Mary J. Wirth

Polyacrylamide brush layer for hydrophilic interaction liquid chromatography of intact glycoproteins

A chromatographic column of nonporous silica particles with a bonded phase of linear polyacrylamide chains is evaluated for hydrophilic interaction liquid chromatography (HILIC) of intact glycoproteins. The column is shown to retain glycoproteins significantly more strongly than non-glycoproteins. A particle diameter of 700nm gives two-fold higher resolution than does a 1.4μm particle diameter, and the column efficiency is found to be mostly limited by packing heterogeneity. LCMS is able to resolve the five glycoforms of ribonuclease B and give high quality mass spectra, but there is loss of resolution of the isomers of glycoforms due to the lower amount of TFA. Compared to two leading commercial HILIC columns operated at 60°C, the polyacrylamide column operated at 30°C provided at least two-fold higher resolution for intact ribonuclease B, and showed peaks for glycoforms of prostate specific antigen, although not resolved.

Trajectory of isoelectric focusing from gels to capillaries to immobilized gradients in capillaries

This review presents the need for replacing gels in 2D separations for proteomics, where speed, high‐throughput, and the ability to characterize trace level proteins or small samples are the current desires. The theme of the review is isoelectric focusing, which is a valuable tool because it pre‐concentrates proteins in addition to separating with high peak capacity. The review traces the technological progress from gel IEF to CIEF to packed capillaries with immobilized gradients for CIEF. Multiple capillary techniques are progressing toward meeting the current desires, providing extremely high sensitivity with regard to concentration and to small samples, integrated automation, and high peak capacity from multiple dimensions of separation. Capillaries with immobilized pH gradients for CIEF are emerging, which will alleviate interference from ampholytes and improve reproducibility in separation times when this valuable technique can be used as one of the dimensions.

Protein UTLC-MALDI-MS using thin films of submicrometer silica particles.

Slides for ultra thin-layer chromatography (UTLC) were made by coating nonporous silica particles, chemically modified with polyacrylamide, as 15 μm films on glass or silicon. Three proteins, myoglobin, cytochrome c and lysozyme, are nearly baseline resolved by the mechanism of hydrophilic interaction chromatography. A plate height as low as 3 μm, with 3900 plates, is observed in 14 mm. Varying silica particle diameter among 900, 700 and 350 nm showed that decreasing particle diameter slightly improves resolution but slows the separation. Matrix-assisted laser desorption/ionization (MALDI)-MS of the proteins after separation is demonstrated by wicking sufficient sinapinic acid into the separation medium.

Insights from theory and experiments on slip flow in chromatography

Slip flow has become a topic of interest in reversed-phase liquid chromatography because it gives a flow enhancement that facilitates the use of submicrometer particles, providing a large improvement in separation efficiency. Moreover, slip flow provides an additional improvement in efficiency by reducing the velocity distribution in the mobile phase. The phenomenon of slip flow in open tubes is described in chromatographically relative terms. A recent paper in this journal is discussed, as it provides the first theoretical study of slip flow in packed beds, in this case for face-centered cubic geometry. The theory paper reveals that the presence of the packed bed introduces a heterogeneity in fluid velocities that is absent in open tubes, reducing the additional improvement in efficiency from slip flow. The recent paper also suggests that there is yet another factor improving efficiency, which is size-exclusion of proteins from regions of stagnant flow. The latter is supported by recently published data on restricted protein diffusion in face-centered cubic silica colloidal crystals. Extremely low plate heights are enabled by use of submicrometer particles, and further improvement appears to be possible when the analyte size is on the order of 1% of the particle diameter or larger.

Modeling of protein electrophoresis in silica colloidal crystals having brush layers of polyacrylamide

Sieving of proteins in silica colloidal crystals of millimeter dimensions is characterized for particle diameters of nominally 350 and 500 nm, where the colloidal crystals are chemically modified with a brush layer of polyacrylamide. A model is developed that relates the reduced electrophoretic mobility to the experimentally measurable porosity. The model fits the data with no adjustable parameters for the case of silica colloidal crystals packed in capillaries, for which independent measurements of the pore radii were made from flow data. The model also fits the data for electrophoresis in a highly ordered colloidal crystal formed in a channel, where the unknown pore radius was used as a fitting parameter. Plate heights as small as 0.4 μm point to the potential for miniaturized separations. Band broadening increases as the pore radius approaches the protein radius, indicating that the main contribution to broadening is the spatial heterogeneity of the pore radius. The results quantitatively support the notion that sieving occurs for proteins in silica colloidal crystals, and facilitate design of new separations that would benefit from miniaturization.

