20 research outputs found
Detection of bovine whey proteins by on-column derivatization capillary electrophoresis with laser-induced fluorescence monitoring
1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS), 4,49-dianilino-1,19-binaphthyl-5,59-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
28 27 27proteins in CE separations was about 6?10 M for BSA, 3?10 M for b-lactoglobulin A (b-LGA), 3?10 M for
26b-lactoglobulin B (b-LGB), and 3?10 M for a-lactalbumin (a-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,
b-LGA and b-LGB were twice as good using LIF than with UV detection. However, the limit of detection for a-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. Ó 1999 Elsevier Science B.V. All rights reserved.This work was supported by CICYT (Spain) under
project ALI97-0630. M.L.M. thanks DGICYT (Spain) for supporting project PB94-0356. The authors also thank Beckman Instruments Espana for their financial support and M.C. Marina for linguistic assistance.Peer reviewe