Sequential injection setup for capillary isoelectric focusing combined with MS detection

Abstract

Capillary isoelectric focusing in the presence of electroosmosis with sequential injection of carrier ampholytes and sample was found to be suitable for MS detection. The separate injection of the sample and the ampholytes provides good condition to suppress and overcome the undesirable effect of the presence of ampholytes in MS. By the appropriate selection of ampholyte solutions, whose pH range not necessarily covers the pI values of the analytes, the migration of the components can be controlled, and the impact of the ampholytes on MS detection is decreased. The unique applicability of this setup is shown by testing several parameters, such as the application of volatile electrolyte solutions, the type of the ampholytes, the order and the number of the ampholyte and sample zones. Broad and narrow pH range ampholytes were applied in experiments using uncoated capillaries with different lengths for the analyses of substituted nitrophenol dyes to achieve optimal conditions for the MS detection. Although the sample components are not leaving the pH gradient, due to the decrease in the ampholyte concentration at the position of the components, and because the sample components migrate in charged state, the ionisation is more effective for MS detection.

Modeling of formation and prevention of a pure water zone in capillary isoelectric focusing with narrow pH range carrier ampholytes

“…CIEF online coupled to MS requires volatile electrode solutions, such as formic acid and ammonium hydroxide . In this simulation work and before , H 3 PO 4 and NaOH were used as anolyte and catholyte, respectively.…”

“…Further work was focused on natural narrow range pH gradients that did not or barely include neutrality, i.e. to gradients such as those formed in the pH 5–7 and 7–9 intervals employed in experimental studies and the pH 6.7–7.7 gradient used for hemoglobin analysis . In this paper, simulation data of narrow range carrier ampholyte based pH gradients that end or begin at, shortly before or shortly after pH 7.00 are presented for the first time.…”

Capillary isoelectric focusing‐mass spectrometry: Coupling strategies and applications

“…Páger et al. demonstrated a successful CIEF‐MS hyphenation using this approach by separate injection of the sample and the ampholyte . All general parameters are summarized in Table .…”

Advanced online mass spectrometry detection of proteins separated by capillary isoelectric focusing after sequential injection

“…The major questions are about the use of coated or uncoated capillaries, the composition of catholyte and anolyte, and most importantly about the amphoteric compounds providing the pH gradient, i.e., the use of ampholytes or other substances. For the MS detection, it is highly advisable to use electrolytes, which are compatible with MS, and one should consider the application of ampholytes (or other compounds) at concentrations, where the suppression of the analyte ionization is not present, or it is low . Páger et al.…”

“…Páger et al. successfully demonstrated hyphenation of CIEF with MS after sequential injection of amphoteric dyes and the carrier ampholytes, and showed that separation can be reached also with one separate ampholyte zone, too (before or after the sample zone). Computer simulations have been performed to see the effect of various sampling strategies in CIEF , studying the behavior of sample components with pI values outside the pH gradient of the ampholytes .…”