Nelu Grinberg

Chiral separations using an immobilized protein—dextran polymer network in affinity capillary electrophoresis

Abstract An improvement in affinity capillary electrophoresis (ACE) was achieved by covalently linking bovine serum albumin (BSA) to a high-molecular- mass dextran ( M r 2 000 000) using cyanogen bromide. The efficiency of the binding reaction was greater than 99%, measured through quantitative separation of the protein and protein—dextran polymer network mixture on a bare capillary using capillary electrophoresis (CE). Baseline separation of leucovorin (LV) enantiomers was obtained under 9 min using a linear polyacrylamide-coated capillary (with an effective length of 20 cm) filled with the BSA—dextran polymer network. The present work offers several significant advantages over other approaches to ligand immobilization, and can be generally applied to a wide variety of ligand—substrate systems.

Capillary electrochromatography: An alternative to HPLC and CE

CEC is a technique that has attracted increased interest in recent years. It combines the advantages of high efficiency of electrophoretic separation methods with the greater selectivity characteristic of HPLC. In this paper we explore the use of CEC as a potential alternative to conventional HPLC, μ-HPLC, and CE. Recent developments, both theoretical and experimental, are reviewed. The practical aspects of application of CEC in various modes of separation are also discussed.

Enantiomeric separation of dansyl amino acids using MECC with a ligand exchange mechanism

Enantiomeric separation of 11 dansyl amino acids is reported using micellar electrokinetic capillary chromatography. The chiral phase consisted of a copper complex of N, N-di-decyl-D-alanine dissolved in the running buffer. Several factors affecting the separation were studied. The partitioning of the copper complex between the micelles and the buffer interface was investigated, and an attempt to describe the mechanism of separation is presented.

Separation of Organophosphonates by Ion Chromatography with Indirect Photometric Detection

Abstract An ion chromatographic method which utilizes lithium trimesate to separate three diphosphonate drugs and two process related organophosphonates is described. The lack of a chromophore on the five species and the high absorptivity of the trimesate anion at 254 nm allows for facile detection using indirect photometry. The effects of mobile phase concentration on the capacity factor of the species were investigated and a logarithmic relationship was established which was found to be dependent on the charge of the analyte anion and the mobile phase anion.

MECHANISTIC ASPECTS OF CHIRAL DISCRIMINATION ON A MOLECULAR IMPRINTED POLYMER PHASE

The mechanism of chiral selectivity of a difunctional polymer imprinted with dansyl-L-phenylalanine was investigated using the polymer as an HPLC stationary phase. Temperature studies revealed that the mass transfer of the imprinted enantiomer with the polymer was sluggish at low temperatures, leading to a non-equilibrium migration down the column. Conversely, retention of the non-imprinted enantiomer was controlled by a thermodynamic equilibrium over the entire temperature range of the study. Variation of the structure of the analyte indicated a single leading interaction between both enantiomers of dansyl-phenylalanine and the polymer phase; a hydrogen bonding interaction between the carboxylic acid group of dansyl-phenylalanine and pyridinyl sites on the polymer. Secondary processes contributing to enantioselectivity were also deduced; a hydrogen bonding interaction occurring between the imprinted enantiomer and carboxyl sites on the polymer and a precise steric fit of the amino acid side chain into th...

ENANTIORECOGNITION MECHANISMS FOR DERIVATIZED CELLULOSE UNDER REVERSED PHASE CONDITIONS

The chiral recognition mechanism for a derivatized polysaccharide chiral stationary phase (CSP) under reversed phase conditions was investigated (Chiralcel OJ-R). While enantiorecognition mechanisms have been proposed for these types of phases under normal phase conditions, it is unlikely that they would apply to reversed phase conditions due to the high polarity of the eluent. Indandiol was utilized as the main probe. The effect of organic modifier, eluent anion, and temperature on enantioselectivity for indandiol was determined. Additionally, a number of structurally related probes were also investigated to define the role of various functional groups on enantioselectivity. The investigation revealed that hydrogen bonding did not have a gross direct effect on enantiorecognition but may contribute through its effect on the tertiary structure of the CSP. It appears that inclusion is the major contributor to enantioselectivity with the size and shape of the probe being the determinant as to the extent of enantioselectivity rather than electrostatic interactions involving the functional groups of the solute.

Enantioseparation of a protease inhibitor, indinavir, by subcritical fluid chromatography

Indinavir is a protease inhibitor which possesses five chiral centers. Enantioseparation of indinavir and its enantiomer was performed on an amylose type stationary phase, Chiralpak® AD, under normal-phase HPLC and subcritical fluid chromatography conditions. Under the utilized chromatographic conditions, it is believed that the leading interactions are hydrogen bonding between one or both hydroxyl groups of the solute with the carbonyl group of the carbamate. An inclusion mechanism appears to control the chiral recognition. The effect of various modifiers, pressure, and temperature were investigated.

Use of subcritical fluid chromatography for the separation of enantiomers using packed cellulose based stationary phase

Investigations into the parameters affecting the enantiomeric separation of an intermediate in the synthesis of a drug targeted for cardiac arrhythmia is presented. The separation was achieved under subcritical fluid chromatography using CO2 modified with methanol and co-modifiers isopropyl amine and methylene chloride. The stationary phase was a cellulose based stationary phase manufactured under the trade name Chiracel OB. The results showed the addition of methylene chloride had a noticeable effect on resolution while the separation factor, α, remained constant. The co-modifier, isopropyl amine, did not considerably influence the resolution, or separation factor. Temperature studies were performed in order to establish the thermodynamic parameters of the interaction between the enantiomers and the stationary phase. Plots of In k′ vs. 1/T proved to be curved, while plots of In α vs. 1/T were linear.

Separation of peptides and proteins by capillary electrochromatography

Abstract Capillary electrochromatography (CEC) is a separation method that has gained increasing attention in recent years. It combines the advantages of high performance liquid chromatography (HPLC) selectivity with the high efficiency of electrophoresis. Due to its promise of high peak capacities, CEC appears to be promising for resolution of complex mixtures, such as those generated during protein and peptide mapping. However, direct transfer of HPLC and capillary zone electrophoresis (CZE) methods to CEC is not trivial due to the unique requirements for CEC in terms of stationary phase and operating conditions. In this paper, we discuss the various approaches undertaken for the CEC separation of peptides and proteins.

The Quantitation of a Residual Quaternary Amine in Bulk Drug and Process Streams Using Capillary Electrophoresis

Abstract A capillary electrophoretic method for the determination of a residual alkyl quaternary amine, tetra-n-butylammonium ion (TBA+), in bulk drug and process streams was developed. Since the analyte does not have a chromophore, detection was performed utilizing indirect photometric detection. The influence of the quinine, tetrahydrofuran and sodium acetate concentrations and pH app in the background electrolyte solution upon the efficiency of the separation and effective electrophoretic mobility of both TBA+ and electro-osmotic flow were studied. The ranges in which the various parameters were examined had considerable effects upon both the efficiency and the effective electrophoretic mobility of the electro-osmotic flow; however, the effective electrophoretic mobility of TBA+ was not significantly affected. The optimized method was validated in terms of detector linearity, sensitivity, precision and accuracy.