Separation of erythrocytes infected with murine malaria parasites in metrizamide gradients

Density gradients with metrizamide, a tri-iodinated benzamido derivative of glucose, have been used to separate erythrocytes infected with 3 species of murine plasmodia. Good separations were obtained of uninfected erythrocytes from erythrocytes containing parasites in different developmental stages. With metrizamide solutions, the densities required for isopyenic separation can be obtained without hypertonicity or high viscosity, and the viability and those aspects of the metabolism of parasites and erythrocytes that we measured are not detectably modified by exposure to these solutions. This type of separation has many possible applications to biochemical and immunological investigations.     

Direct enantioselective HPLC monitoring of lipase-catalyzed kinetic resolution of tiaprofenic acid in nonstandard HPLC organic solvents

The first straightforward lipase-catalyzed enantioselective access to enantiomerically enriched tiaprofenic acid as a versatile method in chiral separation of racemates is demonstrated. The latter was directly monitored by enantioselective HPLC using a 3,5-dimethylphenylcarbamate derivative of cellulose-based chiral stationary phase namely Chiralpak IB (the immobilized version of Chiralcel OD). Non-standard HPLC organic solvents were used as diluent to dissolve the "difficult to dissolve" enzyme substrate (the acid) and as eluent for the simultaneous enantioselective HPLC baseline separation of both substrate and product in one run without any further derivatization. The existence of a non-standard HPLC organic solvent (e.g., methyl tert-butyl ether) in the mobile phase composition is mandatory to accomplish the simultaneous enantioselective HPLC baseline separation of both substrate and product.     

HPLC resolution of atropoisomeric compounds on a csp derived from (1R;2R)-diaminocyclohexane: Thermodynamic data from variable temperature chromatography

The preparation and a preliminary chromatographic evaluation of a novel polymeric chiral stationary phase (CSP) derived from (1R;2R)-diaminocyclohexane (DACH) are presented. A radical copolymerization process has been employed to generate a silica-based chiral sorbent, showing considerable high chemical and thermal stability and stereoselectivity toward racemic compounds capable of H-bonding (3-hydroxy-benzodiazepin-2-ones, chlorthalidone, atropoisomeric sulfur compounds, etc.); in the present paper we present the investigation on the resolution of racemic dihydroxy biarylic atropoisomers; the effects of eluent composition and of temperature on the separation ability of the CSP have been studied in order to elucidate the recognition mechanism operating in these chiral separations. © 1992 Wiley-Liss, Inc.     

Chiral discrimination by ligand-exchange chromatography: A comparison between phenylalaninamide-based stationary and mobile phases

The copper(II) complexes of two new diastereomeric ligands, N²-(R)- and N²-(S)-2′-hydroxypropyl-(S)-phenylalaninamide [(R, S)-1 and (S, S)-1], have been used as additives to the eluent in high-performance liquid chromatography (HPLC) reversed phase for the chiral separation of DNS-amino acids. The aim was that of comparing the separation process obtained by the chiral eluent with that obtained by an analogous bonded stationary phase containing (S)-phenylalaninamide, previously studied [CSP-(S)-Phe-NH₂]. The affinity of the ternary complexes for the C₁₈ column was determined by adsorption experiments in HPLC. It was shown that the two systems (chiral eluent, chiral stationary phase) work according to different mechanisms. Ternary complex formation in solution was studied by fluorescence spectroscopy. It was shown that chiral separation with the Cu(II) complexes added to the eluent was determined by the relative affinities of the ternary complexes for the column-stationary phase rather than by their stabilities in solution. With CSP-(S)-Phe-NH₂ the separation is accounted for by the relative stabilities of the ternary complexes, which depends mainly on the “allowed” geometry of the complex and on the steric repulsion of the amino acid side chain with the spacer. © 1996 Wiley-Liss, Inc.     

