Coupling condensation nucleation light scattering detection with capillary electrophoresis using electrospray.
An improved method for coupling capillary electrophoresis (CE) with condensation nucleation light scattering detection (CNLSD) is described. The method employs an electrospray aerosol source followed by aerosol particle neutralization within a weak plasma established by a polonium-210 alpha emitter. Sensitive, universal detection of underivatized proteins, peptides, and amino acids is demonstrated. The system performance was significantly affected by the operational mode of the electrospray source, characterized by the physical appearance of the droplet at the end of the electrospray capillary. With a so-called pulsating mode, relatively low backgrounds were obtained with 10 mM ammonium acetate or 10 mM ammonium acetate/10 mM ammonia CE buffers using one diffusion screen, allowing detection of proteins at single microgram per milliliter levels and amino acids and peptides at submicrogram per milliliter levels. Linearity of response, expressed as peak height or peak area; mass limits of detection (LODs) for proteins, peptides, and amino acids at the picogram level, corresponding to femtomole levels of peptides and amino acids or subfemtomole levels of proteins; and better detectability compared to UV absorbance at 214 and 200 nm were demonstrated. The separation efficiencies obtained with the CE-electrospray-CNLSD system are much higher than those obtained for a previously reported pneumatic nebulizer-based system and in the range of approximately 20,000-160,000 plates/m using the pulsating electrospray mode. With careful adjustment of the electrospray voltage, a different operating mode, called the silver bullet mode, could be established for which higher signals, lower background and background noise, and higher separation efficiencies were observed compared to the pulsating mode. The lower background levels observed with the silver bullet mode eliminated the need for the use of a diffusion screen for background control with the buffer employed. With the silver bullet mode without a diffusion screen, linear response and LODs at the 15 ng/mL level, corresponding to subpicogram or 1-2 fmol levels of underivatized peptides and amino acids, and plate numbers in the range of 65,000-220,000 plates/m were estimated.
Szostek
B
B
Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale 62901-4409, USA.
Zajac
J
J
Koropchak
J A
JA
D000596:Amino Acids / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D008027:Light; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000032:analysis; D012542:Scattering, Radiation
D000596:Amino Acids; D010455:Peptides; D011506:Proteins