Reversed-phase HPLC Conditions Research Articles

Silica Xerogel as a Continuous Column Support for High-Performance Liquid Chromatography

A preliminary study of the chromatographic performance and permeability of a continuous silica xerogel column under reversed-phase HPLC conditions was performed. A porous chromatographic support was synthesized inside a 0.32 mm i.d. × 13 cm fused silica tube from potassium silicate solution and derivatized with dimethyloctadecylchlorosilane. The plate height at 0.01 cm/s (0.5 μL/min), near the apparent optimum linear velocity, was about 65 μm. The column efficiencies in terms of numbers of plates per meter were 5000 and 13 000 for ethyl benzoate (k = 0.8) and naphthalene (k = 2.0), respectively, at 0.5 μL/min. The major parameter affecting column efficiency was the heterogeneous morphology of the xerogel, modifications to which are expected to improve chromatographic performance. The column provided efficiencies comparable to those reported for continuous polymeric columns but less than that previously reported for a continuous silica column. Gradient elution mode was demonstrated with a mixture of polycyclic aromatic hydrocarbons. The column was highly permeable, exhibiting a linear dependence of pressure to flow rate and a back pressure of only 632 psi at 10 μL/min when a 95% aqueous mobile phase was used.

Synthesis and purification of highly tritiated aryldiazonium photoprobes for cholinergic binding sites

The synthesis and improved purification procedures of two photoactivatable ligands 3H-A and 3H-B (Figure 1) are presented. Particular attention was given to the use of resolutive reversed-phase HPLC conditions which were found to be essential for the characterization and the elimination of contaminating products and for the obtention of pure diazonium probes at high specific radioactivity (up to 50 Ci/mmol).

Rapid and efficient preparative purification of building blocks suitable for solid-phase synthesis of neoglycopeptides: Synthesis and purification of O-(2,3,4,6-tetra-O-acetyl-?-d- galactopyranosyl/glucopyranosyl)-N ?-fluoren-9-yl- methoxycarbonyl-hydroxyproline

As part of scaling-up studies for the preparation of antinociceptive neoglycopeptides, the purification of appropriate glycosylated building blocks has been optimized. The synthesis and purification of O-(2,3,4,6,-tetra-O-acetyl-β- d-galactopyranosyl/glucopyranosyl)-Nα-fluoren-9-yl-methoxycarbonyl-hydroxyproline is described in this paper. The building blocks, suitable for solid-phase synthesis, are obtained in a single chemical step from partially protected hydroxyproline, followed by a rapid and efficient preparative purification. Preparative reversed-phase HPLC conditions have been adjusted to maximize recoveries, while reducing both time and costs of consumables. For the galactosyl conjugate, up to 0.5 g of pure lyophilized building block was obtained from 1.5 g of a crude reaction mixture using plain deionized water and less than 2 l of preparative-grade CH3CN. The glucosyl derivative was even more efficiently purified by spontaneous after-column crystallization from the elution mixture.

Purification and Analysis of Synthetic, Triple-Helical "Minicollagens" by Reversed-Phase High-Performance Liquid Chromatography

To better study collagen-mediated cellular and enzymatic activities, a generally applicable solid-phase methodology has been developed by which aligned triple-helical peptides (designated THPs or "minicollagens") ranging from 79 to 124 residues can be assembled. Reversed-phase HPLC is typically the purification method of choice following chemical synthesis of small proteins of this size, as well as one of the analytical techniques used to verify product purity. We have thus compared the effects of different stationary phases (C 18, C 4, or diphenyl), organic modifiers (acetonitrile or isopropanol), support pore sizes (120 Å 300 Å or nonporous), and counterions for the reversed-phase HPLC analysis of THPs. Large-pore C 18 or C 4 reversed-phase HPLC gave broad THP peaks, resulting in poor resolution of the desired THP from synthetic impurities. Broad peaks were presumably due to conformational instability of THPs to reversed-phase HPLC conditions and subsequent slow cis-trans isomerization of the peptide bonds. Peak sharpness was improved greatly by using large-pore diphenyl reversed-phase HPLC. We found that THPs can be best resolved from synthetic impurities by diphenyl or nonporous C 18 reversed-phase HPLC using water-acetonitrile gradients. These results most likely reflect conditions which maintain the native conformation of collagen-like triple-helices.

Chromatographic Behavior of Deoxyribonucleosides with Respect to Organic Modifier Content in the Mobile Phase

Abstract A chromatographic behaviour of five deoxyribonucleosides (dCyd, dUrd, dGuo, dThd, and dAdo) has been studied under isocratic conditions of reversed-phase HPLC. The Waters column packed by 10 μm μ-Bondapak C18 was used. The volume fraction (F) of organic modifier was changed from 0.05 to 0.30, and to 0.12 for methanol and acetonitrile, respectively. Futhermore, the capacity factors with a mobile phase of 100% water (k'w) have been measured. Linear regression has been carried out for the log k' vs. F data and the intercepts and the slopes of these plots were calculated. The slope magnitudes of different solutes were almost the same for each organic modifier. In all cases, the experimental values of k'w were higher than extrapolated ones. For each solute, extrapolation of log k' vs. F gives practically the same intercept values for both organic modifiers. It was concluded that the intercepts calculated in the region of small content of organic modifier are independent on the nature of organic modifi...