Chiral-recognition chromatography of β-blockers on continuous polymer beds with immobilized cellulase as enantioselective protein

Columns prepared by coupling cellulase as a chiral selector to silica beads are very efficient for the separation of enantiomers. In this paper we show that continuous polymer beds compete favorably with silica beads as chromatographic supports for such separations. The chiral stationary phase is prepared either by entrapment in and simultaneous covalent linkage of ally1 cellulase to the continuous beds during their preparation or by covalent immobilization of cellulase on an epoxy-activated continuous bed. Enantiomers of β-blockers were separated rapidly and with high resolution. The enantiomers of practolol were thus baseline resolved within 45 sec. The recognition center–or at least part of it—coincides with the active center of the enzyme, since the enantiomers could not be separated in the presence of the competitive enzyme inhibitors cellobiose and D-glucose and the separation was also impaired upon addition of the substrate carboxymethyl cellulose to the eluent. Similar observations have been reported for silica columns derivatized with cellulase. The capacity factor and the separation selectivity could be tuned by the pH and the concentration of the mobile phase, a phosphate buffer. No modifier was required, as is sometimes the case with silica-based supports. The continuous beds give faster enantiomer separations than do columns of silica and are more pH-stable and cost effective to prepare. © 1993 Wiley-Liss, Inc.     

Evaluation of a ristocetin bonded stationary phase for subcritical fluid chromatography of enantiomers

A stationary phase derived from ristocetin was evaluated for chiral separation in subcritical fluid chromatography. Separation of various enantiomers having different structures and pK(a) values were investigated using carbon dioxide and polar modifiers. The influence of modifiers, additives, temperature, and mobile phase flow rate on separations is presented. It is concluded that this stationary phase can be used for SFC despite its structural similarity with protein-derived stationary phases that can only be used in HPLC. The separation mechanisms could not be elucidated or predicted using these initial experiments. The separations of warfarin and, especially, efavirenz demonstrate the potential of this type of stationary phase for rapid SFC chiral separations.     

Separating enantiomers by HPLC on silica dynamically coated with a chiral stationary phase of permethylated β-cyclodextrin

The paper demonstrates that the technique of solvent generated liquid--solid chromatography can be used to create normal phase systems for chiral separations. The chiral adsorption layer was generated by pumping a binary hexane:ethanol eluent containing a small fraction of permethylated β-cyclodextrin through a column packed with microparticulate silica. This technique leads to columns with good time stability and reproducibility. The possibility of generating normal phase systems with permethylated β-cyclodextrin as chiral component via the mobile phase broadens the range of phase system which can be used to separate enantiomers by HPLC.     

Simulated moving bed chromatography with supercritical fluids for the resolution of bi-naphthol enantiomers and phytol isomers

The combination of the simulated moving bed (SMB) technique with supercritical fluid chromatography (SFC) leads to a process with unique features. Besides the known advantages of the SMB process, the use of supercritical carbon dioxide as the mobile phase offers the advantages of reduction in organic solvents and an easy eluent/solute separation. Because of the low viscosity and high diffusion coefficients of supercritical fluids, a high efficiency is possible. The steps of process development for SMB SFC are presented using the separations of the bi-naphthol enantiomers and phytol isomers as examples. The development of a packed column SFC method at an analytical scale is shown for the separation of the bi-naphthol enantiomers on a chiral stationary phase and CO(2) with a modifier as the mobile phase. The influence of the modifier, modifier content, and column configuration on productivity of the SMB SFC process was investigated by simulation. The first set of experiments was performed in the SMB separation of phytol isomers at low concentration to test the feasibility of the SMB SFC high purity separation of the binary mixtures. In the second set of experiments, the productivity of the process was increased by increasing the feed concentration up to 54 grams feed per liter stationary phase (SP) and hour (g(feed)/l(SP) h).     

Sugaring out: A new method for removal of acetonitrile from preparative RP-HPLC eluent for protein purification

Reverse phase-high pressure liquid chromatography (RP-HPLC) with an acetonitrile–water mixture as the eluent is widely used for purification of proteins. The separation of acetonitrile (ACN) in RP-HPLC eluent is important for protein recovery. Cooling below subzero temperature and salting out have been used to remove ACN, each with its limitations. In this work we have explored the use of sugaring-out, a new phase separation method developed at University of Illinois for the separation of ACN from a simulated preparative RP-HPLC effluent. The effect of glucose concentration, temperature, and initial amount of ACN in the effluent on phase separation was investigated. Results showed that a good phase separation can be achieved at near room temperature (18 °C). With the optimized conditions, we found that more than 60% (w/w) of ACN was removed and more than 95% (w/w) of water-soluble proteins (bovine serum albumin, trypsin, and pepsin) were recovered.     

