Oral Session: HPLC: Optimization and Chemistry

March 14, 2006

E-Separation Solutions

Ronald E. Majors presided over "HPLC: Optimization and Chemistry," an oral session held Tuesday afternoon that focused on the optimization of analytical- and preparative-scale LC separations.

LCGC North America columnist Ronald E. Majors of Agilent Technologies presided over "HPLC: Optimization and Chemistry," an oral session held Tuesday afternoon that focused on the optimization of analytical- and preparative-scale liquid chromatography (LC) separations. The session covered reversed-phase, chiral, water-only, and orthogonal separations as well as efficiency in LC and prevention of pore dewetting using ultrahigh pressure LC.

Tarab J. Ahmad of Prof. Georges Guiochon's laboratory at the University of Tennessee (Knoxville) discussed single-component adsorption isotherm data in a talk titled "The Effect of Mobile Phase Composition and Temperature on the Isotherm Parameters of Tryptophan in Reversed-Phase Liquid Chromatography." Ahmad described how large concentration band profiles of tryptophan were obtained for three mobile phase compositions at five temperatures. Ahmad used both a frontal analysis method and an inverse (numerical) method of chromatography; she obtained the best values of the isotherm coefficients using the inverse method. The mobile phase organic content and the temperature had different effects on the saturation capacities, the adsorption constants, and the solute-solute interaction parameters. She also discussed the significance of the results with respect to the retention mechanism.

Brian Jones of Selerity Technologies (Salt Lake City, Utah) presented a talk titled "Use and Practicality of Water-Only HPLC Separations." According to Jones, at elevated temperatures hydrogen bonding is decreased and water behaves more like an organic solvent as a mobile phase. Water can be used as a mobile phase with elevated temperature separations with compounds that are soluble in water, alcohol, and glycerin. The use of water as the mobile phase enables the use of flame ionization detection for some separations and is useful for analyzing compounds that do not contain chromophores. He discussed using water-only HPLC for samples such as glycols, diet cola, hydroxylphenols, an alcohol-based mouthwash, and a glycerin-based mouthwash. He also described the optimization of system characteristics and parameters necessary to obtain maximum peak quality, sensitivity, and method robustness.

In a presentation titled "Preparative Chromatography Effects of the Nature and the Sample Solvent," Francois Dardoize of the University of Paris (Paris, France) discussed the use of stainless steel reverse auto compression columns for preparative LC. He reported that the use of an annular injection system reduced band bending in preparative separations. Dardoize presented several examples of small-volume, high-volume, and high-concentration sample injections and spoke about the effects of the sample solvent's elution strength and viscosity.

LCGC North America columnist John W. Dolan (LC Resources Inc., Amity, Oregon) presented "Maximizing the Bang/Buck Ratio: Developing Orthogonal HPLC Separations." In his presentation, Dolan described using a database (Column Match, soon to be available for free on the USP website, according to Dolan) containing the selectivity characteristics of more than 300 reversed-phase columns as an aid in choosing the best column for a particular separation. Dolan then discussed simple strategies for developing orthogonal separations (separations that provide different selectivity for the same sample mixture) and provided practical examples of orthogonal methods for analyzing pharmaceutical samples.

Richard A. Henry of ZirChrom Separations (State College, Pennsylvania) gave a presentation titled "Enantiomer Separations and Fast Chiral Selector Screening." Henry described a method for attaching chiral stationary phases to zirconia through Lewis acid-base chemistry. He discussed chiral zirconia columns created using the Lewis acid-base process and showed data comparing the selectivity and efficiency of the zirconia phases with analogous silica-based chiral phases. He reported the synthesis of various chiral selectors containing anchor groups that can be attached or removed from porous zirconia particles for chiral screening.

In a talk titled "Prevention or Reduction of Chromatographic Pore Dewetting by Employing UPLC at Elevated Pressures," Eric S. Grumbach of Waters Corporation (Milford, Massachusetts) defined pore dewetting as the gradual loss of analyte retention when using 100% aqueous mobile phase. Without adequate pressure, the mobile phase is expelled from the pores of the chromatographic media. He discussed the effect of pore size on the level of dewetting and how the operation of the company's UPLC columns at pressures exceeding 9000 psi forces aqueous mobile phase back into the pores of the stationary phase and thus allows the chromatographic media to maintain its retention characteristics.

Yansheng Liu of Brigham Young University (Provo, Utah) presented "Chromatographic Behavior of Columns Packed with Various Small Particles." Liu described how 75-μm i.d. capillary columns were packed with porous and nonporous small diameter (<2 μm) particles and compared using ultrahigh-pressure LC. Results showed that particles less than 2 μ m in diameter provide a wide linear velocity operating range whether they are porous or nonporous. He also showed separations obtained using 2-4 μ m diamond particles coated with polybutadiene.

Bryan Grimmelt of the University of Prince Edward Island (Charlottetown, Prince Edward Island, Canada) gave a talk titled "Malachite Green: Quantitation Using HPLC" in which he described a method for extracting and analyzing malachite green and its metabolite leuco-malachite green from salmon tissue. Grimmelt used a scaled-down liquid-based extraction method and, for the analysis, monolithic C18 and C8 columns. Results obtained using the monolithic columns were compared with those obtained using particle-based LC columns.