Influence of polyelectrolyte capillary coating conditions on protein analysis in CE (original) (raw)
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Journal of Chromatography A, 2011
The stability of capillaries coated with highly charged polyelectrolytes under various analytical conditions was studied, as well as their performance for the analysis of proteins by Capillary Electrophoreis (CE) over a wide range of pH (2.5-9.3). In this study, fused silica capillaries were modified either with a poly(diallyldimethylammonium) chloride (PDADMAC) monolayer or PDADMAC/poly(sodium 4styrenesulfonate) (PSS) multilayer coatings, using optimal coating conditions previously determined [1-3]. Results show that the coated capillaries are remarkably stable and efficient to limit protein adsorption under a variety of extreme electrophoretic conditions even in the absence of the coating agent in the background electrolyte which is exceptional for non-covalent coatings. Monolayer coated capillaries were demonstrated for the first time to be stable to acidic rinses and to organic solvents which proves that the stability of the capillaries is highly dependent on the coating procedure used. In addition, PDADMAC/PSS multilayer coatings were found to be stable to alkaline treatments. PDADMAC/PSS coated capillaries gave excellent performances for the analysis of proteins covering a large range of pI (4-11) and of molecular weight (14-65 kDa) over a wide pH range (i.e. 2.5-9.3). Even at high pH 9.3, protein analysis was possible with very good repeatabilities (RSD tm < 1% and RSD CPA < 2.6% (n ≥ 8)) and high peak efficiencies in the order of 700,000.
Novel cationic polyelectrolyte coatings for capillary electrophoresis
ELECTROPHORESIS, 2015
The use of bare fused silica capillary in CE can sometimes be inconvenient due to undesirable effects including adsorption of sample or instability of the EOF. This can often be avoided by coating the inner surface of the capillary. In this work, we present and characterize two novel polyelectrolyte coatings (PECs) poly(2-(methacryloyloxy)ethyl trimethylammonium iodide) (PMOTAI) and poly(3-methyl-1-(4-vinylbenzyl)-imidazolium chloride) (PIL-1) for CE. The coated capillaries were studied using a series of aqueous buffers of varying pH, ionic strength, and composition. Our results show that the investigated polyelectrolytes are usable as semi-permanent (physically adsorbed) coatings with at least five runs stability before a short coating regeneration is necessary. Both PECs showed a considerably decreased stability at pH 11.0. The EOF was higher using Good's buffers than with sodium phosphate buffer at the same pH and ionic strength. The thickness of the PEC layers studied by quartz crystal microbalance was 0.83 and 0.52 nm for PMOTAI and PIL-1, respectively. The hydrophobicity of the PEC layers was determined by analysis of a homologous series of alkyl benzoates and expressed as the distribution constants. Our result demonstrates that both PECs had comparable hydrophobicity, which enabled separation of compounds with log P o/w Ͼ 2. The ability to separate cationic drugs was shown with -blockers, compounds often misused in doping. Both coatings were also able to separate hydrolysis products of the ionic liquid 1,5-diazabicyclo[4.3.0]non-5-ene acetate at highly acidic conditions, where bare fused silica capillaries failed to accomplish the separation.
Journal of Chromatography A
Protein adsorption on the inner wall of the fused silica capillary wall is an important concern for capillary electrophoresis (CE) analysis since it is mainly responsible for separation efficiency reduction. Successive Multiple Ionic-polymer Layers (SMIL) are used as capillary coatings to limit protein adsorption, but even low residual adsorption strongly impacts the separation efficiency, especially at high separation voltages. In this work, the influence of the chemical nature and the PEGylation of the polyelectrolyte deposited in the last layer of the SMIL coating was investigated on the separation performances of a mixture of four model intact proteins (myoglobin (Myo), trypsin inhibitor (TI), ribonuclease a (RNAse A) and lysozyme (Lyz)). Poly(allylamine hydrochloride) (PAH), polyethyleneimine (PEI), ε-poly(L-lysine) (εPLL) and αpoly(L-lysine) (αPLL) were compared before and after chemical modification with polyethyleneglycol (PEG) of different chain lengths. The experimental results obtained by performing electrophoretic separations at different separation voltages allowed determining the residual retention factor of the proteins onto the capillary wall via the determination of the plate height at different solute velocities and demonstrated a strong impact of the polycationic last layer on the electroosmotic mobility, the separation efficiency and the overall resolution. Properties of SMIL coatings were also characterized by quartz microbalance and atomic force microscopy, demonstrating a glassy structure of the films.
Polyelectrolyte Multilayers in Capillary Electrophoresis
ChemPlusChem, 2022
Capillary electrophoresis (CE) has been proven to be a performant analytical method to analyze both small and macro molecules. Indeed, it is capable of separating compounds of the same nature according to differences in their charge to size ratios, particularly proteins, monoclonal antibodies and peptides. However, one of the major obstacles to reach high separation efficiency remains the adsorption of solutes on the capillary wall. Among the different coating approaches used to control and minimize solute adsorption, polyelectrolyte multilayers can be applied to CE as a versatile approach. These coatings are made up of alternating layers of polycations and polyanions, and may be used in acidic, neutral or basic conditions depending on the solutes to be analyzed. This Review provides an overview of Successive Multiple Ionic‐polymer Layer (SMIL) coatings used in CE, looking at how different parameters induce variations on the electro‐osmotic flow (EOF), separation efficiency and coat...
