Assessment of the reproducibility of a C18 bonded phase based on ethylene-bridged hybrid organic/inorganic particles.

The batch-to-batch reproducibility of an endcapped trifunctional C18 bonded phase based on ethylene-bridged hybrid particles was assessed using a modified version of a chromatographic test developed by Neue and coworkers. The test involves the isocratic separation of six compounds chosen to probe different characteristics of the stationary phase, including hydrophobicity, hydrogen bonding and cation-exchange. The assessment was based on results for a total of 471 batches manufactured and tested over a 19 year time span. The results were compared to those for an endcapped monofunctional C18 bonded phase on silica particles, based on results generated for 246 batches over 29 years. Overall, both stationary phases show similar reproducibility, with relative standard deviations for the relative retentions ranging from 0.1 to 3.2 %.

Retention and mass transfer properties of the series of unbonded, amide-bonded, and alkylsulfobetaine-bonded ethylene bridged hybrid hydrophilic interaction liquid chromatography columns.

This work investigates the link between the retentivity and the stationary phase to mobile phase mass transfer resistance of hydrophilic interaction liquid chromatography (HILIC) columns packed with the same base ethylene-bridged hybrid particles (BEH). The retention volumes, the plate heights, and the volume of the adsorbed water layer were measured for the ACQUITYTM UPLCTM BEHTM 130 Å HILIC Column (unbonded BEH), ACQUITY UPLC BEH 130 Å Amide Column (amide group attached), and AtlantisTM Premier BEH 95 Å Z-HILIC (zwitterionic group attached) Column. The method of Guo (toluene retention volumes in pure acetonitrile and in the HILIC eluent) was validated from the UNIFAC group-contribution method and applied to measure accurately the water layer volumes in these columns. A strong correlation was found between the retention volumes of most neutral polar analytes and the volume of the water layer adsorbed in the HILIC column. The fraction of the pore volume occupied by the water layer increases significantly from the BEH HILIC Column to the BEH Amide Column, and to the BEH Z-HILIC Column. This is explained by the water solvation of the attached ligands in the pore volume of the BEH Particles and to the smaller average mesopore size of the BEH Z-HILIC Particles. A second and strong correlation is also observed between the water content in the HILIC particle and the stationary phase to mobile phase mass transfer resistance of the HILIC columns at high mobile phase linear velocities. The measured intra-particle diffusivity normalized to the bulk diffusion coefficient decreased from 0.33 (BEH HILIC Column) to 0.10 (BEH Amide Column) and to only 0.03 (BEH Z-HILIC Column) for comparable retention of cytosine. These results are fully consistent with the higher viscosity of the internal eluent (higher water content) and higher internal obstruction for diffusion (smaller mesopores and internal porosity) in the BEH Z-HILIC Particles. Still, in gradient elution mode, the peak capacity was found to be 18% higher for the BEH Z-HILIC Column than that on the BEH Amide Column because the retention factors at elution were smaller when maintaining the same analysis time and starting eluent composition.

Application of a hybrid zwitterionic hydrophilic interaction liquid chromatography column in metabolic profiling studies.

Metabolic phenotyping studies using mouse liver extracts as a model, performed on a novel zwitterionic HILIC UHPLC column, which is based on ethylene-bridged hybrid organic/inorganic particles bonded with sulfobetaine groups and packed into column hardware modified with hybrid surface technology are reported. Initially the chromatographic performance was evaluated under different mobile phase conditions using selected metabolite standards. Following optimization of the chromatographic conditions for 88 hydrophilic metabolites both targeted and untargeted profiling analyses were performed on tissue extracts using LC-MS/MS and LC-TOF/MS, respectively. Chromatographic efficiency parameters such as peak resolution, peak shapes, selectivity and precision in retention and peak areas as well as characteristics that are critical for metabolic profiling analysis such as metabolite coverage and retention time distribution were assessed. The hybrid zwitterionic column exhibited efficient chromatographic separations providing analysis of ca 80 hydrophilic metabolites from different chemical classes and polarities. Utilizing a one-dimensional separation both targeted and untargeted profiling provided comprehensive metabolic signatures that enabled the acquisition of the metabolic phenotypes of the tissue extracts.

