Reversed phase HPLC with high temperature ethanol/water mobile phases as a green alternative method for the estimation of octanol/water partition coefficients.

High temperature ethanol/water was explored as a green eluent in the reversed-phase liquid chromatographic approximation of pure water retention (log kw) and subsequent estimation of the octanol/water partition coefficient (log P) via the Collander equation and the Leave-One-Out method. As part of this work, linear solvation energy relationships were employed to compare the log kw extrapolated systems based on high temperature ethanol/water, ambient acetonitrile/water, and ambient methanol/water mobile phases. Based on the comparisons of the three organic modifiers, high temperature ethanol/water mobile phases were observed to provide the best estimation of log P. This conclusion is based on a high log P correlation of 0.968 R2 and a near unity cos θ value of 0.997 between LSER coefficient vectors of ethanol/water estimated log P and octanol/water log P systems. The method employed in this work, further, provided high correlation for the hydrogen-bonding basicity term between the two systems.

Characterization of the polarity of subcritical water.

The polarity of subcritical water was studied solvatochromically with betaine dye (33) across a temperature range of 30°C-180°C and a pressure range of 13.8bar (200psi) to 124bar (1800psi). It was observed that temperature has a greater effect than pressure on the polarity of subcritical water. In addition, subcritical water was compared with traditional hydro-organic mobile phases and the polarity of subcritical water was found to be comparable to a range of 30%-50% methanol/water and a range of 20%-30% acetonitrile/water mobile phases. It was concluded that subcritical water is more suited to separations involving slightly polar and polar compounds than more nonpolar analytes.

Quantifying injection solvent effects in reversed-phase liquid chromatography.

Peak distortion due to the injection was measured as a function of injection solvent strength, volume, mass, retention factor, and column selectivity. The concept of a method's sensitivity (s) to injection solvent strength was mathematically defined as a vector of theoretical plate counts compared to an ideal vector that does not change with injection solvent strength. Near ideal sensitivity (s>0.90) was measured on all columns with all analytes in low volume injections of 1.25 µL. Increasing the injection volume reduces the measured sensitivity from ideality to a greater extent than increasing the injection mass, with differing values for each column. Using column parameters measured from the hydrophobic-subtraction model and fitting parameters from the acetonitrile excess adsorption isotherm, differences among the columns studied are explained. Decreased ligand density and increased silanol activity provide a consistent peak shape with changes in injection volume or solvent strength. For method development, a quick test is suggested with the ratio of hydrophobic-subtraction column parameters, H/A, to predict the injection solvent sensitivity of a column. As H/A decreases, the sensitivity to injection solvent worsens. Sensitivity to organic modifiers other than acetonitrile are predicted with cited sorbed layer thickness, such that MeOH>EtOH>IPA≈THF≈MeCN, i.e., a strong MeOH diluent is more ideal (better) than a strong MeCN diluent.

Effect of trimethylsilane pre-capping on monomeric C18 stationary phases made from high-purity type-B silica substrates: efficiency, retention, and stability.

Silica-based monomeric C18 stationary phases are the most widely used in reversed-phase liquid chromatography (RPLC), and methods to improve the efficiency and chemical stability of such phases are always being investigated. Previous work showed that stationary phases made by pre-treating the silica surface with small amounts of trimethylsilane (TMS) reagents prior to C18 silanization showed vast improvements in the chromatographic efficiencies of both polar and non-polar analytes. It was concluded that this "pre-capping" step improved efficiency by selectively neutralizing the most reactive highly acidic silanol sites, producing a more energetically homogenous surface prior to exhaustive derivatization that subsequently yielded a more evenly distributed alkyl bonding arrangement. These previous studies were performed on Type-A silica, an older variety of silica gel material that contains higher levels of metal impurities than the Type-B silica used today. The purpose of the work presented here is to investigate the effects of trimethylsilane pre-capping on monomeric C18 stationary phases made from high-purity Type-B silica substrates. The results show little or no efficiency improvements for non-polar compounds, but a more noticeable improvement was observed for some drug compounds and bases under buffered conditions, with the magnitude of the improved efficiencies correlating with metal impurity content and physical parameters of the silica substrate. The results lend supporting evidence that metal impurities are the primary source of highly acidic silanols, but they also suggest a means to improve efficiencies for compounds of interest. Pre-capping also resulted in a decreased retention due to a decrease in carbon loading. In addition, hydrolytic stability of the stationary phases was investigated at high pH and elevated temperature, with results indicating a slight decrease in hydrolytic stability with increased pre-capping.

