Semipermanent Coatings Research Articles

Pyrolyzed cotton balls for protein removal: Analysis of pharmaceuticals in serum by capillary electrophoresis

Because of the inherent affinity of proteins for bare, fused silica capillaries, the analysis of protein-containing samples has proven a challenging task for capillary electrophoresis. The adsorption of proteins to the capillary walls effectively changes the zeta potential and thus affects the electro-osmotic flow leading to significant shifts in migration time, peak broadening, and poor reproducibility. While there are several well-known methods to remove proteins from samples prior to the analysis (including precipitation) or to prevent their adsorption to the capillary (semi-permanent coatings), those approaches are often expensive, time consuming, or simply unreliable. Aiming to address these needs, this manuscript reports on the use of pyrolyzed cotton balls, as a simple and widely accessible hydrophobic material to remove proteins from serum samples. The material retains enough flexibility so it can be placed directly into the sample vials and has enough capacity to capture more than 75% of the proteins in the sample (1% dilution of 1 mL of serum). The advantages of the material are demonstrated by performing the analysis of five representative drugs (in serum) by capillary electrophoresis obtaining a change in migration time of only 5 ± 1%, after 10 consecutive runs.

Evaluation of poly([2-(acryloyloxy)ethyl]trimethylammonium chloride) cationic polymer capillary coating for capillary electrophoresis and electrokinetic chromatography separations.

Capillary electrophoresis and electrokinetic chromatography are typically carried out in unmodified fused-silica capillaries under conditions that result in a strong negative zeta potential at the capillary wall and a robust cathodic electroosmotic flow. Modification of the capillary wall to reverse the zeta potential and mask silanol sites can improve separation performance by reducing or eliminating analyte adsorption, and is essential when conducting electrokinetic chromatography separations with cationic latex nanoparticle pseudo-stationary phases. Semipermanent modification of the capillary walls by coating with cationic polymers has proven to be facile and effective. In this study, poly([2-(acryloyloxy)ethyl]trimethylammonium chloride) polymers were synthesized by reversible addition-fragmentation chain transfer polymerization and used as physically adsorbed semipermanent coatings for capillary electrophoresis and electrokinetic chromatography separations. An initial synthesis of poly([2-(acryloyloxy)ethyl]trimethylammonium chloride) polymer coating produced strong and stable anodic electroosmotic flow of -5.7 to -5.4 × 10-4 cm2 /V⋅s over the pH range of 4-7. Significant differences in the magnitude of the electroosmotic flow and effectiveness were observed between synthetic batches, however. For electrokinetic chromatography separations, the best performing batches of poly([2-(acryloyloxy)ethyl]trimethylammonium chloride) polymer performed as well as the commercially available cationic polymer polyethyleneimine, whereas polydiallylammonium chloride and hexadimethrine bromide did not perform well.

Insight into the stability of poly(diallydimethylammoniumchloride) and polybrene poly cationic coatings in capillary electrophoresis

Polycationic polymers are widely used in capillary electrophoresis (CE) as surface coatings to prevent protein adsorption and control electroosmotic flow (EOF). Such semi-permanent coatings are formed by flushing the capillary with a quaternary amine-based polymer such as poly(diallydimethylammonium chloride) (PDADMAC) or polybrene. Compared to covalent capillary coatings, the claimed advantages of adsorptive polycation coatings are their simple preparation and that they are not limited to the pH 2–8 range as are covalent coatings. However, while the latter is commonly claimed, few studies have demonstrated the stability of polycationic coatings at extreme pH. Herein PDADMAC and polybrene are studied as model cationic coatings. PDADMAC with higher molecular weight (M.W.) demonstrated higher EOF stability at pH 9.5, with PDADMAC of M.W. less than 200,000 being unstable at pH 9.5. X-ray photoelectron spectroscopy (XPS) shows that the quaternary amines of PDADMAC and polybrene were slowly converted to tertiary amines in alkaline solution and more rapidly when adsorbed on a silica surface. The degraded polycation deprotonated at pH >7, resulting in loss of polymer from the surface and diminishing EOF. Successive multiple ionic layer (SMIL) coatings show greater alkaline stability by distancing the polycation from the surface. Separations of inorganic anions at pH 9.5 illustrate the degradation behavior and enhanced stability of higher M.W. polycationic coatings.