Simple high-performance liquid chromatographic method for assessing the deterioration of atropine-oxime mixtures employed as antidotes in the treatment

A set of reversed-phase HPLC conditions for determining the degradation of atropine and the oxime (pralidoxime, obidoxime, or HI-6) in autoinjectors de

HPLC post column derivatization of aromatic amines usingN-methyl-9-chloroacridinium triflate

An HPLC post column chemical derivatization procedure based on the interaction between an acridinium triflate and amines to form highly colored derivatives on-line is described for the determination of aromatic amines. Benzocaine and butesin, local anesthetic agents that contain the aromatic amine group, were used as model compounds. Reversed-phase HPLC conditions were developed for both the separation of analytes and the reaction between analytes and the acridinium triflate in the system. Three-dimensional knitted teflon shape coils and the internal diameter and length of the coils were important parameters in reducing band broadening and background noise.N-Methyl-9-chloroacridinium triflate was shown to be applicable to the determination of primary aromatic amines, selected secondary aromatic amines, hydrazides, and hydrazines. Application of the on-line chemical derivatization procedure to the analysis of pharmaceutical dosage forms containing procainamide (primary aromatic amine), isoniazid (hydrazide), and hydralazine (hydrazine) is also described.

Derivatization procedures for detection of prostaglandins in biological matrices by liquid chromatography/electrochemistry

Conventional reversed-phase HPLC conditions have been optimized for resolution of a mixture containing prostaglandins PGE 1, PGE 2, PGF 1α, and PGF 2α. Electroactive derivative-forming reagents, such as p-nitrobenzyloxyamine, 2-bromo-2′-nitroacetophenone, and 2,4-dinitrophenylhydrazine have been evaluated for use as precolumn reagents for forming prostaglandin derivatives. The results indicate that detection limits of 120 pg are achievable with amperometric detection. The utility of the procedures developed is illustrated by the detection of prostaglandins in human urine and plasma.

Determination of enantiomeric drugs in physiological fluids using on-line solid phase derivatizations and reversed-phase liquid chromatography

A new analytical approach has been developed for the determination of d,l-amphetamine in urine using on-line solid phase derivatizations in HPLC—UV/FL. Several other enantiomeric drugs were also investigated using the same method. The method was validated by several experiments, including: (1) kinetic studies for the reaction of each enantiomeric drug with the solid phase chiral reagent; (2) “single blind spiking” experiments; and (3) polarimetry for confirmation of the enantiomeric composition determined by the solid phase diastereomer formation—HPLC—UV/FL method. The resulting diastereomers were well resolved ( R s = 1.05–1.40) under typical reversed-phase HPLC conditions. Enantiomeric contamination at the 1.1% level could be detected. The lowest amount of d,l-amphetamine that could be simultaneously derivatized and detected was about 50 ng ml −1. The linearity of the overall measurement was 3–4 orders of magnitude. d,l-Amphetamine spiked into urine at different concentration levels and different d,l-ratios, only followed by filtration, were directly injected into the on-line solid phase derivatization—HPLC—UV/FL system for quantitation with relative standard deviations 1.8–6.4% and relative errors 0.6–9.8%.

A study of retention characteristics by functional group analysis using supercritical fluid chromatography

Supercritical fluid chromatography (SFC) has recently been applied to the analysis of agricultural compounds. Studies investigating factors governing retention behavior of the compounds of interest will facilitate SFC method development for new compounds and metabolites. In this study, several compounds containing various functional groups were examined. Initial trends in retention were established; where appropriate, retention behavior was correlated with functional group similarities, differences and regio chemistry of compounds containing the same functional groups. Packed and capillary column SFC were used in these analyses. Results were compared to retention behavior observed under reversed-phase HPLC conditions. In general, the normal phase characteristics of SFC are very desirable in the separation of more polar compounds and metabolites of the agricultural products. Regio chemical effects and additions of methyl, phenyl, nitro, amide, carboxamide, chloro and other functional groups to common base molecules are discussed.

HPLC Method Development for a New Antiarrhythmic Drug

Abstract The present report concerns HPLC method development for chemical samples of a new antiarrhythmic drug 2-methyl-5-phenyl-5-[2-[2-N, N-bis (1-methylethyl amino] ethyl]-1,3 diazabicyclo [4.4.0] octen-4-one (MPMED). Selectivity optimization for MPMED and several synthetic process intermediates with reversed-phase HPLC conditions is described. Also, the use of electrochemical and UV absorption detection for MPMED samples has been evaluated. The developed method has been validated for generation of assay data for chemical lots of MPMED.

Direct assay for phosphotransacetylase and acetyl-coenzyme a carboxylase by high-performance liquid chromatography

A simple and specific assay to measure the activity of two coenzyme A derivative-processing enzymes, i.e., phosphotransacetylase (EC 2.3.1.8) and acetyl-coenzyme A carboxylase (EC 6.4.1.2), is described. The assay is based on the HPLC analysis of the short-chain coenzyme A derivatives formed by the enzymatic reaction, viz., acetyl-CoA and malonyl-CoA. For this purpose, ion-pair reversed-phase HPLC conditions are optimized. Furthermore, the influence of several variables on the enzyme reaction is studied in order to get maximum activity. Due to its short analysis time, good selectivity, and chromatogram information, HPLC proves to be an excellent method for the assay of these enzymes.