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.     

Analysis of 5-hydroxy-7-methoxyflavones by normal-phase high-performance liquid chromatography

The separation and identification of flavones present in a chloroform extract of Baccharis trinervis leaves was investigated. The chromatographic system consisted of a amino-bonded column, gradient elution from hexane-chloroform (85:15) to chloroform-acetonitrile (40:60) and detection at 346 nm. Four flavones were found. From NMR and MS data they were identified as 5-hydroxy-7,4′-dimethoxyflavone (I), 5-hydroxy-7,3′,4′-trimethoxyflavone (II), 5,3′-dihydroxy-7,4′-dimethoxyflavone (III) and 5,4′-dihydroxy-7-methoxyflavone (IV). Flavone II and III have not been found in Baccharis trinervis before. The chromatographic system showed good selectivity for the separation of the flavones. The relation between t_R and the structure is discussed.     

Analysis of purines and pyrimidines by mixed partition-adsorption normal-phase high-performance liquid chromatography

This paper summarizes the results in the development of mixed partition-adsorption (MPA) normal-phase high-performance liquid chromatography published in the last 10 years. The MPA normal-phase systems are an alternative approach not only to the adsorption normal-phase mode but also to the most widely used reversed-phase mode in the separation area of purine and pyrimidine derivatives. It is shown that the MPA systems are applicable in analytical practice.     

New stationary phases for normal-phase high-performance liquid chromatography (NP-HPLC)

3-(2,3-Dihydroxy-propoxy)-propyl-silica (diolsilica) was modified to bonded stationary phases for normal-phase high-performance liquid chromatography (NPLC) carrying phosphorous ester, boronic ester, and nitric acid ester groups. The NPLC phases were investigated with respect to their properties towards separations of polyaromatic hydrocarbons, and chlorinated and nitrated environmental pollutants. Aminopropyl-silica and nitrooxy-propyl-silica (the latter synthesized in our working group) are presented for comparison. Depending on the functionality used for the modification the retention behavior can significantly be changed towards the three analyte classes. Especially 2,2,2-tribromoethyl-phosphoromorpholino-chloridate modified diol-silica, 2,2,2-tribromoethyl-dichlorophosphate modified diol-silica and nitrated diol-silica (nitrooxy-propoxypropyl-silica) show specific properties for the separation of polyaromatic hydrocarbons, chlorinated analytes and nitrated analytes. Preparation and packing of the adsorbents are described. Principal Component Analysis (PCA) is used as a chemometric tool for effective data reduction and visualization of the results in terms of retention behavior of the stationary phases. Received: 4 August 1997 / Revised: 3 December 1997 / Accepted: 7 December 1997     

Determination of dexamethasone in bovine tissues by coupled-column normal-phase high-performance liquid chromatography and capillary gas chromatography-mass spectrometry

A new method for the determination of dexamethasone in bovine liver and muscle tissues has been developed. Crude tissue extracts were obtained by means of a three-phase liquid-liquid extraction scheme. The resulting residue was subjected to coupled-column normal-phase high-performance liquid chromatography which served to isolate the drug for the purpose of screening and quantification. Sample was injected onto the first column of the system, a phenyl column, from which a heart-cut was diverted to a short silica column which retained dexamethasone. The contents of this column were backflushed onto a cyanopropyl column which isolated dexamethasone. Mobile phases consisted of hexane modified with 2-propanol, acetic acid, and water. Analysis of each sample was completed in 15 min. Quantitation was performed by external standard calibration of ultraviolet response at 239 nm. Limits of detection were estimated to be 4 and 6 ppb in muscle and liver, respectively. In addition to screening and quantitation, the coupled-column system purified tissue extracts for gas chromatographic-mass spectrometric analysis which, in the selected-ion monitoring mode, confirmed the identity of the trimethylsilyl-enol-trimethylsilyl derivative of dexamethasone.     

