Characterization of complex hydrocarbon mixtures using on-line coupling of size-exclusion chromatography and normal-phase liquid chromatography to high-resolution gas chromatography

An on-line coupling of size-exclusion Chromatography (SEC), normal-phase liquid Chromatography (NPLC), and gas Chromatography (GC) for the characterization of complex hydrocarbon mixtures is described. The hyphenated system separates according to size, polarity, and boiling point. The use of size exclusion as the first separation step allows for the direct injection of complex (“dirty”) samples withont prior clean-up. SEC-NPLC coupling was realized using an on-line solvent evaporator based on fully concurrent solvent evaporation (FCSE) using a modified loop-type interface, vapor exit and co-solvent trapping. Complete reconcentration of the analytes was realized by the introduction of a cryogenic cold trap. For the subsequent hydrocarbon group-type separation an ammo-silica column with n-heptane as eluent was used. The NPLC-GC coupling was based on an on-column interface using partially concurrent solvent evaporation (PCSE) and an early vapor exit. Initial results obtained on the analysis of a residue from the atmospheric crude-oil distillation (a so-called long residue) are presented as an example of the enormous separation power of the SEC-NPLC-GC system. The application of the system for quantitative analysis has not yet been studied.     

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.     

Separation and determination of nitrobenzenes by micellar electrokinetic chromatography and high-performance liquid chromatography

A micellar electrokinetic chromatographic method and a high-performance liquid chromatographic method are proposed for the separation and determination of a mixture of 12 nitrobenzenes and their reduction products, namely 4-nitro-1,2-phenylenediamine, 4-nitro-1,3-phenylenediamine, 2-nitro-1,4-phenylenediamine, 2-nitroaniline, 3-nitroaniline, 4-nitroaniline, 4-amino-2-nitrophenol, 2-amino-5-nitrophenol, 2-amino-4-nitrophenol, 2-nitrophenol, 3-nitrophenol, and 4-nitrophenol. A solution of 50 mM sodium dodecyl sulfate and 10% ethanol in 23 mM sodium borate buffer was used as the electrophoretic medium. Good resolution could be obtained by the addition of tetrahydrofuran to the liquid chromatographic mobile phase. The retention and migration behavior of the nitrobenzenes are discussed.     

Monitoring of protein conformation by high-performance size-exclusion liquid chromatography and scanning diode array second-derivative UV absorption spectroscopy

Genetic methods now allow the rapid production of mutant proteins for structure-function analysis. To properly interpret any change in biologic activity resulting from modification in primary sequence, it is essential to monitor conformational changes resulting from mutations. Several methods allow low-resolution protein conformational analysis. One method, second-derivative UV absorption spectroscopy, is particularly useful for proteins containing tyrosine and/or tryptophan residues. Using high-performance size-exclusion liquid chromatography and scanning diode array detection we have demonstrated that it is possible to monitor the degree of aggregation as well as conformational perturbation for a series of interleukin-2 structural mutants. Furthermore, the combination of high-performance liquid chromatography and second-derivative UV absorption spectroscopy avoids a potential artifactual contribution in non-chromatographic analysis due to protein aggregation.     

On-line coupling of sol-gel-generated immunoaffinity columns with high-performance liquid chromatography.

The paper demonstrates the possibility to use sol-gel-generated immunoaffinity columns as selective sample preparation step in on-line combination with HPLC. In the past sol-gel-generated immunoaffinity columns have only been included in off-line sample preparation schemes. Compared with conventional RP-materials on-line coupling of sol-gel-generated silica matrices with a pore structure designed to retain antibodies poses additional problems caused by their lower pressure tolerance and by the necessity to match the mobile phases not only to take into account the chromatographic properties but also the conformational stability of the antibodies. These problems have been overcome by an on-line system which can be regarded as a prototype for similar systems which exploit the selectivity of sol-gel immunoaffinity columns. The system consists of a sol-gel-generated immunoaffinity column coupled to an RP enrichment column and an analytical column. The practicality of such systems is demonstrated using the example of anti-pyrene immunoaffinity columns applied for the determination of pyrene in aqueous solutions.     

Separation and determination of phospholipids in biological samples by high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method is developed for the determination of phospholipids in biological samples using a muPorasil silica column and a mobile phase of acetonitrile-methanol-85% phosphoric acid (90:3:1, v/v/v) at a flow rate of 0.80 mL/min. The effluent is monitored by a UV detector at 203 nm. With the method reported in this paper, phosphatidylinostol, phosphatidylserine, phosphatidylethnolamine, and phosphatidylcholine in biological samples are separated and detected successfully. The method is simple, rapid, and has excellent precision.     

Separation of drugs by high-performance liquid chromatography with porous polymer resins.

The separation of cold drugs and neuroleptics by high-performance liquid chromatogarphy with the porous polymer resin DVB-MCL-O (or 11-30-0), which is a styrene-divinylbenzene-methyl methacrylate copolymer substituted with hydroxymethyl groups, was studied. This copolymer was compared with the commercial porous polymers Hitachi gel 3011, 3011-0 and 3030. A very small theoretical plate height was obtained by using DVB-MCL-O and methanol-ammonia solution (99:1) as the stationary and mobile phases, respectively. This combination was found to be the most suitable for the rapid separation of condensed aromatic ring compounds.     