Reequilibration time of superficially porous silica based columns in gradient elution reversed phase liquid chromatography.

Between repetitive analyses using gradient elution liquid chromatography the column must be reequilibrated to the initial conditions, extending run times. We studied the reequilibration time of three superficially porous silica columns compared to one fully porous silica column on a chromatograph with a reduced flush-out volume. Post-gradient acetone injections made at the interface of the pure organic-highly aqueous phase show anomalous, pressure-related band focusing, and increased retention compared to injections on either side of the interface. These anomalies are explained by applying the Buckley-Leverett theory of oil displacement in sands to column reequilibration. Reequilibration was shown to occur quickly, with less than three column volumes of conditioning solvent, and depends on the reproducibility as required by the application. Offline LC-GC was used to quantitate the percent acetonitrile eluting from each column post-gradient. After an initial, large expulsion of acetonitrile, a steady small amount (∼0.03%) of acetonitrile is detected long after the column is considered equilibrated. The limiting variable with column equilibration is not the desorption of organic modifier from the stationary phase, but rather the pressure required to force the aqueous phase into the pores.

Properties of subcritical water as an eluent for reversed-phase liquid chromatography--disruption of the hydrogen-bond network at elevated temperature and its consequences.

The use of subcritical water as an eluent for reversed-phase liquid chromatography is further explored. Shape selectivity as well as thermodynamic values for solute transfer were measured and compared to those seen with traditional ambient methanol/water and acetonitrile/water mobile phases. Linear solvation energy analysis was also used to analyze extrapolated values of the retention factor in pure water at ambient temperatures (k'w) for subcritical water and ambient hydroorganic mobile phases. Results indicate that it is likely that a large disruption in the hydrogen-bonding network of water at high temperatures causes unique chromatographic selectivity, as well as prohibits accurate extrapolation from high temperature to ambient conditions using pure water. Additionally, subcritical water was not found to be a suitable mobile phase for determining k'w for use in estimating octanol/water partition coefficients.

Retention mechanisms in subcritical water reversed-phase chromatography.

Differences in the properties of subcritical water and conventional water/acetonitrile and water/methanol mobile phases for reversed phase separations are explored. Using van't Hoff plots enthalpies and entropies of transfer are compared among the mobile phases while linear solvation energy relationships are used to quantify contributions to retention based on a solute's polarizability, dipolarity, hydrogen bond donating ability, hydrogen bond accepting ability, and molecular size. Results suggest the presence of acetonitrile or methanol in the mobile phase may decrease dispersive interactions of the solute with the stationary phase compared to subcritical water, thereby lowering enthalpic contributions to retention. Enthalpic contributions are found to drive the retention of a methylene group in all systems studied.

Modeling chromatographic dispersion: a comparison of popular equations.

An analyte that is introduced onto a column as a finite band broadens as it moves along the column. This band-broadening is generally attributed to three independent processes, including flow path inequalities, molecular diffusion, and resistance to mass transfer. Many equations have been derived in attempts to mathematically model the process. Some of the more popular of these include the equations of van Deemter, Giddings, Horvath and Lin, and Knox. Although the basis of each equation is theoretically different, the differences among them are minor, and most of the equations can be used to adequately fit plate height data. Chromatographers often collect efficiency data to monitor the performance of their columns, and then use one of the above equations to fit the data. The choice of which equation to use can be daunting, since the theories are conflicting. Using an extensive collection of data, we have compared these equations on the basis of the resulting fit. This study was performed using analytes covering a wide range of retention values and mobile phases of differing strengths. The Foley-Dorsey equation was used to calculate the number of theoretical plates for the efficiency study and special precautions were taken to ensure that the observed broadening was due to only processes occurring in the column and that the peaks were adequately sampled. The variance from extra-column sources was measured and subtracted from the system variance. Although more complex equations gave very slightly better fits, 50 years after its introduction the van Deemter equation remains an incredibly accurate representation of band-broadening processes.

