•Betaine dye (33) used to examine the polarity of subcritical water as temperature and pressure were changed.•Temperature had a greater effect on polarity of subcritical water than pressure.•The polarity of subcritical water is comparable to 30%–50% methanol/water mixtures and 20%–30% acetonitrile/water mixtures.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 13.8 bar (200 psi) to 124 bar (1800 psi). 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.

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.Highlights► Peak distortion from injection solvent effects was measured and quantified. ► Peak distortion occurs with increasing injection volume and solvent strength. ► A predictive test is proposed to estimate column sensitivity to these issues.

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.

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′wk′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′wk′w for use in estimating octanol/water partition coefficients.

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.

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 μmol/m2 and a polymeric phase (nominal surface coverage 4.77 μmol/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 (ΔH°, ΔS°, and ΔG°) 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.

Polyoxyethylene alkyl ethers, CnEm, are nonionic surfactants made of an alkyl chain with n methylene groups and a hydrophilic part with m oxyethylene units. CnEm nonionic surfactants are very useful in chemical analysis. The commercially available products are often a mixture of several CnEm molecules with different m values. Pure CnEm surfactants are now available. The physicochemical parameters: critical micelle concentration (c.m.c.), molar volume, density, cloud-point temperature and hydrophile–lipophile balance value for pure CnEm surfactants were collected from the literature. Regression analyses were carried out on the data. They showed that strong correlations existed between the structure of the molecule (n and m values) and its physicochemical properties. General equations linking the c.m.c., molar volume, density and cloud-point temperature of the CnEm surfactants and their structure (n and m values) are proposed and discussed. The use of these surfactants in chemical analysis is illustrated by the determination of cholesterol in egg yolk. Cholesterol was separated from the bulk yolk by cloud-point extraction using the C12E10 surfactant. It was quantitated using micellar liquid chromatography. The C12E23 surfactant was used to prepare the micellar mobile phase that allowed the separation of cholesterol and the use of an enzymatic detector.

Raman spectroscopy is used to examine the effect of mobile phase composition on the orientation of octadecyl-bonded silica-based reversed-phase liquid chromatographic (RPLC) stationary phase ligands. The effect of ligand bonding density is also investigated. The present experimental set-up utilizes a direct, noninvasive, on-column approach to examine the solvent dependent conformational behavior of the bonded ligands under flow-rate and back pressure conditions similar to those used during conventional RPLC measurements. Neat, single-component, mobile phase solvents including water, acetonitrile, methanol and chloroform are used to investigate the hypothesized collapsing and extension of stationary phase ligands with changes in mobile phase composition. No evidence of phase collapse was observed upon changing the mobile phase composition from an organic to an aqueous content. Also, Raman spectroscopic measurements allowed the differentiation between associated and free acetonitrile solvent.