Determination of the enantiomeric composition of samples of cocaine by normal-phase high-performance liquid chromatography with UV detection

A high-performance liquid chromatographic method has been developed for the quantitation of the enantiomers of cocaine. Mixtures of the naturally occurring (−)-cocaine and synthetically produced (+)-cocaine were hydrolysed in water to (+) and (−)-benzoyl ecgonine. Esterification with an optically pure 2-octanol resulted in diastereoisomers that could be separated on bare silica gel using an acetonitrile-aqueous ammonium phosphate mobile phase.     

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.     

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.     

Separation of purines and pyrimidines by normal-phase high-performance liquid chromatography using dimethyl sulfoxide in binary and ternary eluents

Chromatographic systems with a silica sorbent and mobile phases containing dimethyl sulfoxide have been studied. It has been established that the substitution of isopropanol by dimethyl sulfoxide in binary eluents results in a specific selectivity of the chromatographic system and shows an improvement of the peak shape for the solutes under study. When mobile phases consisting of hexane, isopropanol and dimethyl sulfoxide (solvents with a limited mutual solubility) are used, changes in retention characteristics and peak symmetry are caused by a transition from adsorption to partition sorption mechanism. The stationary liquid phase is generated dynamically in the pores of silica, even in the mobile phases not saturated with a polar component. If the phase ratio of the column reaches 0.1, partition dominates over adsorption and such mixed partition-adsorption (MPA) systems show very good peak symmetry for the solutes under study. The investigation has shown that dimethyl sulfoxide-containing MPA systems are applicable in analytical practice.     

Pirmenol determination by high-performance liquid chromatography

A method for the determination of pirmenol in serum is presented in this paper. The method consists of extraction of pirmenol and chlorodisopyramide (internal standard) from serum at an alkaline pH using methylene chloride. The organic extract was analysed using high-performance liquid chromatography. The mobile phase consisted of 0.01 M K2HPO4 (pH 2.4)-acetonitrile (94:6, v/v) delivered at ambient temperature and 2 ml/min through a 25 cm x 0.4 mm C18 reversed-phase column. Detection of the compounds of interest was achieved at 210 nm. The analytical method demonstrated low intra- and inter-assay variation. During the analysis of patient samples and a therapeutic drug mixture test serum, no substances that interfered with pirmenol detection were found. The method is shown to be stable, accurate, selective and sensitive enough to be utilized for the analysis of multiple samples such as may be encountered in clinical or research situations.     

Separation and determination of aminohalogenbenzophenones by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection has been developed for the determination of three aminohalogenbenzophenones: 2-amino-2',5-dichlorobenzophenone, 2-amino-5-chlorobenzophenone and 2-amino-5-bromo-2'-fluorobenzophenone, metabolites of benzodiazepinooxazoles and other psychotropic drugs. A mobile phase of methanol-water (65:35), containing 5 mM KH2PO4 appeared to be the optimal when a 4-microns, 60-A Nova-Pak C18 column and a flow-rate of 0.75 ml/min (130 bar) were used. The temperature was optimized at 30 degrees C. The amperometric detector, equipped with glassy carbon electrode, was operated at 1.3 V versus Ag/AgCl in the DC mode. The method was applied to the determination of these compounds at two concentration levels: ppm and ppb (ng/cm3) using 2-amino-5-chlorobenzophenone as internal standard. The limit of determination was 750 pg/ml of biological fluid for each compound, and recoveries greater than 97% were obtained for spiked samples of urine and serum, using C18 Sep-Pak cartridges in the sample clean-up procedure.     

Separation and determination of quaternary ammonium compounds by high-performance liquid chromatography with a hydrophilic polymer column and conductometric detection

An HPLC method has been developed for separation and determination of long alkyl chain quaternary ammonium compounds. A column packed with a hydrophilic polymer packing, Shodex Asahipak GF-310 HQ, and a water–acetonitrile mixture containing 4,4′-bipyridyl and hydrochloric acid were used to depress hydrophobic adsorption of the quaternary ammonium compounds and increase the sensitivity of the conductometric detection with a micromembrane suppressor. Dodecyltrimethylammonium, cetyltrimethylammonium, tetradecyldimethylbenzylammonium and stearyltrimethylammonium ions can be completely separated from one another and quantified at 0.1 nmol level.     

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.     

Separation of porphyrin isomers by high-performance liquid chromatography

A high-speed reversed-phase high-performance liquid chromatographic method using an octadecylsilyl 3 cm long (3 microns particle size) column to separate the free acids of uroporphyrins I and III and coproporphyrins I and III from each other, and from the type I isomers of several other porphyrin carboxylic acids, is described. Separation of the porphyrins was achieved in less than 8 min, and injections were possible every 12 min. The detection limits of uroporphyrin, coproporphyrin, and mesoporphyrin were 75, 45, and 35 fmol (at a signal-to-noise ratio of 2), respectively. Application of the method to the determination of urinary and liver porphyrin patterns is shown.