Fluorescein-based pImarkers for capillary isoelectric focusing with laser-induced fluorescence detection

Abstract

We prepared a series of low-molecular-mass fluorescent ampholytes with narrow pI range. These fluorescein-based ampholytes are detection compatible with argon laser-induced fluorescence (LIF) detection. The selected properties, important for their routine use as fluorescent pI markers, were examined. The pI values of new fluorescein-based pI markers were determined by capillary isoelectric focusing (CIEF) using currently available low-molecular-mass pI markers for CIEF with photometric detection. The examples of CIEF with fluorometric detection of new compounds together with fluorescein isothiocyanate (FITC) derivatized proteins are presented.

Capillary isoelectric focusing of proteins and microorganisms in dynamically modified fused silica with UV detection

“…All chemicals were analytical grade. Low-molecular-mass pI markers [16], 3.9, 5.3, 6.4, 7.5, 8.5, 10.1, and 10.4, were synthesized in the Institute of Analytical Chemistry Academy of Sciences of the Czech Republic, Brno. Other chemicals were obtained from Pliva-Lachema a.s., Brno, Czech Republic.…”

“…The suggested solutions include dynamic modification of the inner capillary surface [12,13] by soluble polymers such as hydroxymethylpropyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol, polyacrylamide, poly(acryl-amide-coacrylic acid), polyvinylpyrrolidone, etc. [12,14,15]; coating of the capillary with the hydrophilic polymer such as an acrylamide derivative; and modification of the capillaries by ␥-glycidoxypropyltrimethoxysilane [16,17] or the sol-gel technique [18]. The lifetime of the polymeric film on the inner surface of the capillary is often shortened by its degradation due to the strong acids and bases used as the anolytes and catholytes, respectively [12].…”

“…In order to obtain reproducible results we also suggest the washing of the capillaries between analyses by the mixture of organic solvents. For the tracing of the pH gradients the low-molecular pI markers [16] were used. The mixed cultures of microorganisms and proteins were reproducibly focused and separated by CIEF method suggested.…”

Two‐dimensional capillary electrophoresis: Capillary isoelectric focusing and capillary zone electrophoresis with laser‐induced fluorescence detection

“…The sample was spiked with a fluorescent pI standard (4.0) synthesized and donated by Šlais [36]. Figure 3 presents the two-dimensional electropherogram.…”

Advanced Capillary and Microchip Electrophoretic Techniques for Proteomics

“…Three different common detection schemes are conducted in IEF, including single-point detection (Slais et al, 2002), whole column scanning detection , and whole column detection by using a chargecoupled device (CCD) camera (Liu and Pawliszyn, 2003). A Teflon AF 2400 capillary was used as both the separation channel and the axially illuminated liquid core waveguide (Liu and Pawliszyn, 2003).…”