Quantitative determination of underivatized anti-convulsant drugs by high resolution gas chromatography with support-coated open tubular columns.

A method is described to prepare routinely high resolution support-coated open tubular (SCOT) columns. A siliceous support material (Cab-O-Sil) is deactivated with benzyltriphenylphosphonium chloride and deposited on the inside wall of a glass capillary column. After additional coating with a polar stationary phase (OV-225) a number of anticonvulsant drugs can be analyzed without prior derivatization. The columns described show high plate numbers and do not deteriorate in use. The repeatability of the gas chromatographic analysis is better than 1.2% (Orel). The minimum detectable quantity is of the order of 10(-10) g. An improved procedure for isolating the drugs from serum is given, which results in an extraction recovery of better than 90% for the drugs of interest.

A new method for the determination of L-dopa and 3-O-methyldopa in plasma and cerebrospinal fluid using gas chromatography and electron capture negative ion mass spectrometry.

L-3-(3,4-Dihydroxyphenyl)alanine (DOPA) and its 3-O-methyl metabolite (OMD) were measured in plasma and cerebrospinal fluid by a new assay which combines N,O-acetylation of amino acids in aqueous media, preparation of pentafluorobenzyl esters under anhydrous conditions, and analysis by gas chromatography-electron capture negative ion mass spectrometry. The N,O-acetyl, carboxy-PFB derivatives gave abundant carboxylate anions ([M-CH2C6F5]-) which were suitable for sensitive analysis using selected ion monitoring. Plasma and CSF samples were sufficiently purified by a simple organic solvent extraction. Analytical recovery for DOPA was 100.2 +/- 3.7% at the level of 100 nmol/l. Analysis of DOPA in plasma was performed with a relative standard deviation of 5%. The limit of quantitation in plasma and CSF was at the sub-nmol/l level. In healthy adults, DOPA concentration in plasma was 9.0 +/- 2 nmol/l (n = 11) and in CSF 3.5 +/- 0.9 nmol/l (n = 9). The concentration of OMD in plasma was 99.1 nmol/l (pool of 24 samples) and 15.3 nmol/l in CSF (pool of 12 samples). Measurement of 5-[2H]DOPA and 5-[2H]OMD in plasma of a healthy individual who had been orally loaded with 3,5-[2H2]tyrosine (150 mg kg body wt) was possible for several hours after the load.

Screening of UV-absorbing solutes in uremic serum by reversed phase HPLC--change of blood levels in different therapies.

In order to screen UV-absorbing solutes in large numbers of uremic serum samples, an automated liquid chromatographic method was developed. The method proved to be reliable and reproducible in more than 500 analyses. HPLC separation was performed using gradient elution on a 25-cm Ultrasphere Octyl reversed phase column, with 5 microns particles. Characteristic profiles for the uremic state were obtained in the analyses of serum samples of 43 uremic patients before and just after artificial kidney treatment; hemodialysis (n = 14), hemodiafiltration (n = 13) and hemofiltration (n = 16). In these profiles 20-40 peaks were resolved of which nine were 'quantitated' by peak height relative to a standard. Of these solutes creatinine, uracil, uric acid, hypoxanthine, indoxylsulfate, tryptophan and hippuric acid were identified. The heterogeneity of the population of uremic patients, with respect to the UV-absorbing solutes, was estimated. Significant differences of solute blood level changes during hemodialysis, hemodiafiltration and hemofiltration, were observed.

On-line coupling of equilibrium-sorptive enrichment to gas chromatography to determine low-molecular-mass pollutants in environmental water samples.

On-line combination of equilibrium sorptive enrichment and gas chromatography is used for the analysis of a group of pollutants varying widely in polarity and volatility in aqueous samples at trace levels. For the ESE process open-tubular traps were used. The newly developed hyphenated method shows a high sensitivity for all the compounds under study. The detection limits were typically between 0.1 and 1 microg/l. The sample volumes required for the compounds to reach equilibrium with the stationary phase are in the range of 20 ml for the aromatic hydrocarbons included in the study (benzene, toluene and p-xylene), to 200 ml for epichlorohydrin and dichlorohydrin. Within- and between-day precision of the absolute peak areas varied between 3 and 16%. The performance of the new method was tested by the analysis of different environmental water samples.

Chromatographic properties of reversed-phase stationary phases under pressure- and electro-driven conditions.

