Fast, high‐resolution (capillary) electrophoresis in buffers designed for high field strengths

Abstract

Capillary electrophoresis in conventional buffers and in 50 microns capillaries permits field strengths as high as 300-500 V/cm with acceptably low thermal zone deformation. However, still higher field strengths (up to at least 2000 V/cm) can be applied without a decrease in resolution if the experiments are performed in the buffers described in this paper. Characteristic of these buffers is their low electrical conductivity and yet satisfactory buffering capacity accomplished either (i) by selecting buffer constituents of relatively high molecular weight and small net charge or (ii) by fractionation of carrier ampholytes (originally introduced for isoelectric focusing experiments) into a series of narrow pH range fractions and using these fractions as buffers, or (iii) by selecting an ampholyte with two acidic groups and one basic group (or one acidic group and two basic groups) and with a pI value close to two of its pK values. In such buffers, aromatic carboxylic acids and proteins used as model substances could be analyzed rapidly. For instance, albumin and transferrin were separated at 30,000 V (1.99 microA) in 15 cm long fused silica capillaries (50 microns ID) within 40 s and the carboxylic acids within 25 s. The resolution was similar to that obtained at standard voltage (5000 V; 0.33 microA), but the analysis time was reduced sixfold. Although not verified experimentally we also suggest the use of relatively high-molecular-weight polyoxyethylene derivatized with one acidic group (for instance, boric acid) and one basic group (an amine), both having the same pK value, which should afford both a very high buffering capacity and very low electrical conductivity (at low buffer concentrations).

Protein tryptic digests analyzed by carrier ampholyte‐based capillary electrophoresis coupled to ESI‐MS

“…Among others, amino acids and carrier ampholytes (CAs), usually used in IEF, were tested. Blanco et al [2] studied the bandspreading mechanism in these kinds of buffers and, by computation, found results in accordance with [1]. Compared to the CA-based buffers, amino acids have been studied more frequently as low conductivity buffers, and this especially by the team of Pier Giorgio Righetti.…”

Lysine acetylation can generate highly charged enzymes with increased resistance toward irreversible inactivation

“…A Tris-Glycine buffer was used as the running buffer (10 mM Tris, 192 mM glycine, pH 8.5). This buffer is ideal for experiments involving capillary electrophoresis because it has a low absorbance at 214 nm, and has a low conductivity (which allows it to be subjected to high voltages without overheating the apparatus) (Hjerten et al 1995;Righetti et al 2000).…”

“…To determine whether any loss of activity was reversible, the solutions of BLA were cooled to 25°C for 1 h at the end of the 520-min, 90°C incubation, and the activity was measured. We chose to use Tris-glycine buffer because of its strong buffer capacity at alkaline pH (many enzyme-surfactant systems operate at alkaline pH) and also because Tris is an ideal buffer for electrophoretic experiments (Hjerten et al 1995;Righetti et al 2000).…”

Selectivity change in the separation of proteins and peptides by capillary electrophoresis using high-molecular-mass polyethyleneimine

“…At very low or very high pH values the interaction would be smaller, since at low pH the number of negative groups in the proteins decreases, while the number of positive groups in PEI decreases at high pH values. Moreover, some other effects, such as hydrophobic interactions [22] and hydrogen binding, may play a role as well.…”