Detailed analysis of petroleum hydrocarbon attenuation in biopiles by high-performance liquid chromatography followed by comprehensive two-dimensional gas chromatography

Enhanced bioremediation of petroleum hydrocarbons in two biopiles was quantified by high-performance liquid chromatography (HPLC) followed by comprehensive two-dimensional gas chromatography (GCXGC). The attenuation of 34 defined hydrocarbon classes was calculated by HPLC–GCXGC analysis of representative biopile samples at start-up and after 18 weeks of biopile operation. In general, a-cyclic alkanes were most efficiently removed from the biopiles, followed by monoaromatic hydrocarbons. Cycloalkanes and polycyclic aromatic hydrocarbons (PAHs) were more resistant to degradation. A-cyclic biomarkers farnesane, trimethyl-C13, norpristane, pristane and phytane dropped to only about 10% of their initial concentrations. On the other hand, C29–C31 hopane concentrations remained almost unaltered after 18 weeks of biopile operation, confirming their resistance to biodegradation. They are thus reliable indicators to estimate attenuation potential of petroleum hydrocarbons in biopile processed soils.     

Antioxidant analysis in edible oils and fats by normal-phase high-performance liquid chromatography

Following a simple dilution in the appropriate phase, the sample is injected directly onto either of two normal-phase high-performance liquid chromatography systems (3,5-di-tert.-butyl-4-hydroxytoluene or 3-tert.-butyl-4-hydroxyanisole-tert.-butylhydroquinone) with UV detection at 280 nm. An isocratic ternary mobile phase, incorporating acetonitrile as the polar modifier, has been found to facilitate such an approach, thereby avoiding the discriminatory and recovery problems inherent in other techniques requiring prior sample manipulations. The three most commonly used antioxidants may be estimated at levels down to 3 ppm (3,5-di-tert.-butyl-4-hydroxytoluene or 3-tert.-butyl-4-hydroxyanisole) and 10 ppm (tert.-butylhydroquinone) within 30 min.     

Ion-pair reversed-phase high-performance liquid chromatography for trace metal preconcentration followed by ion-interaction chromatography

Ion-interaction chromatography of Plasmocorinth B (a disulphonated azo dye) complexes of Co(III), Cu(II), Fe(III), Ga(III), In(III), Ni(II), V(V) and Zr(IV) was studied. The behaviour of two different reversed-phase C₁₈ columns (5 and 10 μm) was compared and an on-line enrichment procedure was developed following the optimization of eluent (pH, ligand concentration, ionic strength and organic modifier). The described technique, applied to the analysis of metal ions at μg/1 levels in natural waters, gave satisfactory precision and accuracy in comparison with inductively coupled plasma atomic emission spectroscopic results.     

Determination of serum estradiol by normal-phase high-performance liquid chromatography with peroxyoxalate chemiluminescence detection

Estradiol extracted with an ODS minicolumn from serum (500 μl) is derivatized with 5-dimethylamino-1-naphthalenesulfonyl (dansyl) chloride at room temperature for 80 min, purified on the ODS minicolumn, separated on silica gel columns (150 × 1.9 mm i.d. and 100 × 1.9 mm i.d.) with n-hexane/chloroform/ethanol (70:30:0.1) as eluent at 3 ml min^?1 and detected by the chemiluminescence emission produced in a post-column reaction with bis-(2,4,6-trichlorophenyl) oxalate (8 mM in chloroform containing 100 mM triethylamine) and hydrogen peroxide (600 mM in methanol containing 6% (v/v) sodium acetate buffer at pH 4.0). The overall recovery of estradiol from serum is ca. 90% and the detection limit is ca. 50 pg.     

Chromatographic behaviour of low molar-mass polyesters in normal-phase high-performance liquid chromatography

Summary The normal-phase chromatographic retention behaviour of polyesters on bare silica and on a polymer-based polyamine (PA) column has been studied with a variely of binary mobile phases under isocratic conditions. The dependence of experimental retention data on the degree of polymerization (p) and on mobile phase composition (φ) was characterized by to an approach developed by Jandera et al. The bulky repeating unit and the relatively highly polar end groups of the polyesters both had a large influence on retention behaviour. The two effects in combination explain the molar-mass-independent retention observed experimentally at a particular mobile phase composition for all the mobile phase—stationary phase combinations investigated. These conditions were found to be independent of the type of end group. End group separation on a silica column improves when the polarity of the less polar solvent is increased. End group separation is better on the PA column because of a greater difference between the adsorption energy of the alcohol and acid end groups. Better prediction of retention data on the PA column was achieved by use of an approach which assumes two different types of adsorption site. Results enabled further understanding of retention behaviour in normalphase gradient polymer-elution chromatography (NPGPEC) and explained both the dependence of the order of elution onp and differences between the end-group selectivity of different systems.     