Nonionic surfactant enhanced semipermanent coatings for capillary electrophoresis of inorganic anions without use of organic additives

Separation of inorganic anions by capillary electrophoresis (CE) is usually conducted in co-electroosmotic mode due to the large electrophoretic mobilities of inorganic anions. Semipermanent surfactant coatings have been shown to be effective for CE of inorganic anions due to their strong capability of electroosmotic flow (EOF) manipulation. However, semipermanent coatings often suffer from their unsatisfactory stability. In addition, organic solvent additives are usually required to adjust the selectivity, which also aggravate the degradation of coating. In this work, a novel semipermanent coating consisting of cationic Gemini surfactant 18-10-18 and nonionic surfactant Tween 20 was developed to separate inorganic anions in CE. This coating is easy to prepare and more stable than pure Gemini coating. The introduction of nonionic surfactant in the coating not only suppresses the reversed EOF but can also adjust the selectivity of separation. Good separations of six model anions were achieved, the separation efficiency was as high as 65040-169700 plates/m and the RSDs of the migration times were less than 0.5 and 2.5% for run-to-run and day-to-day assays, respectively. Calibration curves were linear in the range of 0.05-5.0 mM; the detection limits ranged from 20 to 50 μM. More importantly, no organic solvents are required in the background buffer to achieve the satisfactory separations. This guarantees the coating stability and makes the method greener than most of other methods for CE of inorganic anions.

Effect of coating electrolytes on two-tailed surfactant bilayer coatings in capillary electrophoresis

Surfactants such as dioctadecyldimethylammonium bromide (DODAB) form semi-permanent coatings that effectively prevent adsorption of cationic proteins onto the fused silica capillary in capillary electrophoresis (CE). The bilayer coating is generated by flushing the capillary with a 0.1 mM surfactant solution. However, formation of the bilayer is strongly dependent on the coating electrolyte. The effect of counter-ions, electrolyte concentrations and buffer co-ions were monitored based on: the separation of basic model proteins; the adsorption kinetics of DODA + onto fused silica; and dynamic light scattering (DLS) to determine vesicle size. Low concentrations (≤10.0 mM) and/or weakly associating buffers such as phosphate (pH 3.0), acetate (pH 4.0) and chloride should be used for DODAB coating solutions. Dissolving the surfactant in strongly associating electrolyte, such as phosphate at pH 7.0, results in poor coating of the capillary surface. Effective cationic bilayer coatings are formed if the buffer conditions favor formation of vesicles with diameters < 300 nm. Monitoring turbidity at 400 nm provides a convenient means of verifying vesicle diameter variation of <5 nm; that is, that the coating solution is effective.

Surfactant bilayer coatings in narrow-bore capillaries in capillary electrophoresis

The cationic surfactants didodecyldimethylammonium bromide (DDAB) and dioctadecyldimethyl-ammonium bromide (DODAB) have previously been shown to form semi-permanent coatings that effectively prevent adsorption of cationic proteins in fused silica capillaries with inner diameters of 25-75 µm. This paper investigates the impact that narrower capillary diameters (≤25 µm) have on the stability of surfactant bilayer coatings and the efficiency of separations of model cationic proteins and neurotransmitters. Using a DODAB-coated 5 µm i.d. capillary 210 consecutive protein separations (1050 min) were performed without recoating the capillary between runs. Separation efficiencies of 1,400,000-2,000,000 plates per m (340,000-430,000 plates) were obtained. Migration time reproducibilites of 6.8% RSD were observed for 300 injections performed over a 30 day period without any regeneration of the coating. Neurotransmitters were separated with efficiencies ranging from 470,000-610,000 plates per m (110,000-140,000 plates) in a 5 µm capillary.

Enhanced stability of surfactant-based semipermanent wall coatings in capillary electrophoresis using oppositely charged surfactant

Semipermanent surfactant coatings are effective for the prevention of wall adsorption of proteins in CE. However, they often suffer from their unsatisfactory coating stability as they essentially degrade from the capillary walls after the surfactants are removed from the buffer. In this paper, we proposed a facile and universal method to improve the stability of semipermanent surfactant coatings based on addition of an oppositely charged surfactant into the coating. Didodecyldimethylammonium bromide (DDAB) and a gemini surfactant, 18-6-18, were used as the model semipermanent coatings, and sodium dodecyl sulfate (SDS) was chosen as their oppositely charged surfactant. SDS can strongly alter the packing parameter P of the cationic surfactants, and consequently mediates the coating stability. With the increase of SDS concentration in coating, the coating stability first dramatically increases due to the enlarged P, and then decreases due to the weakness of electrostatic interaction between the capillary wall and surfactant coating. At the proper SDS concentration, very stable coatings can be obtained that, even after rinsing under 138 kPa for 60 min, the reversed electroosmotic flow (EOF) only decreases by 3.6%. These SDS-enhanced coatings show excellent stability and reproducibility in protein separation (RSD of migration time <1.1% for run-to-run assay, n = 9). Also, the high separation efficiency (>500,000 plates/m) and fine recovery of tested proteins indicate that these coatings are powerful in wall adsorption suppression. Finally, we found that the separation efficiency of protein was a more exact indicator for the coating stability than the traditional EOF magnitude.

