotometer. Using bovine serum albumin (BSA) as a model protein, it was demonstrated that 2-( N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 8 and pH 10.2) and the fluorescent probe 2,6-TNS gave rise to the highest increase in fluorescence for BSA. When the composition of these separation buffers was optimized for the electrophoretic separations, CHES buffer, pH 10.2 was chosen as the most suitable buffer to detect bovine whey proteins. The limit of detection obtained for some whey proteins in CE separations was about 6·10 −8 M for BSA, 3·10 −7 M for β-lactoglobulin A (β-LGA), 3·10 −7 M for β-lactoglobulin B (β-LGB), and 3·10 −6 M for α-lactalbumin (α-LA). These detection limits were compared to those achieved using UV detection under the same separation conditions. The results showed that the detection limits of BSA, β-LGA and β-LGB were twice as good using LIF than with UV detection. However, the limit of detection for α-LA was better when UV was used. The applicability of LIF detection to CE separation of whey proteins in bovine milk samples was also demonstrated.