The electrolytic efficiency of amperometric detection in normal-phase high-performance liquid chromatography

The electrolytic efficiency of the wall-jet detector in normal-phase high-performance liquid chromatography (between 0.1 and 5%) is significantly less than its efficiency in reversed-phase separations. A large-volume wall-jet cel with a Ag/Ag⁺ reference serves as the detector system in evaluating the effects of various chromatographic conditions on efficiency. Anthraquinone, phenanthrenaquinone (both at 10^-3–10^-4 M) and three estrogen gen steroids (at 10^-2–10^-3 M) are used as samples with ethanol/ hexane eluents. The effect of pH depends on the sample; increased ethanol concentrations and lower flow rates improve the efficiency.     

Determination of melengestrol acetate in bovine tissues by automated coupled-column normal-phase high-performance liquid chromatography

A method has been developed for the determination of melengestrol acetate in bovine tissues at lower levels than previously reported. Liquid-liquid extraction of tissue homogenates provided crude clean-up while final isolation, screening, and quantification was done on-line with an automated, normal-phase, coupled-column high-performance liquid chromatographic system. The chromatographic system included phenyl and silica analytical columns for the purposes of isolation and final separation, respectively. These columns provided a large difference in selectivity when operated under normal-phase conditions which allowed for the efficient isolation of melengestrol acetate from the complex tissue extracts. Mobile phases were composed of hexane and dichloromethane modified with methanol and water. Transfer and enrichment of the analyte from the primary phenyl column to the silica column was via a short (12 mm x 4 mm I.D.) silica column. Regeneration and equilibration of the phenyl column was performed after the injection of each tissue extract and was accomplished simultaneously while analytical separation occurred on the final silica column. Routing of the mobile phases and regeneration solvent was performed with automated switching valves. The total time required for each analysis was 12 min. Quantification is demonstrated using external standards with UV detection at 287 nm. The overall recovery of the method was 86% with a coefficient of variation of 9.84% at the 10 ppb [the American billion (10(9] is used in this article] level in bovine liver extracts.     

Determination of oestriol in pregnancy urine by normal-phase high-performance liquid chromatography with electrochemical detection

A method for the determination of oestriol in pregnancy urine by normal-phase high-performance liquid chromatography with electrochemical detection is described. A large-volume wall-jet cell with an Ag-Ag+ reference electrode was used as the detector system. The limit of detection obtained is comparable to that of electrochemical detection following reversed-phase liquid chromatography. One of the advantages of electrochemical detection with normal-phase systems is that adsorption problems are minimized.     

Simultaneous determination of four lignan compounds in Herpetospermum caudigerum by normal-phase high-performance liquid chromatography

A normal-phase high-performance liquid chromatographic method with diode array UV detection is developed for the simultaneous quantitation of four lignan compounds in Herpetospermum caudigerum. This analysis provides a good resolution and reproducibility. Chromatography is carried out with a mobile phase of N-hexane-dichlormethane-methanol (42.5:42.5:5, v/v) at a flow rate of 1.0 mL/min. UV detection is performed at 280 nm. The calibration curve for lignans concentration is linear over the range of 2.10 to 42.0 microg/mL, 15.26 to 305.2 microg/mL, 6.15 to 123.0 microg/mL, and 6.24 to 124.8 microg/mL, respectively. The limit of quantitation and detection for compounds 1, 2, 3, and 4 is 1.31, 2.74, 2.63, and 2.17 microg/mL and 0.28, 0.25, 0.27, and 0.31 microg/mL, respectively. The validation data show that the assay is sensitive, specific, accurate, and reproducible for the simultaneous quantitation of four compounds. This rapid method is therefore appropriate to quantitate these lignans in Herpetospermum caudigerum.     

