Multiparallel microfluidic high-performance liquid chromatography for high-throughput normal-phase chiral analysis

The suitability of the Eksigent Express 800 microfluidic eight-channel HPLC instrument for multiparallel normal-phase chiral analysis in support of high-throughput pharmaceutical process research was investigated. Analysis of test mixtures containing the two enantiomers of benzoin and the closely related (R,S)-dihydrobenzoin, was carried out in a 96-well microplate, affording rapid (<2 h) and accurate assessment of enantiopurity. In a second example, use of the instrument to support high-throughput catalyst screening of the asymmetric hydrogenation of a prochiral unsaturated ester is presented, in which method development (gradient screening of four columns and two eluents, followed by optimization to afford a fast analytical method) and analysis of a 96-well microplate was carried out within a single working day. This represents a considerable improvement over conventional analysis techniques that usually take several days to complete.     

Development of silica-based stationary phases for high-performance liquid chromatography

Stationary phases are the basis of the development and application of high-performance liquid chromatography (HPLC). In this review we focused on the development of silica-based stationary phases, including the synthesis of silica gel and the application of silica in hydrophilic interaction chromatography (HILIC), reversed-phase liquid chromatography (RPLC), chiral separation chromatography, and ion chromatography. New stationary phases, advances in ionic liquid-modified silica, silica-based core-shell materials, and silica-based monolithic columns for HPLC are introduced separately.     

Faster analysis by reversed-phase high-performance liquid chromatography

Summary Many analysts are not taking full advantage of the high speed possibilities of modern LC. Some analytical procedures reported in the literature, and many in regular use in control laboratories, could be achieved in less time without loss in precision. Some factors which affect retention times are discussed and the advantages and disadvantages of employing shorter column lengths and finer packing materials in reversed-phase HPLC are examined. The effect on efficiency of increased flow rates with 10,5 and 3 m ODS materials is shown. The ability to couple shorter column lengths without loss of efficiency is also demonstrated. This allows a minimum length to be selected that gives adequate resolution. Examples of high speed separations are shown and limitations in state of the art HPLC equipment and chromatographic data systems are discussed briefly.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Development of some stationary phases for reversed-phase high-performance liquid chromatography

The availability of a variety of stable organic stationary phases for columns has been a key factor in the development of HPLC as a major scientific tool. This paper explores the history and rationale used in the development of some important stationary phases and attempts to identify some of the strengths and limitations of these materials. Some of the author's experiences in stationary phase development illustrate approaches leading to present-day columns that exhibit a broad range of selectivity coupled with a high degree of reproducibility. Suggestions also are made for additional stationary phases that may be needed to complete column selectivity potential for HPLC separations.     

Rapid analysis of phentolamine by high-performance liquid chromatography

A rapid liquid chromatographic method is validated for the quantitative analysis of phentolamine. Phentolamine exists in three forms for this investigation: as a mesylate salt, hydrochloride salt, and free base. In solution, phentolamine dissociates from its salt and is chromatographed as free phentolamine. This validation confirms the analysis of each form, which is simply based upon molar mass differences encountered in weighing. As such, both the United States Pharmacopeia hydrochloride and mesylate standards are used throughout this validation to demonstrate this equivalency. The validation demonstrates that this method may be used to quantitate phentolamine, regardless of its salt form.     

A generic fast solid-phase extraction high-performance liquid chromatography/mass spectrometry method for high-throughput drug discovery

High-performance liquid chromatography/mass spectrometry (HPLC/MS) is increasingly perceived to be an essential tool in drug discovery at many key steps, like drug screening, lead identification, ADME profiling, and drug metabolism and pharmacology studies. High-throughput screenings in the early phase for metabolic stability, protein binding, permeability (ADME) and bioavailability are widely used to weed out compounds that do not exhibit the necessary characteristics. For such high-throughput LC/MS studies, a generic LC/MS method that can be used for a variety of compounds is desired. In this study, we used a small set of compounds with a wide range of properties to guide method development, and achieved a sample throughput of 1.7 min/sample. Here, we present a generic fast method that achieves good peak separation and peak shape on conventional HPLC systems using a column-switching mechanism for on-line solid-phase extraction (SPE)-HPLC/MS analysis. The method has a linear response range from 1 to 500 nM for the tested compounds. When a larger set of 658 randomly picked small molecules were analyzed using this method, 612 were observed with good signal intensity and HPLC peak shapes. This generic fast SPE-LC/MS method has been used to screen more than 1.5 million compounds repetitively against over 200 protein targets for hit confirmation and semi-quantitation of binding constants from biological assays. Over 7000 different compounds for a variety of protein-binding assays have been studied using this method for quantitative analysis as well.     

New application of high-performance liquid chromatography for analysis of urinary C-peptide

A successful application of high-performance liquid chromatography for analysis of urinary C-peptide is described. Samples (1.0 ml of human urine) were first subjected to gel chromatography to remove interfering substances, and then applied to a reversed-phase column (LiChrosorb RP-18, 7 micron). The detection of C-peptide was performed using a highly specific radioimmunoassay. With the newly developed techniques, at least four forms of immunoreactive C-peptide were detected in human urine. One of these peptides was indistinguishable from authentic C-peptide. The present study has clearly demonstrated the heterogeneity of urinary C-peptide.     