Linear voltage profiles and flow homogeneity in pressurized planar electrochromatography.

Planar electrochromatography (PEC) is an emerging technique for thin-layer chromatography (TLC) where electroosmosis is the driving force for the solvent, not capillary action. This allows for much faster and constant flow rates in turn yielding increased zone capacities and efficiencies. Instrumental designs have changed greatly over the last few years solving many of the initial instrumentation challenges. We have previously shown that low applied pressure (or no applied pressure) PEC instruments do not give linear voltage drops across the separation path length of a TLC plate, which in turn results in non-stable electroosmotic flow (EOF). By the use of our unique reader electrode grid we have the ability to monitor the potential at eight discrete positions throughout the 10-cm separation path length. We now show that high-pressure PEC instruments, most commonly referred to as pressurized planar electrochromatography (PPEC) do show a linear voltage drop and constant EOF. We compare plate equilibration times of PPEC and low-pressure PEC, use of increased field strengths, as well as sample application designs. In addition, we discuss the use of rhodamine B as a visual marker for reproducible migration and calculation of theoretical plates.

Influence of stationary phase solvation on shape selectivity and retention in reversed-phase liquid chromatography.

In the past few decades, shape selectivity has drawn a great deal of attention from chromatographers. The chemistry and characteristics of bonded stationary phases such as phase type, length of bonded phase, surface coverage, and silica surface material have an effect on the shape selectivity of the columns. Although the effects of bonded phase shape selectivity are relatively well understood, one remaining question is the effect of intercalated solvent on shape selectivity. The intercalation of organic modifier and water molecules into the stationary phase is believed to introduce more rigidity into bonded alkyl chains in RPLC. The use of gas chromatography (GC) opens a new dimension to approach this question. C18 columns 4 cm in length were prepared in our laboratory and used in both LC and GC experiments. Shape selectivity and thermodynamic constants for the transfer of a solute from the mobile phase to the stationary phase have been determined as a function of monomeric octadecyl stationary phase bonding densities over the range of 1.44-3.43 micromol/m2 and a polymeric phase (nominal surface coverage 4.77 micromol/m2). Comparing LC and GC experiments, we observed: (a) similar relationships between shape and phenyl selectivities with monomerically bonded C18 phase densities; (b) different correlation of thermodynamic quantities (DeltaH degrees , DeltaS degrees , and DeltaG degrees ) versus bonded phase densities. The effects of high temperature and residual silanol groups are sources of difficulty in elucidation of the intercalated mobile phase role in selectivity and retention for GC measurements.

Characterization of flow and voltage profiles in planar electrochromatography.

Planar electrochromatography (PEC) is a new technology for thin layer chromatography (TLC) where the separation is driven by electroosmotic forces, not capillary action. This allows for much faster and more efficient chromatography in a planar format. Care needs to be taken when performing these experiments because voltage and flow characteristics can change through a single run, due to buffer gradients, temperature changes (Joule heating) and localized plate heterogeneity. We have designed a PEC instrument and cover grid to allow investigation of flow and voltage characteristics as solvent moves across a TLC plate. Our unique cover grid allows monitoring voltage at eight discrete points between the positive and negative reservoirs. A linear relationship between voltage and distance should be seen, giving a constant voltage drop across a plate, but this did not occur. This non-linear function changes over time, following the plate equilibration. Once a plate is equilibrated, voltage and flow characteristics remain fairly constant. Theoretical calculations support the physical observations. Larger plate widths (5 cm) were also briefly investigated and it is concluded that large width plates could be easily implemented to maintain multiple sample capability.

Column selection for liquid chromatographic estimation of the k'w hydrophobicity parameter.

Newer reversed-phase column technologies that incorporate polar groups either by an endcapping procedure or by embedding them into the stationary phase ligand have been receiving much attention in the literature for their robustness when highly aqueous conditions are used. We investigated their ability to accurately determine the chromatographic hydrophobicity value log k'w. The non-linear deviations of retention data as mobile phase conditions approach zero percent modifier are a large source of error when extrapolating to log k'w values using the linear solvent strength model. Here, we compare a conventional reversed-phase stationary phase with others that have incorporated either polar embedded or polar endcapped phases, along with a hybrid-based particle derivatized with a polar embedded ligand. Our results show that polar endcapped phases perform very similarly to the conventional phase and do not show any improved ability for determining log k'w, but polar embedded phases have reduced curvature in the data, and therefore result in less error in extrapolation. We also investigated the solubility parameter model and the [ET(30)] model for their extrapolation efficiency, and have concluded that the [ET(30)] model shows the least error when extrapolating the data.