Characterization of alkyl polyglycosides by both reversed-phase and normal-phase modes of high-performance liquid chromatography

Alkyl polyglycosides today represent the most important sugar surfactant. Nonionic sugar surfactants produced via different synthetic routes are mixtures of alkyl homologues, oligomers, anomers and isomers. Alkyl homologues and oligomers of alkyl mono- and diglucosides were separated by reversed-phase high-performance liquid chromatography (HPLC) with methanol-water as the mobile phase using a gradient elution. The gradient was optimized in respect to a simultaneous separation of alkyl glycosides according to their alkyl chain length and alkyl polyoxyethylene glucosides with regard to their length of the polyoxyethylene spacer. The separation of alkyl glycosides into alpha- and beta-anomers was carried out by normal-phase HPLC with isooctane-ethyl acetate (60:40, v/v)-2-propanol in the gradient mode. Light scattering detection was used. Matrix-assisted laser desorption ionization time-of-flight mass spectra of alkyl glucosides and dodecyl glucosides with oxyethylene spacer groups are presented.     

Identification of medicinal additives in animal feedingstuffs by high-performance liquid chromatography

A method is described for the detection of 25 drugs used as prophylactics or as growth promoters in commercial animal feedingstuffs. The sample was extracted with aqueous acetonitrile, the co-extractives were removed with a silica cartridge and the eluate was examined by high-performance liquid chromatography, using two columns and five mobile phases. Thirteen animal ++feedingstuffs containing different combinations of drugs were prepared and used to test the method.     

Fractionation of apple procyanidins according to their degree of polymerization by normal-phase high-performance liquid chromatography

A new method was developed for the fractionation of procyanidin oligomers according to their degree of polymerization. Monomeric flavan-3-ols and low molecular mass procyanidins were selectively extracted from the lyophilized powder of apple condensed tannins (ACTs) by methyl acetate extraction. Sequentially, the separation of each oligomer from dimer to pentamer in this extract was carried out by normal-phase high-performance liquid chromatography using a silica-beads packed column. The best separation was achieved with a mobile phase system containing hexane; (1) hexane–methanol–ethyl acetate, (2) hexane–acetone. These sequential treatments can be easily adapted to large-scale fractionation.     

Determination of 3-nitrobenzanthrone in surface soil by normal-phase high-performance liquid chromatography with fluorescence detection

A sensitive method for determining 3-nitrobenzanthrone in surface soil was developed. 3-Nitrobenzanthrone was reduced to 3-aminobenzanthrone by refluxing at 60 degrees C with hydrazine and Raney nickel for 20 min, and 3-aminobenzanthrone was determined by normal-phase high-performance liquid chromatography (HPLC) with fluorescence detection. We used a cyanopropyl stationary phase and an n-hexane-ethyl acetate (3:1, v/v) mobile phase, since 3-aminobenzanthrone exhibits fluorescence in a low-polarity solvent such as n-hexane or ethyl acetate, but not in a polar solvent such as water or methanol. The calibration graph showed good linearity (r2>0.9999) in the range of 0.002-2 ng, and the detection limit was 0.002 ng (S/N=3). 3-Nitrobenzanthrone in extracts from surface soil collected in the Chubu area (central area) of Japan was determined after clean-up using silica gel chromatography and high-performance liquid chromatography on a pyrenylethyl stationary phase. The concentration of 3-nitrobenzanthrone in surface soil was determined in the range of 1.2-1020 pg/g soil.     

Automated normal-phase preparative high-performance liquid chromatography as a substitute for flash chromatography in the synthetic research laboratory

An automated normal-phase preparative HPLC system was developed in order to omit time-consuming flash column chromatography in the synthetic research laboratory. The system is equipped with steel columns packed with spherical 12 microm silica and is able to separate samples in a range of 0.1-10 g depending on the column diameter and chromatographic problem. It was designed to be used as an open access instrument in the research department. The general users select from binary gradient programs after running an analytical TLC with the raw product. The HPLC instrument was fully controlled by the Chromeleon software from Dionex. A Gilson 215 robot served as injector/collector.