We describe a segmental filling method for the analysis of SYPRO Red labeled sodium dodecyl sulfate (SDS)-proteins (SRSPs) by capillary electrophoresis-laser induced fluorescence (CE-LIF) with electroosmotic counterflow of poly(ethylene oxide) (PEO). It is shown that SDS and salt play a crucial role in determining the fluorescence intensity of the SRSP Although the fluorimetric measurements reveal that the SRSPs fluoresce strongly in Tris-borate (TB) buffer containing 0.1 % SDS and high concentrations of NaCl (100 mM), these conditions are not appropriate to CE in view of Joule heating. To overcome that impediment, we applied a plug of 0.1 % SDS (1/5 to 1/3 of the injection volume) prior to injection of samples (0.64 μL) prepared in TB buffer containing 50 mM NaCl and SYPRO Red. When using a background electrolyte of 0.6% PEO in TB buffer containing NaCl, electroosmotic counterflow of the analytes allows one to concentrate large sample volumes (up to 1/3 of effective capillary length) in 21 min, with detection of 0.35 and 0.10 nM for bovine serum albumin and casein, respectively. With a linear dynamic range from 10 nM to 5 μM this method provides the capability of determining the concentration of casein in cow's milk as 0.45 ± 0.03 mM (n = 5).
All Science Journal Classification (ASJC) codes
- Analytical Chemistry
- Clinical Biochemistry