Annealing of silica to reduce the concentration of isolated silanols and peak tailing in reverse phase liquid chromatography

Non-porous, colloidal silica particles were annealed at three different temperatures, 800, 900 and 1050 °C. The adsorption of lysozyme, a probe of surface roughness, was consistent with progressively reduced surface roughness as temperature increased. The heat treated silica particles were rehydroxylated and then used to pack UHPLC columns. The cationic protein lysozyme was used to probe silanol activity, which exhibited progressively less tailing as the annealing temperature increased. FTIR spectroscopy confirmed that the abundance of isolated silanols on the surface was reduced by annealing at 900 °C or 1050 °C. FTIR also revealed that there was markedly increased hydrogen bonding of the isolated silanols to neighbors after rehydroxylation. These results combine to support the hypothesis that (a) isolated silanols on silica cause tailing in RP-LC and (b) nonplanar topography gives rise to isolated silanols.

Silica Colloidal Crystals as Emerging Materials for High-Throughput Protein Electrophoresis

Silica colloidal crystals are a new type of media for protein electrophoresis, and they are assessed for their promise in rapidly measuring aggregation of monoclonal antibodies. The nature of silica colloidal crystals is described in the context of the need for a high-throughput separation tool for optimizing the formulations of protein drugs for minimal aggregation. The fundamental relations between molecular weight and mobility in electrophoresis are used to make a theoretical comparison of selectivity between gels and colloidal crystals. The results show that the selectivity is similar for these media, but slightly higher, 10%, for gels, and the velocity is inherently lower than that for gels due to the smaller free volume fraction. These factors are more than compensated for by lower broadening in colloidal crystals. These new media give plate heights of only 0.15xa0μm for the antibody monomer and 0.42xa0μm for the antibody dimer. The monoclonal antibody is separated from its dimer in 72xa0s over a distance of only 6.5xa0mm. This is five times faster than size-exclusion chromatography, with more than tenfold miniaturization, and amenable to parallel separations, all of which are promising for the design of high-throughput devices for optimizing protein drug formulations.

Alleviating nonlinear behavior of disulfide isoforms in the reversed-phase liquid chromatography of IgG2.

Reversed-phase chromatography is an established method for characterizing the disulfide isoforms of IgG2. This work explores the effect of mobile phase gradient profile and sample concentration on the separation of disulfide isoforms. The acidic mobile phase can alter the relative proportions of disulfide isoforms, but only when the level of the reactive A1 isoform is much higher than for typical conditions of separation and typical IgG2 samples. Otherwise, there is minimal disulfide scrambling. A slower gradient and flow rate modestly improve resolution, but the peaks remain heavily overlapped. Resolution is further improved and nonlinear chromatography lessened when injection is performed under non-stacking conditions. Non-stacking conditions also keep the concentration from spiking at the head of the column, reducing noncovalent associations that can promote disulfide scrambling. The higher resolution from non-stacking injection reveals the presence of at least seven species.

In-column bonded phase polymerization for improved packing uniformity

It is difficult to pack chromatographic particles having polymeric-bonded phases because solvents used for making a stable slurry cause the polymer layer to swell. Growth of the polymer inside the column (in situ) after packing was investigated and compared with conventional, ex situ polymer growth. The method of activators generated by electron transfer, along with atom-transfer radical polymerization, enabled polymerization under ambient conditions. Nonporous, 0.62xa0μm silica particles with silane initiators were used. Polyacrylamide films with a hydrated thickness of 23xa0nm in 75:25 water/isopropanol grew in 55xa0min for both in situ and ex situ preparations, and the same carbon coverage was observed. Higher chromatographic resolution and better column-to-column reproducibility were observed for in situ polymer growth, as evaluated by hydrophilic interaction liquid chromatography for the model glycoprotein, ribonuclease B. In situ polymer growth was also found to give lower eddy diffusion, as shown by a narrower peak width for injected acetonitrile in 50:50 acetonitrile/water. When columns were packed more loosely, bed collapse occurred quickly for ex situ, but not for in situ, polymer growth. The higher resolution and stability for in situ polymer growth is explained by packing with hard, rather than soft, contacts between particles.

Chromatographic efficiency and selectivity in top-down proteomics of histones

Histones are involved in epigenetic control of a wide variety of cellular processes through their multiple post-translational modifications. Their strongly cationic nature makes them challenging to separate with reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS), where trifluoroacetic acid is avoided due to adduct formation. Columns with higher resolution are needed. In this work, RPLC-MS is performed on a histone sample using difluoroacetic acid and a 20-min gradient. Columns with C18 surfaces are compared for two different types of particle morphologies: 1) fully porous particles of 5μm in diameter, 2) superficially porous particles of 3μm in diameter with a shell of 0.2μm. The resolution for the histone separation is better for the latter column, but only when the modifier is trifluoroacetic acid, which is used with UV absorbance detection. When difluoroacetic acid is used for LCMS, the peaks broaden enough to erase the advantage in efficiency for the superficially porous particles. The fully porous and superficially porous cases show similar performance in RPLC-MS, with slightly higher resolution for the fully porous particles. The expected advantage of the shorter diffusion distances for the superficially porous particles is shown to be outweighed by the lower selectivity of its bonded phase.