Electrophoresis, 2008
Polyelectrolytes are widely used in capillary electrophoresis as coating agents of silica capillaries to prevent adsorption phenomena and improve the repeatability of peptide and protein analysis. A systematic study of the coating experimental conditions has been carried out to optimize coating stability and performance. The main experimental parameters studied were the type and concentration of polyelectrolytes used in several monolayer and multilayer coatings, the ionic strength of coating and stabilizing solutions, and the procedures used for coating and capillary storage. Electroosmotic flow magnitude, direction and repeatability were used to monitor coating stability. Coating ability to limit adsorption was investigated by monitoring variations of migration times, time-corrected peak areas and separation efficiency of test peptides. Capillary-to-capillary and batch-to-batch reproducibility was also studied. In addition, the separation performance of polyelectrolyte coatings were compared to those obtained with bare silica capillaries.
ELECTROPHORESIS, 2005
A simple polyelectrolyte multilayer (PEM) coating procedure was used for the development of stable modified capillaries. PEM coatings were constructed in fused-silica capillaries using alternating rinses of cationic and anionic polyelectrolytes. The multilayer coatings investigated in this study consisted of two and twenty layer pairs, or bilayers. A bilayer is one layer of a cationic polymer and one layer of an anionic polymer. Poly(diallyldimethylammonium chloride) was used as the cationic polymer, and the polymeric surfactant poly(sodium N-undecanoyl-L-leucylvalinate) was used as the anionic polymer. Previous studies for both chiral and achiral separations have shown that PEM-coated capillaries have excellent reproducibilities, remarkable endurance, and strong stabilities against extreme pH values when used in open-tubular capillary electrochromatography (OT-CEC). In this study, the stability of the coatings was further investigated after exposure to 0.1 M and 1.0 M NaOH. Structural changes of these coatings were monitored using laser scanning confocal microscopy (LSCM) after flushing the capillaries with NaOH. This technique allowed observation of the degradation of the coatings. Observations are discussed in terms of separations using OT-CEC. Electropherograms obtained from the chiral separation of 1,1'-binaphthyl-2,2'dihydrogenphosphate in OT-CEC showed a decrease in selectivity and an increase in electroosmotic mobility after long exposure to NaOH. The ability to recover the capillaries by exposure to NaOH was also demonstrated. Measurements of electroosmotic mobility and selectivity showed that 2-bilayer and 20-bilayer PEM coatings could be completely removed from the capillary surface after approximately 3.5 and 9.5 h, respectively, of continuous exposure to 1 M NaOH. Abbreviations: BNP, (6)-1,1'-binaphthyl-2,2'-dihydrogenphosphate; LSCM, laser scanning confocal microscopy; PDAD-MAC, poly(diallyldimethylammonium chloride); PEM, polyelectrolyte multilayer; poly(L-SULV), poly(sodium N-undecanoyl-Lleucylvalinate) Electrophoresis 2005, 26, 783-789 783
ELECTROPHORESIS, 2008
Novel polyelectrolyte multilayer (PEM) coatings for enhanced protein separations in open tubular CEC (OT-CEC) are reported. Use of four cationic polymers (poly-L-lysine, poly-Lornithine, poly-L-lysine-serine, and poly-L-glutamic acid-lysine), and three anionic molecular micelles, sodium poly(N-undecanoyl-L-leucyl-alaninate) (poly-L-SULA), sodium poly(Nundecanoyl-L-leucyl-valinate) (poly-L-SULV), and sodium poly(undecylenic sulfate) (poly-SUS) were investigated in PEM coatings for protein separations. The simultaneous effects of cationic polymer concentration, number of bilayers, temperature, applied voltage, and pH of the BGE on the separation of four basic proteins (a-chymotrypsinogen A, lysozyme, ribonuclease A, and cytochrome c) were analyzed using a Box Behnken experimental design. The influence of NaCl on the run-to-run reproducibility was investigated for PEM coatings containing each cationic polymer. All coatings exhibited excellent reproducibilities with a %RSD of the EOF less than 1% in the presence of NaCl. Optimal conditions were dependent on both the cationic and anionic polymers used in the PEM coatings. Poly-Lglutamic acid-lysine produced the highest resolution and longest migration time. The use of molecular micelles to form PEM coatings resulted in better separations than single cationic coatings. Chiral poly-L-SULA and poly-L-SULV resulted in higher protein resolutions as compared to the achiral, poly-SUS. Furthermore, the use of poly-L-SULV reversed the elution order of lysozyme and cytochrome c when compared to poly-L-SULA and poly-SUS.
Chromatographia, 2003
High performance liquid chromatography (HPLC) was used to study the mechanism of formation of polyelectrolyte multilayers on porous silicas. The coatings were produced by alternating the adsorption of positively and negatively charged polymers. The stationary phases formed by adsorbing a single layer, double layers and triple layers were tested by studying the elution behavior of model proteins. The double polymer coating was achieved by adsorbing first a polycation such as hexadimethrine bromide (HB) on the HPLC silica support and then a polyanion such as dextran sulfate (DS) on the cationic layer formed. The retention properties of this support are mainly those of a cation exchanger as the negatively charged proteins were strongly retained while positively charged ones were weakly adsorbed. This work demonstrated the importance of the first underlying layer as the retention behavior of proteins was greatly affected by the properties of this coating. The triple polymer coating was achieved by adsorbing the polycation (HB) on the double layer coating (HB-DS). Its retention behavior was that of an anion exchange support. The HB-DS stationary phase displayed good chromatographic performances, with an adsorbed layer relatively stable. The polyelectrolyte multilayer coating procedure was useful to easily synthesize cation-exchange supports for the separation of basic proteins.