Seven different reversed-phase (RP) stationary phases were examined under high-performance liquid chromatographic (pressure-driven, HPLC), and capillary electrochromatographic (electro-driven, CEC) conditions. Characterization of the stationary phases was performed following well-established test procedures providing a number of distinct column descriptors: hydrophobicity, hydrophobic selectivity and silanol activity. These parameters were used to describe the behavior of the RP-columns under both HPLC and CEC conditions. It is shown that chromatographic characteristics of porous RP-phases greatly depend on the mode of operation. By contrast, column descriptors of a non-porous viz. solid RP-phase material hardly differed for HPLC and CEC conditions.

Preparation and characterization of monolithic polymer columns for capillary electrochromatography.

A series of micro-monolithic columns with different porosities were prepared for capillary electrochromatography (CEC) by in-situ copolymerization of butyl methacrylate, ethylene glycol dimethacrylate, and 2-acrylamido-2-methyl-1-propane-sulfonic acid in the presence of a porogen in fused-silica capillaries of 100 microm I.D. Different column porosities were obtained by changing the ratios of monomers to porogenic solvents. Columns were investigated and evaluated under both pressure-driven (high-performance liquid chromatography, HPLC) and electro-driven (capillary electrochromatography, CEC) conditions. Each column exhibited different efficiency and dependency on flow velocity under electro-driven conditions. Abnormally broad peaks for some relatively bulky molecules were observed. Possible explanations are discussed. The differences in column efficiency and retention behavior between the two eluent-driven modes were studied in detail. In addition, other column properties, such as morphology, porosity, stability and reproducibility, were extensively tested.

Novel preconcentration technique for on-line coupling to high-speed narrow-bore capillary gas chromatography: sample enrichment by equilibrium (ab)sorption. I. Principles and theoretical aspects.

In recent years, there has been substantial progress in the field of high-speed narrow-bore capillary gas chromatography (GC) in general, and in the development of (trans)portable gas chromatographs for fast and accurate analysis in field applications in particular. Due to the limited (concentration) sensitivity of instrumentation for high-speed (portable) GC, environmental applications of this technique frequently require a preconcentration step. The equilibrium (ab)sorption technique described in this article was found to be very promising for on-line coupling to high-speed narrow-bore capillary GC and field portable GC instruments. Enrichment factors up to at least 100 can be obtained rapidly without the use of complicated instrumentation. The new preconcentration technique is shown to have clear advantages over enrichment based on conventional adsorption, i.e. thermal desorption techniques. It can be carried out at ambient trapping temperatures, gives uniform desorption profiles, reduces water effects, uses inert adsorption materials and does not require a (cryogenic) refocusing step. Moreover, the new preconcentration method allows the enrichment factors to be predicted from tabulated chromatographic data, thereby facilitating calibration.

Analysis of higher polyamide-6 oligomers on a silica-based reversed-phase column with a gradient of formic acid as compared with hexafluoroisopropanol.

The analysis of polyamide-6 oligomers and polymer is usually performed with expensive fluorinated alcohols like 2,2,2-trifluoroethanol (TFE) or 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). Formic acid is well known as a mobile phase additive to adjust pH in reversed-phase high-performance liquid chromatography. However, formic acid is seldom used as a modifier to perform gradient elution chromatography on octadecyl-modified silica-based columns. Here we demonstrate the determination of cyclic and linear polyamide-6 oligomers using formic acid as a modifier on an octadecyl-modified silica-based column. This column was shown to be stable for more than 5000 column volumes, even when a mobile phase of 65-95% formic acid in water at a flow of 1 ml/min is applied. With formic acid under the conditions used (65-95% formic acid in water) the oligomers are retained on the column, while the polymer does not precipitate. In comparison, during adsorption and separation with a HFIP gradient, precipitation of the polymer occurs. The implications of the different separation mechanisms, i.e., adsorption vs. precipitation chromatography are discussed. Loadability is shown to be much better with the formic acid system. However, with formic acid as a modifier UV detection below 250 nm is not feasible. The less sensitive evaporative light scattering detector is used to detect the polyamide oligomers in the formic acid phase. In addition it is shown that capillary zone electrophoresis (CZE) with UV-absorbance detection using HFIP is an attractive combination as HFIP is UV-transparent and CZE allows low modifier consumption.

Evaluation of time-of-flight mass spectrometric detection for fast gas chromatography.

Separations below 1 s of a mixture of organic compounds ranging from C5 to C8 have been performed to investigate the performance of a time-of-flight mass spectrometer in fast gas chromatography. The gaseous samples were focussed on a cold trap, and then injected after thermal desorption to obtain the required narrow input band-widths. Also, to obtain a very fast separation, a short narrow bore column was used, operated at above-optimum inlet pressures. With this system, it was possible to identify ten compounds within 500 ms, showing peak-widths (2.354sigma) as narrow as 12 ms. The spectral acquisition rate used for these analyses was 500 Hz. The quality of the recorded spectra and the comparison with library spectra was very high. Deconvolution algorithms offer the possibility of identifying overlapping peaks. It is shown that the spectral scan speed of the time-of-flight mass spectrometer is high enough for very fast separations.

Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method. III. Separation mechanism and gradient optimization.

The first six linear and cyclic oligomers of polyamide-6 can be quantitatively determined in the polymer using HPLC with the sandwich injection method and an aqueous acetonitrile gradient. In this final part of the triptych concerning the determination of the oligomers in polyamide-6, the irregular elution behavior of the cyclic monomer compared to the cyclic oligomers was investigated. We also optimized the separation of the involved polyamide oligomers, with respect to gradient steepness, stationary phase, column temperature and mobile phase pH. The irregular elution behavior of the cyclic monomer could be caused by its relatively large exposed/accessible hydrophobic surface, which permits relatively easy penetration into the hydrophobic stationary phase giving extra retention. The dipole moment of the different oligomers was used as a measure for this exposed/accessible hydrophobic area to correlate the retention factors using quantitative structure-retention relationships. We also studied the retention behavior of the polyamide, which is injected each run directly onto the column and modifies the stationary phase. Using a 250-microl post gradient injection zone of formic acid on a 250x3 mm Zorbax SB-C18 column, the polyamide could be effectively removed from the stationary phase after each separation. The linear solvent strength (LSS) model was used to optimize the separation of the first six linear and cyclic oligomers. As the LSS model assumes a linear correlation between the modifier concentration and the logarithm of the retention factor and the cyclic monomer and dimer show extreme curvation of this relation in the eluting region, we investigated different models to predict gradient elution from isocratic data. A direct translation of the isocratic data to gradient retention times did not yield adequate retention times using the LSS model. It was found that the LSS model worked acceptably if gradient retention times were used as input data. Even for fast non-linearly eluting components, an average error of 0.4 resolution units of 4sigma was obtained. Using the LSS model in combination with different column temperatures and mobile phase pH values, a separation of the first six linear and cyclic oligomers was accomplished.

Endgroup-based separation and quantitation of polyamide-6,6 by means of critical chromatography.

Polyamide-6,6 is a polycondensation product from the two monomers adipic acid and 1,6-hexamethylenediamine. Depending on the reacted amount of these monomers, different ratios of amine and carboxylic acid endgroups can be formed. Besides linear chains, cyclic polyamides will also be formed. Using critical chromatography, polyamide-6,6 can be separated independently of molar mass. Retention is based solely on endgroup functionality. It is demonstrated that high-molecular-mass polyamide-6,6 (Mw approximately 20,000-30,000) can be separated using this approach. The separation was optimized by using different parameters, such as percentage modifier, temperature and pressure. The concentration of phosphoric acid was used for selective retention of the different endgroup functionalities. Using this property, a new method called critical gradient chromatography was performed where the mobile phase changes from a weak to a strong solvent with respect to the endgroup functionality, while retaining the critical conditions of the backbone unit. Quantification using UV detection is discussed.

Non-aqueous capillary electrophoresis using non-dissociating solvents. Application to the separation of highly hydrophobic oligomers.

The application of non-aqueous capillary electrophoresis for the separation of very hydrophobic oligomers has been studied. N-Phenylaniline oligomers having degrees of polymerisation (n) of 2, 4, 6, and 8 were taken as model compounds. Capillary electrophoresis could be performed using a mixture of non-aqueous solvents with a high percentage of solvents with a low dielectric constant. These solvents, such as tetrahydrofuran (THF), chloroform or dichloromethane, are needed to solubilise the hydrophobic solutes in the electrolyte. The composition of the solvent mixture and the nature of the acid added to the electrolyte, which is needed to obtain electrophoretic motion of the N-phenylaniline oligomers, are discussed in detail. Next, other parameters such as ionic strength, injection time, electric field, and temperature were investigated too and their influence on the separation is discussed as well. The existence of a reversed (anodic) electroosmotic flow in a fused-silica capillary containing a THF-methanol mixture under acidic conditions is reported.

Hydrodynamic aspects of slurry packing processes in microcolumn liquid chromatography.

A Stokesian dynamics computer simulation based method is presented for the estimation of the bed porosity of slurry-packed capillary liquid chromatography (LC) columns. A colloidally well-described reversed-phase stationary phase-slurry liquid suspension was used as a model system. The applied simulation method takes into account the velocity of the slurry and colloidal interaction forces, as well as inter-particle hydrodynamic interactions. The predicted bed porosities suggest that a lower slurry velocity leads to a denser packing structure due to the increased effect of colloidal repulsion effects. The results of the simulations were compared with the external porosity and chromatographic performance of capillary LC columns that were packed at different filtration and compaction pressures. However, the trends that were observed in the experimental results suggest that hydrodynamic packing parameters have no or little effect on the chromatographic performance of capillary LC columns. Within the experimental parameter window, the chromatographic performance and the column porosity were not influenced by the filtration and compaction pressure, nor by the duration of the compaction process.

