John A. Blackwell

Effect of mobile phase additives in packed-column subcritical and supercritical fluid chromatography.

The effect of mobile phase additives is investigated for a variety of compounds under subcritical and supercritical conditions using packed columns. Retention of hydrogen bond donor/acceptor analytes was found to be more dependent on the presence of mobile phase additives than weak hydrogen bond acceptor analytes. The temperature and pressure of the mobile phase are major factors in the extent of this dependence. Consequently, selectivity between homologous compounds is dependent on both the additive used and the state of the mobile phase. Efficiency is nearly always improved by the presence of mobile phase additives, more so under supercritical conditions than under subcritical conditions. These observations suggest that surface molar excesses of mobile phase additives play a large part in the resulting character of the supercritical chromatographic system.

Ion- and ligand-exchange chromatography of proteins using porous zirconium oxide supports in organic and inorganic Lewis base eluents

The applicability of an eluotropic scale pertaining to the desorption of low molecular weight Lewis base solutes from zirconium oxide is examined for its ability to rationalize the retention of proteins on this substrate. The strongest Lewis base eluents (phosphate and fluoride) are able to bring about elution of nearly all proteins provided that their initial mobile phase concentration almost saturates the eluents adsorption isotherm. In contrast, weaker Lewis bases such as borate, sulfate and bromide are able to elute only those proteins which are retained primarily by ionic interactions. In weak eluents, proteins that contain a large number of accessible Lewis base sites are not eluted from the support. The effect of ionic strength and a variety of Lewis base eluents were also examined.

Empirical relationship between chiral selectivity and mobile phase modifier properties

An empirical relationship was derived which relates properties of the mobile phase modifier to the chiral selectivity factor for a given analyte/chiral selector combination. Using carbon dioxide and heptane-based mobile phases, the effect of various mobile phase modifiers on Pirkle-type stationary phases may be accurately modeled using a two-parameter equation. Similar results are obtained using cellulosic stationary phases with carbon dioxide-based mobile phases. Modeling separations performed using heptane-based mobile phases with cellulosic stationary phases were not successful. The predictive ability of this modeling approach was demonstrated using novel modifiers and chiral analytes.

TEMPERATURE EFFECTS FOR CHIRAL SEPARATIONS USING VARIOUS BULK FLUIDS IN NEAR-CRITICAL MOBILE PHASES

As supercritical fluid chromatography becomes more accepted as a facile means for the separation of chiral compounds, the need for mobile phases that can readily solubilize these polar compounds grows. Prior studies suggest that HFC-134a may prove suitable due to its very high eluotropic strength compared to carbon dioxide-based mobile phases. A comparison is made between ethanol-modified carbon dioxide, HFC-134a, and decafluoropentane as to their relative eluotropic strength, selectivity, and efficiency for three chiral compounds using a Whelk O-1 chiral bonded phase. The bulk component of the mobile phase was found to have relatively little effect on chiral selectivity over the range of 5° to 95°C. Chirality 9:693–698, 1997.

Study of the effect of mobile phase additives on retention in reversed phase HPLC using linear solvation energy relationships

Linear solvation energy relationships (LSERs) were used to delineate which specific intermolecular interactions are responsible for changes in retention for a variety of well characterized analytes when acidic and basic additives were used in reversed phase HPLC. The effects of trifluoroacetic acid, triethylamine and a combination of trifluoroacetic acid and triethylamine on the LSERs were compared to those observed in the absence of additives. These effects were examined using four different mobile phase modifiers and five different stationary phases. Trifluoroacetic acid alone and in combination with triethylamine produced LSER regression coefficients nearly identical to those obtained with no additive present in the mobile phase. Triethylamine alone produced different LSER regression coefficients from the other systems unless the mobile phase contained trifluoroethanol as the mobile phase modifier, or the stationary phase consisted of a polymeric support.

