Size Exclusion and Ion Exchange Chromatographic Hardware Modified with a Hydrophilic Hybrid Surface.

Certain biomolecules have proven to be difficult to analyze by liquid chromatography (LC), especially under certain chromatographic conditions. The separation of proteins in aqueous mobile phases is one such example because there is the potential for both hydrophobic and ionic secondary interactions to occur with chromatographic hardware to the detriment of peak recovery, peak shape, and the overall sensitivity of the LC analysis. To decrease non-specific adsorption and undesired secondary interactions between column hardware and biomolecules, we have developed and applied a new hydrophilically modified hybrid surface (h-HST) for size exclusion chromatography (SEC) and anion exchange (AEX) separations of proteins and nucleic acids. This surface incorporates additional oxygen and carbon atoms onto an ethylene bridge hybrid siloxane polymer. As a result, it exhibits reduced electrostatic properties and hydrophilicity that facilitates challenging aqueous separations. Flow injection tests with a phosphate buffer showed superior protein recovery from an h-HST frit when compared to unmodified ethylene-bridged hybrid HST, titanium, stainless steel, and PEEK frits. When applied to SEC of rituximab, ramucirumab, and trastuzumab emtansine with a 50 mM ammonium acetate buffer, this new hydrophilic chromatographic hardware yielded improved monomer and aggregate recovery, higher plate numbers, and more symmetrical peaks. AEX columns also benefited from h-HST hardware. An acidic mAb (eculizumab) showed improved recovery, more stable retention, and a sharper peak when eluted from an h-HST versus SS column. Moreover, AEX separations of intact mRNA samples (Cas9 and EPO mRNA) were improved, where it was seen that h-HST column hardware provided higher sensitivity and more repeatable peak areas from injection to injection. As such, there is significant potential in the use of h-HST chromatographic hardware to facilitate more robust and more sensitive analyses for a multitude of challenging separations and analytes.

Using Hybrid Organic-Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC.

Interactions of analytes with metal surfaces in high-performance liquid chromatography (HPLC) instruments and columns have been reported to cause deleterious effects ranging from peak tailing to a complete loss of the analyte signal. These effects are due to the adsorption of certain analytes on the metal oxide layer on the surface of the metal components. We have developed a novel surface modification technology and applied it to the metal components in ultra-HPLC (UHPLC) instruments and columns to mitigate these interactions. A hybrid organic-inorganic surface, based on an ethylene-bridged siloxane chemistry, was developed for use with reversed-phase and hydrophilic interaction chromatography. We have characterized the performance of UHPLC instruments and columns that incorporate this surface technology and compared the results with those obtained using their conventional counterparts. We demonstrate improved performance when using the hybrid surface technology for separations of nucleotides, a phosphopeptide, and an oligonucleotide. The hybrid surface technology was found to result in higher and more consistent analyte peak areas and improved peak shape, particularly when using low analyte mass loads and acidic mobile phases. Reduced abundances of iron adducts in the mass spectrum of a peptide were also observed when using UHPLC systems and columns that incorporate hybrid surface technology. These results suggest that this technology will be particularly beneficial in UHPLC/mass spectrometry investigations of metal-sensitive analytes.

Highly efficient capillary columns packed with superficially porous particles via sequential column packing.

Highly efficient capillary columns packed with superficially porous particles were created for use in ultrahigh pressure liquid chromatography. Superficially porous particles around 1.5µm in diameter were packed into fused silica capillary columns with 30, 50, and 75µm internal diameters. To create the columns, several capillary columns were serially packed from the same slurry, with packing progress plots being generated to follow the packing of each column. Characterization of these columns using hydroquinone yielded calculated minimum reduced plate heights as low as 1.24 for the most efficient 30µm internal diameter column, corresponding to over 500,000plates/m. At least one highly efficient column (minimum reduced plate height less than 2) was created for all three of the investigated column inner diameters, with the smallest diameter columns having the highest efficiency. This study proves that highly efficient capillary columns can be created using superficially porous particles and shows the efficiency potential of these particles.

Chromatographic evidence of silyl ether formation (SEF) in supercritical fluid chromatography.

In this article, we propose that silyl ether formation (SEF) is a major contribution to retention and selectivity variation over time for supercritical fluid chromatography (SFC). In the past, the variations were attributed to instrumentation, but high performance SFC systems have shed new light on the source of variation. As silyl ethers form on the particle surface, the hydrophilicity is decreased, significantly altering the retention and selectivity observed. SEF is expected to occur with any chromatographic particle containing silanols but is slowed on hybrid inorganic/organic particles. The SEF reaction is between alcohols on the particle surface and in the mobile phase solvent. We have found that storage conditions of a column are paramount, which can either prevent or accelerate the process. Because SEF exists as an equilibrium between the liquid phase and the particle surface, the process is also reversible. The silanols can be hydroxylated (regenerated) to their original state upon exposure to water. The next generation of stationary phases will either advantageously utilize SEF or effectively mitigate its effects. Mitigation of SEF would be a significant improvement in SFC that has the potential to vault their performance to levels of similar reproducibility and reliability observed for high performance liquid chromatography (HPLC). Further research in SEF may lead to a better understanding of the mechanism of interaction between the solutes and chromatographic surface.

Characterization and evaluation of C18 HPLC stationary phases based on ethyl-bridged hybrid organic/inorganic particles.

The characterization and evaluation of three novel 5-microm HPLC column packings, prepared using ethyl-bridged hybrid organic/inorganic materials, is described. These highly spherical hybrid particles, which vary in specific surface area (140, 187, and 270 m(2)/g) and average pore diameter (185, 148, and 108 A), were characterized by elemental analysis, SEM, and nitrogen sorption analysis and were chemically modified in a two-step process using octadecyltrichlorosilane and trimethylchlorosilane. The resultant bonded materials had an octadecyl surface concentration of 3.17-3.35 micromol/m(2), which is comparable to the coverage obtained for an identically bonded silica particle (3.44 micromol/m(2)) that had a surface area of 344 m(2)/g. These hybrid materials were shown to have sufficient mechanical strength under conditions normally employed for traditional reversed-phase HPLC applications, using a high-pressure column flow test. The chromatographic properties of the C(18) bonded hybrid phases were compared to a C(18) bonded silica using a variety of neutral and basic analytes under the same mobile-phase conditions. The hybrid phases exhibited similar selectivity to the silica-based column, yet had improved peak tailing factors for the basic analytes. Column retentivity increased with increasing particle surface area. Elevated pH aging studies of these hybrid materials showed dramatic improvement in chemical stability for both bonded and unbonded hybrid materials compared to the C(18) bonded silica phase, as determined by monitoring the loss in column efficiency through 140-h exposure to a pH 10 triethylamine mobile phase at 50 degrees C.