Reversed-phase retention thermodynamics of pure-water mobile phases at ambient and elevated temperature.

The use of pure water at superheated temperatures, between 100 and 200 degrees C, as a mobile phase for reversed-phase separations is explored. The thermodynamics of the retention process at low temperature (15-55 degrees C) are compared to the thermodynamics at elevated temperature (125-175 degrees C). Significant differences in the enthalpy of the retention process are observed between the two temperature ranges. This is possibly due to changes in the hydrogen-bond network of the pure-water mobile phase, which would change the solvation, and therefore retention, of non-polar solutes. The change in thermodynamic values between the two temperature regions invalidates extrapolation of retention as a function of temperature between the two temperature regions for the prediction of room-temperature pure-water retention factors. The thermodynamic changes observed as the temperature is increased are similar to those seen when mobile phase composition is changed (by adding organic modifier) at constant temperature.

Comparison of frits used in the preparation of packed capillaries for capillary electrochromatography.

The performance of several types of frits, including sintered frits, photopolymerized frits, and frits made by sol-gel technologies, is evaluated. The frits are formed in open capillaries, and the electroosmotic mobilities are calculated and compared to those obtained in an open capillary. Run-to-run, day-to-day, and column-to-column reproducibilities are evaluated. In all cases, the run-to-run reproducibility varied by < 4%. The greatest mobility was through the capillaries with the sol-gel frits (1.29 x 10(-3) cm2/V x s) which also exhibited the best day-to-day reproducibility. The capillaries with the photopolymerized frits had the best column-to-column reproducibility, yet the slowest mobility (1.00 x 10(-3) cm2/V x s). The second central moment of benzene as a solute is calculated and plotted as a function of time to estimate the differences in peak dispersion among the various frits studied. Although the mobilities through the capillaries with the sintered frits were the least reproducible, these frits are associated with the least amount of band-broadening. An estimation of the dispersion caused by each of the different types of frits is reported.

Reducing residual silanol interactions in reversed-phase liquid chromatography. Thermal treatment of silica before derivatization.

This study describes the thermal pretreatment of a silica gel between 150 and 800 degrees C before derivatization with dimethyloctadecylchlorosilane as a means of reducing residual silanol activity in HPLC bonded stationary phases. A time study was done from 12 to 48 h to find the optimum time needed for dehydroxylation. With increasing pretreatment temperatures, the number of reactive silanols is reduced from 8 micromol/m2 to essentially zero at 1000 degrees C (where sintering occurs). The effects of the thermal pretreatments were observed with diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and solid state cross polarization magic angle spinning (CP-MAS) 29Si NMR. Following derivatization, residual silanol activity and pH stability were tested by packing columns with the derivatized silica and carrying out a series of reversed-phase liquid chromatography (RPLC) experiments. Residual silanol activity was greatly reduced when the silica was pretreated at 800 degrees C, leading to less peak tailing for basic solutes. In a strongly basic mobile phase (pH 11.5) the pretreated silica was surprisingly stable, although bond cleavage of C18 groups from the surface was observed.

Synthesis and characterization of C13 to C18 stationary phases by monomeric, solution polymerized, and surface polymerized approaches.

A series of bonded phases were synthesized from consecutive length alkylsilanes ranging from C13 through C18, with three different bonding chemistries (monomeric, solution polymerized, and surface polymerized) at each phase length. The phases were characterized in terms of methylene selectivity, shape selectivity, and band broadening. No significant chromatographic differences were found to result from the synthetic routes, except that the different bonding chemistries provide a different range of bonding densities. For bonding densities ranging from 2 to 8 micromol/m2, a linear increase in methylene selectivity was observed with increasing bonding density. Over the narrow range of bonded phase lengths investigated, shape selectivity is more dependent on density than phase length.