Effect of Mobile Phase Components on the Separation of Polypeptides Using Carbon Dioxide-Based Mobile Phases

The effect of mobile phase modifier and additive on the chromatographic properties of various small polypeptides was explored under subcritical conditions. A polymeric column was used to separate various enkephalin analogs, bradykinin, and oxytocin using a carbon dioxide-based mobile phase with either an ethanol or a 2-methoxyethanol modifier. The role of the modifier was found to be secondary to that of the mobile phase additive. As progressively stronger acidic mobile phase additives were used, the peak profiles of the various polypeptides improved and retention decreased. Heptadecafluorooctanesulfonic acid was found to be the most useful additive for these types of solutes under near-critical conditions, while the potassium salt of heptadecafluorooctanesulfonic acid failed to elute any of the polypeptides. At low temperatures, pressure gradients with a carbon dioxide/ethanol/heptadecafluorooctanesulfonic acid ternary mobile phase produced reasonably good peak profiles with the polymeric column.

Ligand Exchange Chromatography of Free Amino Acids and Proteins on Porous Microparticulate Zirconium Oxide

Abstract The Lewis acid sites present on the underlying zirconium oxide particles are responsible for the unusual elution sequence for amino acids on copper(II) loaded, phosphated zirconium oxide supports reported in an earlier study. To more thoroughly examine the effect of these strong Lewis acid sites, we have studied ligand exchange chromatography on copper(II) loaded zirconium oxide particles. It is shown here that carboxylate functional groups on amino acid solutes strongly interact with surface Lewis acid sites. Addition of competing hard Lewis bases to the eiuent attenuates these specific interactions. The result is a chromatographic system with high selectivity which is also suitable for ligand exchange chromatography of proteins.

Influence of mobile phase composition on methylene group selectivity and homologous compound retention in near-critical mobile phases

A number of near-critical mobile phases were evaluated with respect to methylene group selectivity and overall eluotropic strength on a polymeric column. Binary mobile phases, consisting of mixtures of carbon dioxide and 1,1,1,2-tetrafluoroethane (HFC-134a), showed strong temperature and compositional dependence on both retention and selectivity. Binary mobile phases, consisting of mixtures of carbon dioxide and conventional modifiers such as alcohols, acetonitrile and tetrahydrofuran, also showed a strong temperature dependence on methylene group selectivity and relative retention. In contrast to the behavior observed using a polymeric column, a bonded silica-based stationary phase displayed significant deviations from the Martin rule, even with high order homologs. Of all the systems evaluated in this study, a binary mixture of carbon dioxide and 2-methoxyethanol proved to be optimal for obtaining a high degree of methylene group selectivity and strong eluotropic strength.

Chemometric characterization of Lewis base-modified zirconia for normal phase chromatography

Abstract Microporous zirconia was evaluated for its chromatographic properties as a normal phase support. Since previous studies in aqueous media had indicated a strong effect of eluent Lewis base on chromatographic properties, a number of Lewis base pretreatments of the support were evaluated as to their effect on normal phase selectivities. The retention characteristics of over thirty well characterized probe solutes were determined on eight different “acid”- or “base”-washed zirconia supports. These results were compared with those obtained for a silica column. Chemometric methodology was used to characterize the similarities and differences between the “acid”- and “base”-washed supports. The lack of chromatographic reproducibility previously observed under normal-phase conditions on zirconia appears to be a result of the lack of specific descriptions regarding the “acid” or “base” pretreatment.

Comparison of 1,1,1,2-tetrafluoroethane and carbon dioxide-based mobile phases for packed column supercritical fluid chromatography

Abstract Highly polar, homogeneous mobile phases have a number of advantages over carbon dioxide-based mixed mobile phases for supercritical fluid chromatography. While these fluids have been demonstrated to have very high eluotropic strengths in capillary systems, their utility for packed columns lies in their ability to overcome the deleterious effects of residual silanol groups present on the bonded-phase surface. A particularly promising fluid, 1,1,1,2-tetrafluoroethane (HFC-134a), is characterized with respect to eluotropic strength, selectivity, efficiency and temperature effects. These results are compared to those obtained with carbon dioxide and methanol-modified carbon dioxide mobile phases on a silica column. HFC-134a displays different selectivities, efficiencies, eluotropic strength and temperature dependencies on retention than either of the two reference mobile phases.