Contribution of ionic interactions to stationary phase selectivity in hydrophilic interaction chromatography.

We compared the separation selectivities of 19 different hydrophilic interaction chromatography columns. The stationary phases included underivatized silica and hybrid particles, cyano-bonded silica, materials with neutral ligands such as amide, diol, pentahydroxy, and urea, zwitterionic sorbents, and mixed-mode materials with amine functionalities. A set of 77 small molecules was used to evaluate the columns. We visualized the retention behavior of the different columns using retention time correlation plots. The analytes were classified as cations, anions, or neutral based on their estimated charge under the separation conditions. This involved adjusting the dissociation constants of the analytes for the acetonitrile content of the mobile phase and experimentally determining the pH of the mobile phase containing 70% acetonitrile. The retention correlation plots show that the selectivity differences strongly depended on ionic interactions. Comparisons of the neutral stationary phases (e.g., diol vs. amide) showed more similar selectivity than did comparisons of neutral columns versus columns with cation or anion exchange activity (bare silica or amine columns, respectively). The zwitterionic columns did not behave as perfectly neutral. The correlation plots indicated that they exhibited either cation or anion exchange activity, although to a lesser degree than the silica and amine-containing stationary phases.

Characterization of a highly stable zwitterionic hydrophilic interaction chromatography stationary phase based on hybrid organic-inorganic particles.

We have characterized a sulfobetaine stationary phase based on 1.7 µm ethylene-bridged hybrid organic-inorganic particles, which is intended for use in hydrophilic interaction chromatography. The efficiency of a column packed with this material was determined as a function of flow rate, demonstrating a minimum reduced plate height of 2.4. The batch-to-batch reproducibility was assessed using the separation of a mixture of acids, bases, and neutrals. We compared the retention and selectivity of the hybrid sulfobetaine stationary phase to that of several benchmark materials. The hybrid sulfobetaine material gave strong retention for polar neutrals and high selectivity for methyl groups, hydroxy groups, and configurational isomers. Large differences in cation and anion retention were observed among the columns. We characterized the acid and base stability of the hybrid sulfobetaine stationary phase, using accelerated tests at pH 1.3 and 11.0, both at 70°C. The results support a recommended pH range of 2-10. We also investigated the performance of columns packed with this material for metal-sensitive analytes, comparing conventional stainless steel column hardware to hardware that incorporates hybrid surface technology to mitigate interactions with metal surfaces. Compared to the conventional columns, the hybrid surface technology columns showed a greatly improved peak shape.

Electrospray ionization mass spectrometry ion suppression/enhancement caused by column bleed for three mixed-mode reversed-phase/anion-exchange high-performance liquid chromatography columns.

RATIONALE: Mixed-mode reversed-phase/anion exchange liquid chromatography is useful for separations of mixtures containing anions (e.g. ionized acids). However, when using this form of liquid chromatography with mass spectrometry detection, the bleed of amine-containing hydrolysis products from the columns may cause ion suppression or enhancement. METHODS: Using electrospray ionization tandem quadrupole mass spectrometry detection, we determined the ion suppression or enhancement caused by column bleed for three mixed-mode reversed-phase/weak anion-exchange columns containing stationary phases that differ in chemical structure. Two of the stationary phases are based on silica particles, while the third uses ethylene-bridged hybrid organic/inorganic particles, which have improved hydrolytic stability. Mixtures of acidic and basic analytes were combined with the chromatography flow postcolumn, both with and without a column, and their mass spectrometry ion signal responses (peak areas) were determined. The ratio of signal response with and without a column is the matrix factor. Positive ion electrospray measurements were carried out using 0.1% formic acid (pH ~ 2.7) as a mobile phase additive, and 10mM ammonium formate (pH ~ 6.4) was used for negative ion electrospray detection. RESULTS: The matrix factors under both positive and negative ionization modes were closest to 1 (0.74-1.16) for the hybrid particle-based columns, showing minimal ion suppression or enhancement. In contrast, the silica-based columns gave matrix factors ranging from 0.04 to 1.86, indicating high levels of ion suppression or enhancement. These results may be explained by the differences in the structures of the stationary phases, which affect the relative amounts of hydrolysis products that elute from the columns. CONCLUSIONS: The low levels of mass spectrometry ion suppression or enhancement caused by column bleed from the hybrid particle-based columns should allow for accurate quantitative mass spectrometric detection combined with mixed-mode reversed-phase/weak anion-exchange chromatography.

