Use of quasi‐isoelectric buffers as anolyte and catholyte to improve capillary isoelectric focusing performances

Abstract

The use of quasi-isoelectric anolytes and catholytes has been investigated to improve CIEF performances. Narrow pH cuts of carrier ampholytes (NC) have been compared to more conventional couples of anolytes/catholytes (phosphoric acid/sodium hydroxide and glutamic acid/lysine). First, a CIEF setup that consists in a bare silica capillary and 70:30 water/glycerol separation medium has been used. The experiments have shown that when using NC instead of more classical anolytes and catholytes, an increase in the protein detection time was observed and the resolutions obtained for neutral and acidic proteins were doubled. Moreover, according to the NC fraction used, the resolution was modified. In order to investigate further the mechanisms involved, a second setup using a capillary coated with hydroxypropylcellulose was used. With this setup no difference has been observed when changing anolyte and catholyte nature. A simple methodology has then been developed to evaluate EOF during focusing and mobilization steps of CIEF experiments. It highlighted the crucial role played by EOF when using a bare silica capillary. EOF indeed decreased by 33% during mobilization step when using NC instead of classical anolytes and catholytes.

Surface isoelectric focusing (sIEF) with carrier ampholyte pH gradient

“…Fig. illustrates that pH 3–10 CAs were compressed near the cathode, a common phenomenon reported previously . In a large slab gel with small reservoirs that allowed electrolytes to diffuse into the gel, the pH gradient was observed to compress due to differing anion and cation migration rates within the gel .…”

“…Ideally, commercial carrier ampholytes should generate a linear pH gradient that spans the anode to cathode gel length of 300 m. Fig. 3 illustrates that pH 3-10 CAs were compressed near the cathode, a common phenomenon reported previously [6,44,[50][51][52][53][54]. In a large slab gel with small reservoirs that allowed electrolytes to diffuse into the gel, the pH gradient was observed to compress due to differing anion and cation migration rates within the gel [55].…”

“…4A. Curved bands are common in IEF applications including flat gels [56][57][58][59], channels [6,7,54] and other platforms [52,53]. Distortion in smaller electric fields, such as 100 V/cm, are more susceptible to gel non-uniformity effects because protein migration is not strong enough to overcome pore geometry differences [60].…”

Effect of electrolyte pH on CIEF with narrow pH range ampholytes

“…Lopez‐Soto‐Yarritu et al enhanced the resolution of sample components by titrating the sodium hydroxide catholyte with phosphoric acid. In the presence of EOF, Poitevin et al observed altered resolution by using quasi‐isoelectric amphoteric anolytes and catholytes, i.e., narrow pH cuts of carrier ampholytes or amino acids. Cui et al modified the pH gradient with applying narrow range ampholytes as electrolytes in multistage on‐chip IEF experiments in a polymeric (PDMS) microchip, whereby segments of the samples separated in the first stage with broad‐range ampholytes were refocused within narrower pH ranges in order to achieve higher resolving power.…”

Recent developments in electrophoretic separations on microfluidic devices

“…This technique has been adapted to capillaries (cIEF) [40] and microdevices [1]. Capillary IEF designs from the last 2 years utilized capillaries in the tens of centimeters range [41,42], falling outside the scope of this review. Although there are several applications of IEF in microdevices in the last 2 years, these designs still use channels whose lengths are in the centimeter range [43][44][45][46][47][48].…”