Long‐chained gemini surfactants for semipermanent wall coatings in capillary electrophoresis of proteins

In this paper, we presented the first example of using gemini surfactants as semipermanent coatings in CE for protein separation. These coatings are based on the self-assembly of a series of cationic gemini surfactants, alkanediyl-alpha,omega-bis(dimethylalkylammonium bromide) (m-s-m), on the capillary wall. The coatings can keep stable for a long time without surfactant in the buffer, e.g., after the surfactants were removed from the buffer, the reversed EOF only decreased by 3.6 and 3.9% for 18-2-18 and 16-2-16 coatings over 60 min under continuous electrophoretic conditions. The coating stability increased with the alkyl chain length m. The double long chains of geminis (m > or = 14) yielded a good coating stability; meanwhile, the spacer group acted as an EOF modifier. Thus, this bifunctional surfactant coating provided a new buffer-independent method for EOF control. For 18-s-18 series, the best coating stability and largest EOF were obtained at s = 10. Ranging s from 3 to 10 yielded a linear fine-tuning of EOF and thereby allowed the adjustment of the protein apparent mobility. Highly efficient separation (>500 000 plates/m) was achieved with all the 18-s-18 coatings. Excellent run-to-run and day-to-day reproducibility (RSD of migration time </=0.7 and 4.3% for run-to-run and day-to-day assays, respectively) and fine recoveries (97.5-100.1%) of the protein samples indicated that the gemini semipermanent coatings were quite effective for the wall adsorption suppression of proteins.

Oligomerized Phospholipid Bilayers as Semipermanent Coatings in Capillary Electrophoresis

Double-chained surfactants form semipermanent coatings that prevent protein adsorption in capillary electrophoresis (CE). To make such coatings more permanent, vesicles of the unsaturated phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine were prepared and subjected to free-radical-initiated polymerization, both inside the capillary and in free solution. The latter generated oligomers of 2-5 units based on ESI-TOF MS, and formed the more stable coating in CE. Rinsing the capillary with a solution of the ex situ oligomerized DOPC suppressed EOF (0.8 x 10(-)(8) m(2)/V.s) for more than 20 h, whereas in situ oligomerized electroosmotic flow (EOF) suppressed the EOF for only 10 h. Mixtures of anionic and cationic proteins were separated under neutral pH and low ionic strength buffer with efficiencies of 480,000-930,000 plates/m and recoveries of 75-99%.

Enhanced Stability Self-Assembled Coatings for Protein Separations by Capillary Zone Electrophoresis through the Use of Long-Chained Surfactants

Semipermanent coatings were generated within fused-silica capillaries by flushing the capillary with a 0.1 mM solution of the double-chained cationic surfactants didodecyldimethylammonium bromide, dimethylditetradecylammonium bromide (2C(14)DAB), dihexadecyldimethylammonium bromide, and dimethyldioctadecylammonium bromide (2C(18)DAB) and the triple-chained surfactant tridodecylmethylammonium iodide. All of these coatings were semipermanent, whereby the coating remained intact after the unadsorbed surfactant was removed from the capillary. The separation efficiencies for four model cationic proteins ranged from 1.2 to 1.4 million plates/m for the 2C(14)DAB coating to 0.3-0.4 million plates/m for the 2C(18)DAB coatings. The stability of the coating increased with increasing hydrophobicity of the surfactant (i.e., increasing chain length and decreasing cmc). Over 60 successive separations were performed on a 2C(18)DAB-coated capillary over 12 days, without any regeneration of the coating. The migration times varied by less than 2.3% over this period with no loss in efficiency.

Semi-permanent surfactant coatings for inorganic anion analysis in capillary electrophoresis

Capillary electrophoretic separations of inorganic anions are performed using a capillary coated with a mixture of the cationic surfactant didodecyldimethylammonium bromide (DDAB) and the zwitterionic surfactant 1,2-dilauroyl- sn-phosphatidylcholine (DLPC). These double-chained surfactants form semi-permanent coatings on the capillary wall, which allows the excess surfactant to be removed from the buffer prior to separation. Interactions between surfactant aggregates in the buffer and analyte anions are thus eliminated. The electroosmotic flow (EOF) can be altered from fully reversed (100% DDAB) to near zero (100% DLPC) using different ratios of DDAB and DLPC. Controlling the EOF allows for improved resolution of the anions while maintaining a rapid, co-EOF separation, free from analyte–surfactant additive interactions.