Protein based microarrays: A tool for probing the proteome of cancer cells and tissues

A novel proteomic approach for probing cell and tissue proteome, which combines liquid phase protein separations with microarray technology has been developed. Proteins in cell and tissue lysates or in cellular subfractions are separated using any one of a number of separation modes which may consist of ion exchange liquid chromatography (LC), reverse phase LC, carrier ampholyte based separations, e.g. the use of Rotofor, affinity based separations, or gel based separations. Each first-dimension fraction obtained using one separation mode can be further resolved using one or more of the other separation modes to yield either purified protein in solution or liquid fractions with substantially reduced protein complexity. The advantage of a liquid based separation system is that proteins in hundreds of individual fractions can be arrayed directly and used as targets for a variety of probes. Constituent proteins in reactive fractions are identified by mass spectrometry and may be further resolved to determine the nature of the reactive protein(s). We present in this report initial data based on microarray analysis of individual Rotofor fractions obtained from lung adenocarcinoma cell line A549 lysates which have been probed with antibodies against specific proteins.     

High‐performance liquid chromatography separation of enantiomers of mandelic acid and its analogs on a chiral stationary phase

The enantiomers of mandelic acid and its analogs have been chromatographically separated on a chiral stationary phase (CSP) derived from 4‐(3,5‐dinitrobenzamido) tetrahydrophenanthrene. The rationale of separations of these compounds is discussed with respect to the method development for determining enantiomeric purity and possibility of obtaining enantiomerically pure materials by high‐pressure liquid chromatography. The relationship of analyte structure to the extent of enantiomeric separation has been examined and separation factors (α) are presented for various groups of structurally related compounds. Chiral recognition models have been suggested to account for the observed separations. These models provide mechanistic insights into the chiral recognition process. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Gel permeation chromatography of neutral hydroxy lipids on Sephadex LH-20

Gel-permeation chromatography on Sephadex LH-20, using ethanol as eluent, permits the resolution of neutral hydroxy lipids according to molecular size. The influence of molecular shape, functional groups, chain lengths, and degree of unsaturation, as well as the effect of the eluent on the elution pattern are discussed. The usefulness of the method for the separation of classes of hydroxy lipids which cannot be resolved by other chromatographic procedures, is demonstrated. Examples include the separations of 1,2- and 1,3-diglycerides from long-chain alcohols, and of alkyl ethanediol monoethers from cholesterol.     

Enantiomeric separation of mineralocorticoid receptor (hMR) antagonists using the Chiralcel OJ-H HPLC column with novel polar cosolvent eluent systems

This study demonstrates the increased versatility of the Chiralcel OJ-H stationary phase when using various alcohol/acetonitrile mobile phases. This chiral stationary phase has traditionally been employed in the normal phase mode and more recently with neat alcohols as eluents. Selected isomeric human mineralocorticoid receptor (hMR) antagonist pharmaceutical candidates and synthetic intermediates were separated using the Chiralcel OJ-H HPLC column with novel polar cosolvent eluent systems. The capacity factors, resolution, and selectivity of the chiral separations were assessed while varying the alcohol/acetonitrile composition and alcohol identity. The mixed polar eluents provide separations that are nearly always superior to both the traditional hexane-rich and single-alcohol "polar organic" eluents for the compounds tested in this article.     

Effect of buffer pH and peptide composition on the selectivity of peptide separations by capillary zone electrophoresis

A series of 10 synthetic peptides containing varying degrees of charge and hydrophobicity was used to study the effects of peptide composition and buffer pH on the selectivity of separations by capillary zone electrophoresis (CZE). A simple model is used to explain the effect of buffer pH on the separation. It was found that pH is an important parameter affecting the selectivity of CZE separations. Furthermore, it is shown that the selectivity of the separation is such that peptides differing in neutral amino acid composition can be resolved, and that even differences in a peptide's amino acid sequence can be detected. A protease digest of beta-lactoglobulin A is shown as a practical example of a separation of a complex peptide mixture.     