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.

Characterization of a highly stable mixed-mode reversed-phase/weak anion-exchange stationary phase based on hybrid organic/inorganic particles.

We have characterized Atlantis ethylene-bridged hybrid C18 anion-exchange, a mixed-mode reversed-phase/weak anion-exchange stationary phase designed to give greater retention for anions (e.g., ionized acids) compared to conventional reversed-phase materials. The retention and selectivity of this stationary phase were compared to that of three benchmark materials, using a mixture of six polar compounds that includes an acid, two bases, and three neutrals. The compatibility of the ethylene-bridged hybrid C18 anion-exchange material with 100% aqueous mobile phases was also evaluated. We investigated the batch-to-batch reproducibility of the ethylene-bridged hybrid C18 anion-exchange stationary phase for 27 batches across three different particle sizes (1.7, 2.5, and 5 µm) and found it to be comparable to that of one of the most reproducible C18 stationary phases. We also characterized the acid and base stability of the ethylene-bridged hybrid C18 anion-exchange stationary phase and the results show it to be usable over a wide pH range, from 2 to 10. The extended upper pH limit relative to silica-based reversed-phase/weak anion-exchange materials is enabled by the use of ethylene-bridged hybrid organic/inorganic particles. The improved base stability allows Atlantis ethylene-bridged hybrid C18 anion-exchange to be used with a wider range of mobile phase pH values, opening up a greater range of selectivity options.

Evaluating MISER chromatography as a tool for characterizing HILIC column equilibration.

Hydrophilic Interaction Liquid Chromatography (HILIC) is a technique for retaining polar analytes that uses polar stationary phases and acetonitrile-rich mobile phases. While this technique has several advantages over reversed-phase liquid chromatography (RPLC), one main drawback is the reported need for longer column equilibration. The reason for this is not fully understood and is a topic of current investigation. In order to better understand and reduce the equilibration needs, accurate characterization of column equilibration under varying conditions is required. The current method of characterizing HILIC column equilibration produces limited data points per test, or low time resolution, and is highly dependent on the column and probe compounds being used. There is a need for an improved method for characterizing HILIC column equilibration, especially if trends across stationary phases are to be observed. In this work, MISER, or Multiple Injections in a Single Experimental Run, is evaluated as a possible tool for characterizing HILIC column equilibration. MISER improves time resolution by allowing for replicate injections without interruption of data collection, enabling a more thorough evaluation of column equilibration compared to traditional techniques. Experimental results gathered using MISER show that equilibration of a BEH Amide column is notably shorter when equilibrating from acetonitrile to mobile phases containing higher percentages of water. Column equilibration to a 10% aqueous mobile phase was found to be approximately 5-fold faster than equilibration to a 3% aqueous mobile phase.

Retention loss of reversed-phase chromatographic columns using 100% aqueous mobile phases from fundamental insights to best practice.

