Rodger W. Stringham

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.

Memory effect of mobile phase additives in chiral separations on a Chiralpak AD column.

Using chiral probes shown to be sensitive to the presence of mobile phase additives, a memory effect for these additives by an amylosic column was demonstrated. Exposure to these additives gave prolonged chromatographic performance changes even after their removal from the mobile phase. This finding is consistent with strong binding of the additives to the stationary phase. A procedure to remove bound additives was developed.

Enantioseparation of amino acids on a polysaccharide-based chiral stationary phase

Sulfonic acids have been shown to be more effective than the commonly used trifluoroacetic acid (TFA) in the chiral resolution of underivatized aromatic amino acids on an amylosic column. Sulfonic acid additives give a more UV transparent mobile phase, possibly allowing the detection of non-aromatic analytes. Work presented demonstrates that through the combination of sulfonic acid mobile phase additives, amine mobile phase additives and solvent modifier variations, the enantiomers of 20 of 25 probe amino acids are fully resolved, four are partially resolved with only one failing to be separated on a common amylosic column.

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.

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.

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.

RELATIONSHIP BETWEEN RESOLUTION AND ANALYSIS TIME IN CHIRAL SUBCRITICAL FLUID CHROMATOGRAPHY

A model is presented that predicts a defined relationship between chiral SubFC resolution and analysis time. This model is based upon ideal chromatographic behavior and requires column efficiency and selectivity to be independent of mobile phase modifier level and flow rate. The validity of these assumptions was found to be imperfect but acceptable for two model compounds on two commonly used chiral columns. A major implication of the model is that the maximum resolution obtainable with a particular column and mobile phase modifier may be predicted from one injection. The retention time required to obtain a desired resolution is also calculable. This information enables the practitioner to discern quickly the futility of method development efforts. Insufficient maximum resolution predicted from the first injection would require an increase in selectivity to achieve a useful separation. Selectivity may then be altered by temperature, modifier, or stationary phase. The increased column efficiency of SubFC at typical flow rates rescues separations that fail by HPLC, thus shrinking the practitioners required library of chiral columns. This work demonstrates that SubFC also allows the practitioner to skim through that library very quickly.