Phase Separations in Binary and Ternary Cholesterol-Phospholipid Mixtures

A pair of recent studies has reopened debate on the subject of phase separations in model bilayer mixtures of cholesterol (Chol) and dipalmitoyl-phosphatidylcholine (DPPC). Fluorescence microscopy methods have not been able to detect phase separations in binary DPPC-Chol mixtures that have been inferred from NMR studies. However, micron-scale phase-separated liquid domains are observed by fluorescence in ternary mixtures of DPPC, Chol, and diphytanoyl-phosphatidylcholine (DiPhyPC). Here, a model of condensed complexes of Chol and DPPC is used to account for these results. In particular, it is shown that the orientation of tie-lines in ternary mixtures of DiPhyPC/DPPC/Chol is compatible with phase separation in binary DPPC/Chol mixtures.     

Direct chiral separations of the enantiomers of phenylpyrazole pesticides and the metabolites by HPLC

The enantiomeric separation of the enantiomers of three phenylpyrazole pesticides (fipronil, flufiprole, ethiprole) and two fipronil metabolites (amide‐fipronil and acid‐fipronil) were investigated by high‐performance liquid chromatography (HPLC) with a CHIRALPAK^® IB chiral column. The mobile phase was n‐ hexane or petroleum ether with 2‐propanol or ethanol as modifier at a flow rate of 1.0 mL/min. The influences of mobile phase composition and column temperature between 15 and 35°C on the separations were studied. All the analytes except ethiprole obtained complete enantiomeric separation after chromatographic condition optimization. Fipronil, flufiprole, amide‐fipronil, and acid‐fipronil obtained complete separation with the best resolution factors of 2.40, 3.40, 1.67, and 16.82, respectively, but ethiprole showed no enantioselectivity under the optimized conditions. In general, n‐ hexane with 2‐propanol gave better separations in most cases. The results showed decreasing temperature and content of modifier in the mobile phase resulted in better separation and longer analysis time as well. The thermodynamic parameters calculated according to linear the Van't Hoff equation indicated the chiral separations in the study were enthalpy‐driven. Fipronil and its two chiral hydrolyzed metabolites obtained baseline separation simultaneously under optimized conditions.     

Analysis of oligogalacturonic acids with 50 or fewer residues by high-performance anion-exchange chromatography and pulsed amperometric detection

Underivatized oligogalacturonic acids with a degree of polymerization (DP) ranging from 2 to 50 have been separated for the first time on a high-performance CarboPac PA1 pellicular anion-exchange stationary phase column. Baseline separation of these pectic fragments was accomplished using a nonlinear gradient of pH 6 potassium oxalate buffer as the mobile phase. Acetate buffer linear gradients were also useful as mobile phases, but only for separations of oligogalacturonic acids that were soluble in this solvent (DP less than 20). Additionally, oligogalacturonic acid separations were accomplished on a lower capacity AS4A stationary phase column. Triple pulse amperometric detection was selective, sensitive, and reproducible, nevertheless, oligogalacturonic acid response factors were affected by DP and compositional changes in the mobile phase.     

Reproducibility of gel-chromatography

We think it worthwhile to other experimenters to mention the following observations. Recently it was noted that gel chromatographic separations of the five fragments resulting from the degradation of cytochrome c by bromocyanogen became dependent on the batch number of the gel. This dependency concerns only separations of unprotected fragments, using 7% (v/v) formic acid as the eluent. The elution profile of protected compounds, which in turn run in 50% (v/v) formic acid, was unaffected.     

Retention factors and resolutions of amino benzoic acid isomers with some lonic liquids

Ionic liquids in the form of organic salts are being widely used as new solvent media. In this paper three positional isomers,o-amino benzoic acid,m-amino benzoic acid, andp-amino benzoic acids were separated with four different ionic liquids as mobile phase additives using high performance liquid chromatography (HPLC). The following ionic liquids were used: 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF₄]), 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIm][BF₄]), 1-ethyl-3-methylimidazolium methylsulfate ([EMIm][MS]), and 1-octyl-3-methylimidazolium methylsulfate ([OMIm][MS]). The effects of the alkyl group length on the imidazolium ring and its counterion, and the concentrations of the ionic liquids on the retention factors and resolutions of amino benzoic acid isomers were tested. The results of the separations with ionic liquids as the eluents were better than those without ionic liquids. Excellent separations of the three isomers were achieved using 2.0≈8.0 mM/L [OMIm][MS] and 1.0≈8.0 mM/L [EMIm][MS] as the eluent modifiers.