This work deals with experimental investigations pertaining to the impact of chemical (electrolyte concentration from 0 to 100 mM, dissolved nitrogen gas from 0 to 6.7  ×  10-4 M in water; surface chemistry including hexylphenyl, polyphenyl, C30, C18, and C8; surface coverage in C18-bonded chains from 1.5 to 3.5 µmol/m2; presence of surface dopant), physical (hydrostatic pressure of water from 50 to 500 bar; temperature from 27 ∘C to 75 ∘C), and structural parameters (average pore size from 50 Å to 400 Å; pore connectivity) on the dewetting kinetics of water from the hydrophobic mesopores of particles packed in RPLC columns. The results are explained from physico-chemical viewpoints involving intrusion and extrusion Laplace pressures, advancing and receding contact angles, surface tension of water, vapor pressure of water, 3D reconstruction of the actual mesoporous structure, pore connectivity, and the hysteresis in nitrogen adsorption and desorption isotherm onto reversed-phase chromatographic materials. A model of water dewetting consistent with the observations and the physical interpretations is then proposed. Finally, the most relevant practical solutions (pressurizing the column in absence of flow, pore size enlargement, using phenyl-bonded phase, polar embedded or surface doped C18-bonded phases, reducing the C18 surface coverage, doping the silica surface, lengthening of the alkyl-bonded chains, applying low temperatures, purging and degassing the mobile phase with helium gas) are suggested in order to eliminate or at least minimize the retention loss of RPLC columns when using fully aqueous mobile phases.

Kinetic mechanism of water dewetting from hydrophobic stationary phases utilized in liquid chromatography.

An experimental protocol was designed to accurately measure the dewetting kinetics of aqueous mobile phases from reversed-phase liquid chromatography (RPLC) columns. The protocol enables the determination of the losses in the wetted surface area and internal pore volume (leading to undesirable retention losses) of RPLC columns as a function of the dewetting time. It is used to evaluate the impact of the buffer/salt concentration in water (0-100 mM), nitrogen concentration dissolved in water (0-6.7 × 10-4 M), column temperature (300-358 K), and of the internal structure (pore connectivity) of the stationary phase on the dewetting kinetics of various RPLC packing materials. From a fundamental viewpoint, the experimental facts demonstrate that dewetting kinetics are not solely driven by the pore size of the stationary phase and the contact angle with water. Temperature has a major influence on dewetting kinetics as it controls the nucleation rate of isolated water vapor bubbles over the entire mesoporous network. Additionally, the internal microstructure of the stationary phase (characterized by its internal porosity, pore size distribution, and pore connectivity) influences the rate at which the water vapor bubbles grow and coalesce in the entire particle volume. From a more practical viewpoint, the retention loss of RPLC columns due to water dewetting can be eliminated or at least minimized by (1) adjusting the surface and bonding chemistries to reduce the receding contact angle, (2) elevating the column outlet pressure, (3) operating at the lowest possible temperature, (4) minimizing the pore connectivity of the stationary phase (e.g., by increasing the degree of surface functionalization from C8 to C18-bonded phases), and (5) by degassing the aqueous mobile phase from any dissolved gases.

Mechanism of retention loss when C8 and C18 HPLC columns are used with highly aqueous mobile phases.

We describe investigations into the cause of retention losses encountered when C8 and C18 HPLC columns are used with highly aqueous (> 90% water) mobile phases. A procedure for quantifying these losses is described, involving stopping and restarting the flow. This procedure was used to study the dependence of retention loss on the pore size, surface concentration, and chemical structure of the bonded phase. Experiments were also carried out to determine how to restore the original retention of the columns by changing the composition of the mobile phase, or by increasing the pressure applied to the column. The results are shown to be consistent with a mechanism based on the theory of pore filling by non-wetting liquids, as employed in Mercury Porosimetry. The retention losses are attributed to the highly aqueous mobile phase being forced out of the pores when the flow is stopped and the pressure released. Retention is lost because the mobile phase is no longer in contact with the interior surface of the particles, where most of the surface area is located. The implications of this phenomenon for maximizing the reversed phase retention of polar analytes are discussed.

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.