In CE practice, conditioning of the capillary tube with strong base, acid, or both in sequence, has been recognized as essential to obtain reasonable precision in electroosmotic mobility (EOM), and consequently, in the migration times of analytes. This report focuses on the comparative study of three different approaches for capillary conditioning: etching with HF, etching with NaOH, and leaching with HCl after NaOH treatment. EOM-based measurements, including the hysteresis effect that characterizes the dependence of EOM with pH, were used to evaluate the conditioned silica surface. Additionally, indirect inspection of the conditioned surface was carried out by examining the electrophoretic profile of the cationic probe Ru(bpy)(3) (2+), known to strongly interact with the ionized silica surface while displaying no significant acid-base activity. It was shown that, once conditioned and prior to any CE measurement, extensive rinse of the capillary with the running electrolyte at high flow rate was essential to attain relatively rapid re-equilibration of the tube with the fluid. Insufficient rinse with the running electrolyte would result in negatively biased EOM measurements and significant drift in migration times. It was also established that relatively high flow rates of 1 M NaOH conditioning solution (4-5 column volumes per minute) was required to attain capillaries with reproducible electrophoretic performance within a reasonable conditioning time (typically, 1 h). In addition to relatively more extensive rehydroxylation of the silica surface, evidenced by the highest EOM values obtained, the sequential use of NaOH-etching and HCl-leaching provided better precision than HF-etching or NaOH-etching alone.

Two synthetic schemes to produce a hydride-modified support that serves as an intermediate for the preparation of bonded phases for liquid chromatography (LC) and capillary electrophoresis (CE) are investigated. The strategies differ in the silane reagent utilized (trichlorosilane (TCS) or triethoxysilane (TES)) and the manner water is incorporated into the reaction. In the first approach, TCS in toluene reacts with a previously humidified silica substrate so that the reaction is confined to the silica surface. In the second approach, TES and a small amount of aqueous HCl are dissolved in THF, and this hydrolysate is diluted by a great factor in cyclohexane, prior to reaction with the silica substrate. Atomic force microscopy (AFM) images of the hydride film on wafers revealed that, unlike the traditional approach that produced a patchy coating, both new methods provided a homogeneous layer on the substrate's surface. IR and NMR spectra from porous silica particles clearly confirmed a successful surface modification. AFM and water contact angles (WCA) were used to examine the effect of dilution of the TES hydrolysate in cyclohexane on the trend of the film to polymerize on wafers and found that a dilution factor of at least 100 is required to attain a molecularly thin hydride layer. WCA and CE also revealed a strong susceptibility of the hydride silica intermediate to hydrolyze, even at low pH. Compared to TCS, the lower reactivity and volatility of TES resulted in a much more desirable experimental approach.

Analyte recovery is an important figure to assess protein adsorption on fused-silica capillaries. In 1991 Regnier and coworkers estimated recovery by assuming the loss of analyte from adsorption and thus the decrease in peak area measured by two detectors to be proportional to the length of the capillary section between them. In this report we closely examine this concept and its adaptation to commercial CE instruments to determine protein recovery. We hypothesize that, once a steady-state migration is reached, protein adsorption is a first order process with respect to protein concentration and surface density of adsorbing sites. This hypothesis is shown to be valid over a reasonably wide range of capillary effective length and, as a result, protein recovery decreases exponentially with the migrated distance. However, unlike the traditional recovery figure obtained through a conventional spike process, protein recovery measured by this approach does not have the same merit since it is strongly dependent from capillary dimensions and applied electric field. Nevertheless, protein recovery and the slope of the logarithmic protein peak area vs. length plot are useful figures to compare protein adsorption on different capillary surfaces. Several literature reports dealing with the application of Regnier concept to calculate protein recovery are discussed.

A novel 3-hydroxypropyl (propanol) bonded silica phase has been prepared by hydrosilylation of allyl alcohol on a hydride silica intermediate, in the presence of platinum (0)-divinyltetramethyldisiloxane (Karstedt's catalyst). The regio-selectivity of this synthetic approach had been correctly predicted by previous reports involving octakis(dimethylsiloxy)octasilsesquioxane (Q8M8H) and hydrogen silsesquioxane (T8H8), as molecular analogs of hydride amorphous silica. Thus, C-silylation predominated (~ 94%) over O-silylation, and high surface coverages of propanol groups (5±1 µmol/m2) were typically obtained in this work. The propanol-bonded phase was characterized by spectroscopic (IR and solid state NMR on silica microparticles), contact angle (on fused-silica wafers) and CE (on fused-silica tubes) techniques. CE studies of the migration behavior of pyridine, caffeine, tris(2,2’-bipyridine)Ru(II) chloride and lysozyme on propanol-modified capillaries were carried out. The adsorption properties of these select silanol-sensitive solutes were compared to those on the unmodified and hydride-modified tubes. It was found that hydrolysis of the SiH species underlying the immobilized propanol moieties leads mainly to strong ion-exchange based interactions with the basic solutes at pH 4, particularly with lysozyme. Interestingly, and in agreement with water contact angle and electroosmotic mobility figures, the silanol-probe interactions on the buffer-exposed (hydrolyzed) hydride surface are quite different from those of the original unmodified tube.

In the present study, porous silica particles as well as impervious fused-silica wafers and capillary tubes were modified with hydrophilic polymers (hydroxylated polyacrylamides and polyacrylates), using a surface-confined grafting procedure based on atom transfer radical polymerization (ATRP) which was also surface-initiated from α-bromoisobutyryl groups. Initiator immobilization was achieved by hydrosilylation of allyl alcohol on hydride silica followed by esterification of the resulting propanol-bonded surface with α-bromoisobutyryl bromide. Elemental analysis, IR and NMR spectroscopies on silica micro-particles, atomic force microscopy, ellipsometry and profilometry on fused-silica wafers, as well as CE on fused-silica tubes were used to characterize the chemically modified silica substrate at different stages. We studied the effect of monomer concentration as well as cross-linker on the ability of the polymer film to reduce electroosmosis and to prevent protein adsorption (i. e., its non-fouling capabilities) and found that the former was rather insensitive to both parameters. Surface deactivation towards adsorption was somewhat more susceptible to monomer concentration and appeared also to be favored by a low concentration of the cross-linker. The results show that hydrophilic polyacrylamide and polyacrylate coatings of controlled thickness can be prepared by ATRP under very mild polymerization conditions (aqueous solvent, room temperature and short reaction times) and that the coated capillary tubes exhibit high efficiencies for protein separations (0.3–0.6 million theoretical plates per meter) as well as long-term hydrolytic stability under the inherently harsh conditions of capillary isoelectric focusing. Additionally, there was no adsorption of lysozyme on the coated surface as indicated by a complete recovery of the basic enzyme. Furthermore, since polymerization is confined to the inner capillary surface, simple precautions (e.g., solution filtration) during the surface modification process are sufficient to prevent capillary clogging.