Edward R. Grover

Novel chiral surfactant for the separation of enantiomers by micellar electrokinetic capillary chromatography.

A novel chiral surfactant was prepared as both enantiomeric forms, (R)- and (S)-N-dodecoxycarbonylvaline, and employed for the separation of enantiomeric mixtures by micellar electrokinetic capillary chromatography (MECC). The enantioselectivities (alpha) obtained for twelve typical pharmaceutical amines using the (S)-surfactant were compared to those obtained with (S)-N-dodecanoylvaline, a chiral surfactant described in the literature. Higher enantioselectivities were seen for ten of the twelve compounds using (S)-N-dodecoxycarbonylvaline. Furthermore, (S)-N-dodecoxycarbonylvaline had significantly less background absorbance in the low UV. It is shown that exact enantiomer migration order reversal can be obtained by individually employing both enantiomeric forms of the surfactant. For ionizable compounds like the amines examined here, enantioselectivity can be optimized by changing the pH of the MECC buffer. Partitioning is optimized through surfactant concentration, organic additives and pH. The ability to achieve fast chiral separations is shown. A separation of ephedrine enantiomers in urine is shown, with the only sample preparation being filtration.

Validation of enantiomeric separations by micellar electrokinetic capillary chromatography using synthetic chiral surfactants

Abstract The first report on the feasibility of method validation for the separation of enantiomeric mixtures by chiral micelle electrokinetic capillary chromatography is described. Method validation data elements were investigated according to U.S. Pharmacopeia protocol and are reported for the separation of ephedrine enantiomers using the synthetic chiral surfactants ( S )- and ( R )-N-dodecoxycarbonylvaline as buffer additives. Excellent linearity from 1–150% of the target concentration was obtained. A 1.0% limit of quantitation for an enantomeric impurity with acceptable precision is shown. Method robustness and ruggedness is also presented. Migration order reversal was used for the determination of a trace enantiomeric impurity, and to identify the enaniomeric compound in a multiple active ingredient formulation. The capability to reverse migration order is shown to be a valuable tool to satisfy the U.S. Pharmacopeia specificity requirement as well as improving detection and quantitation limits.

Separation of aldose enantiomers by capillary electrophoresis in the presence of optically active N-dodecoxycarbonylvalines

Abstract The 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of aldose enantiomers were well separated from each other by capillary zone electrophoresis using a neutral phosphate buffer containing an optically active N-dodecoxycarbonylvaline (DCV). The l -enantiomers migrated first and the d -enantiomers followed, when R-DCV was added to electrophoretic solution, and the migration order was reversed for the addition of S-DCV. This separation was inherent to PMP derivatives; other derivatives such as 2-aminopyridine, 8-aminonaphthalene-1,3,6-trisulfonate and 4-nitrobenz-2-oxa-1,3-diazole-tagged glycamine derivatives were not separated at all. l -Lauroylvaline and sodium dodecyl sulfate were also not separated. Based on these results, together with the 1H NMR signal change (upfield shift of the carbohydrate-proton signals in PMP–aldoses) on addition of R-DCV, we speculated this separation to be due to the difference in the ease of intermolecular ring formation by hydrogen bonding.

Separation of piperidine-2,6-dione drug enantiomers by micellar electrokinetic capillary chromatography using synthetic chiral surfactants

Abstract The enantioselective separation of five racemic piperidine-2,6-dione compounds was accomplished using chiral micelle electrokinetic capillary chromatography. This class of drugs includes glutethimide, aminoglutethimide, cyclohexylaminoglutethimide, pyridoglutethimide, and phenglutarimide. The resolution of all five compounds with simultaneous enantioselective separation of four of the five was obtained in a single run, using the synthetic chiral surfactant (S)-N-dodecoxycarbonylvaline. An enantioselective separation of the fifth compound was obtained by a second synthetic chiral surfactant, (R)-dodecoxycarbonylproline. It was observed that surfactant type and concentration, pH, and sample matrix all effect enantiomeric resolution. The migration order of cyclohexylaminoglutethimide enantiomers was confirmed by injecting a sample comprised mostly of the (−)-enantiomer. In addition, the separation of a sixth related sample (a thalidomide mixture) is shown to be achiral and not enantiomeric, as verified by the lack of enantiomer migration order reversal.

Purification of proteins on an epoxy-activated support by high-performance affinity chromatography

Abstract The use of a rigid silica-based packing material with large particle and pore size, 37–55 μm and 500 A pore, for affinity chromatography makes it possible to combine high selectivity with short analysis times. Both large and small molecules have been covalently bonded to the Protein-Pak TM Affinity Epoxy-Activated bulk packing for purification of glycoproteins, immunoglobulins, enzymes, lectins and other proteins. Recombinant protein A, GammaBind TM G, heparin, Cibacron Blue F3G-A, sulfanilamide, N-acetyl- D -glucosamine, concanavalin A and aminophenylboronic acid were covalently attached to the affinity packing for selective purification of proteins.

3 HPLC columns and packings

Abstract In this chapter on HPLC columns, we are discussing both the surface chemistry of a packing as well as column design and performance. In the section that covers column chemistry, we cover modern options of base materials as well as the commonly used approaches towards the surface chemistry of a packing. Specific subsections are dedicated to the selectivity of reversed-phase packings, HILIC, monolithic structures, and the reproducibility of modern packings. In the section on speed and resolution, we familiarize the reader with the principles of how to choose a column. In the section on specialty columns, we cover briefly preparative chromatography and columns with a very small diameter.