Stacking and separation of protein derivatives of naphthalene‐2,3‐dicarboxaldehyde by CE with light‐emitting diode induced fluorescence detection

We describe the stacking and separation of proteins by CE under discontinuous conditions in conjunction with light‐emitting diode induced fluorescence (LEDIF) detection using a violet LED at 405 nm. The proteins were derivatized with naphthalene‐2,3‐dicarboxaldehyde (NDA) to form NDA–protein derivatives prior to CE‐LEDIF analysis. During the separation, poly(ethylene oxide) (PEO) solution containing CTAB enters from the cathodic inlet to the capillary via electroosomotic flow (EOF). The optimum conditions are: the capillary was filled with 50 mM glycine buffer (pH 9.0) containing 1.0 mM CTAB, NDA–protein derivatives were prepared in deionized water containing 1.0 mM CTAB, and 0.6% PEO was prepared in 50 mM glycine (pH 9.0) containing 2.0 mM CTAB. The analysis of four NDA–protein derivatives is fast (<3 min), with RSD <1.5% in terms of migration time. In order to improve the sensitivity of NDA–protein derivatives, a stacking approach based on increases in viscosity and electric field, as well as sieving was applied. The efficient stacking approach provides LODs (S/N = 3) of 2.41, 0.59, 0.61, and 4.22 nM for trypsin inhibitor, HSA, β‐lactoglobulin, and lysozyme, respectively. In addition, we also applied the stacking approach to determination of the concentration of HSA in one urine sample, which was determined to be 0.31 ± 0.05 μM ( n  = 3).