Novel stationary phases for high-performance liquid chromatography analysis of cyclodextrin derivatives

Novel stationary phases were prepared for separation of cyclodextrins and cyclodextrin derivatives by bonding substituted aromatic groups (phenyl and naphthyl) to the silica gel matrix. Both the electron-withdrawing (nitro) and the hydrogen-donor/acceptor (amide or carbamide) substituents of the phenyl group play essential role in the separation of cyclodextrins and cyclodextrin derivatives. On the basis of the comparison of experimental data obtained on different columns the N-(4-nitrophenyl)-carbamide group bonded silica gel stationary phase was selected as the most effective one for analysis of cyclodextrin derivatives. Improved separation potency was observed for hydroxypropylated, methylated and several other cyclodextrin derivatives compared with the previously and presently used stationary phases. Owing to the strong retention of cyclodextrins and its derivatives on the selected column, detection of their decomposition products was easily achieved. Determination of unsubstituted, natural cyclodextrin as an impurity in the cyclodextrin derivatives was implemented. Identification and characterization of cyclodextrin derivatives in industrial products could also be performed.     

Direct analysis of food samples by high-performance liquid chromatography

A short review on the sample analysis of food samples by high-performance liquid chromatography is presented. The paper is focused on direct injection of liquid samples, automated solid-phase extraction and column switching techniques, on-line dialysis and application of restricted-access media.     

Quantitative analysis of Pentamax tablets by high-performance liquid chromatography

A procedure was proposed for quantitative analysis of Pentamax tablets, a new multicomponent drug, by high-performance liquid chromatography. Model mixtures containing all active and auxiliary components of the tablets were analyzed by the introduced/found method; the absence of a systematic determination error was verified. Preproduction tablet samples were analyzed. The results of the analysis match the requirements of normative technical documentation and technologic loads.     

Porphyrins as ligands for trace metal analysis by high-performance liquid chromatography

The role of porphyrins in the determination of metal ions by high-performance liquid chromatography (HPLC) is examined. In assessing the current status of the area, recent advances and developments are discussed and future potential is pointed out.     

Intact-protein trapping columns for proteomic analysis in capillary high-performance liquid chromatography

A new type of monolithic trapping columns with high mechanical strength was prepared by thin-layer sol–gel coating method and applied to trapping intact proteins for on-line capillary liquid chromatography. Monolithic trapping columns were fabricated by entrapping C8 reversed-phase particles into the capillary columns through a sol–gel network, which was formed by hydrolysis and polycondensation of methyltriethoxysilane. Hundreds times of trapping/untrapping for intact proteins were carried out. The trapping columns showed long-term stability up to 300 bar. Recovery, loading capacity and reproducibility of trapping columns were evaluated using four proteins. The recovery of four protein mixtures for the C8 monolithic trapping columns was 99.3% on average. The loading capacity of 5 mm × 320 μm i.d. C8 trapping columns for the protein mixtures was 30 μg. Day-to-day relative standard deviation (RSD) values for recoveries of protein mixtures on the same C8 trapping column ranged from 2.34 to 5.87%, column-to-column RSD values were from 3.01 to 6.81%. The C8 trapping columns were used to trap normal mouse liver intact proteins in a capillary liquid chromatography system. Results demonstrated high efficiency of the monolithic trapping columns for trapping intact proteins for proteomic analysis in on-line capillary liquid chromatography system.     

A phosphorus-sensitive molecular emission cavity analysis detector for high-performance liquid chromatography

Molecular emission cavity analysis is adapted to monitor organophosphorus compounds emerging from both normal-phase and reversed-phase columns. Reproducibility of detection was ca. 3% (using peak area measurements) and sub-microgram quantities were easily detectable. The detector consists of a water-cooled duralumin disc with 40 cavities drilled into its circumference. Eluent is collected in the cavities and rotated stepwise into a H₂—N₂—air flame where the HPO emission is monitored at 528 nm. During rotation, the bulk mobile phase is completely evaporated. Examples are given of separations of phosphate esters, phosphonates, pyrophosphonates, phosphonic acids and pesticides. The detector also operates as a sulphur-selective detector at 384 nm.     

Corticosteroid analysis in biological fluids by high-performance liquid chromatography

A sensitive, specific, and reproducible high-performance liquid chromatographic assay for the simultaneous determination of prednisone, prednisolone and cortisol in biological fluids was developed with dexamethasone as the internal standard. Samples are extracted with methylene chloride, washed with sodium hydroxide and then water, and chromatographed on a microparticulate silica gel column with UV detection at 254 nm. Sensitivity was greater than 15 ng for all four steroids. Specificity was supported by use of dual wave-length UV detection and/or radioimmunoassay. The assay has been applied in pharmacokinetic studies and a typical plasma concentration--time profile for the three steroids is presented for one subject who received 50 mg of prednisone.     

Metal-organic frameworks for reverse-phase high-performance liquid chromatography

Metal-organic framework MIL-53(Al) is explored for reverse-phase high-performance liquid chromatographic separation of a wide range of analytes from non-polar to polar, and acidic to basic solutes with high resolution, good selectivity, stability and reproducibility.     

Sub-microlitre permittivity detectors for high-performance liquid chromatography

A theoretical model is developed for the response of permittivity detectors working at radiofrequency for h.p.l.c. applications. Three types of cell were tested: a coil cell, a parallel-ring cell and a two-wire intrusive cell. All offer small cell volumes (ca. 700 nl for the coil cell, 9 nl for the parallel-ring cell and 40 nl for the intrusive two-wire cell). The last cell, with a 20-MHz Franklin oscillator, is shown to be most useful for conventional and microbore h.p.l.c., offering low instrument noise and reasonable sensitivity. The model is shown qualitatively to describe the observed response well. The coil cell also offers the possibility of monitoring conductivity changes in electrolyte solutions through frequency-change measurements. The theoretical response function indicates that the oscillator frequency is proportional to the square root of the eluate conductivity.