Optimisation and characterisation of silica-based reversed-phase liquid chromatographic systems for the analysis of basic pharmaceuticals.

Reversed-phase liquid chromatography using silica-based columns is successfully applied in many separations. However, also some drawbacks exist, i.e. the analysis of basic compounds is often hampered by ionic interaction of the basic analytes with residual silanols present on the silica surface, which results in asymmetrical peaks and irreproducible retention. In this review, options to optimise the LC analysis of basic pharmaceutical compounds are discussed, i.e. eluent optimisation (pH, silanol blockers) and stationary phase optimisation (development of new columns with minimised ionic interactions). The applicability of empirical based, thermodynamically based and test methods based on a retention model to characterise silica-based reversed phase stationary phases, as well as the influence of the eluent composition on the LC analysis of basic substances is described. Finally, the applicability of chemometrical techniques in column classification is shown.

High-speed gas chromatography: an overview of various concepts.

An overview is given of existing methods to minimise the analysis time in gas chromatography (GC) being the subject of many publications in the scientific literature. Packed and (multi-) capillary columns are compared with respect to their deployment in fast GC. It is assumed that the contribution of the stationary phase to peak broadening can be neglected (low liquid phase loading and thin film columns, respectively). The treatment is based on the minimisation of the analysis time required on both column types for the resolution of a critical pair of solutes (resolution normalised conditions). Theoretical relationships are given, describing analysis time and the related pressure drop. The equations are expressed in reduced parameters, making a comparison of column types considerably simpler than with the conventional equations. Reduction of the characteristic diameter, being the inside column diameter for open tubular columns and the particle size for packed columns, is the best approach to increase the separation speed in gas chromatography. Extremely fast analysis is only possible when the required number of plates to separate a critical pair of solutes is relatively low. Reducing the analysis time by reduction of the characteristic diameter is accompanied by a proportionally higher required inlet pressure. Due to the high resistance of flow of packed columns this seriously limits the use of packed columns for fast GC. For fast GC hydrogen has to be used as carrier gas and in some situations vacuum-outlet operation of capillary columns allows a further minimisation of the analysis time. For fast GC the columns should be operated near the conditions for minimum plate height. Linear temperature programmed fast GC requires high column temperature programming rates. Reduction of the characteristic diameter affects the sample capacity of the "fast columns". This effect is very pronounced for narrow-bore columns and in principle non-existing in packed columns. Multi-capillary columns (a parallel configuration of some 900 narrow-bore capillaries) take an intermediate position.

Molecular mechanism of retention in reversed-phase high-performance liquid chromatography and classification of modern stationary phases by using quantitative structure-retention relationships.

Quantitative structure-retention relationships (QSRRs) were derived for logarithms of retention factors normalised to a hypothetical zero percent organic modifier eluent, log kw, determined on 18 reversed-phase high-performance liquid chromatography (RP-HPLC) columns for 25 carefully designed, structurally diverse test analytes. The study was aimed at elucidating molecular mechanism of retention and at finding an objective manner of quantitative comparison of retention properties and classification of modern stationary phases for RP-HPLC. Three QSRR approaches were employed: (i) relating log kw to logarithms of octanol-water partition coefficient (log P); (ii) describing log kw in terms of linear solvation-energy relationship-based parameters of Abraham; (iii) regressing log kw against simple structural descriptors acquired by calculation chemistry. All the approaches produced statistically significant and physically interpretable QSRRs. By means of QSRRs the stationary phase materials were classified according to the prevailing intermolecular interactions in the separation process. Hydrophobic properties of the columns tested were parametrized. Abilities of individual phases to provide contributions to the overall retention due to non-polar London-type intermolecular interactions were quantified. Measures of hydrogen-bond donor activity and dipolarity of stationary phases are proposed along with two other phase polarity parameters. The parameters proposed quantitatively characterize the RP-HPLC stationary phases and provide a rational explanation for the differences in retention patterns of individual columns observed when applying the conventional empirical testing methods.

Determination of phenylurea herbicides in water samples using online sorptive preconcentration and high-performance liquid chromatography with UV or electrospray mass spectrometric detection.

A recently developed method for the extraction of organic micropollutants from aqueous samples based on sorptive enrichment in columns packed with 100% polydimethylsiloxane (PDMS) particles was coupled on-line with HPLC analysis. The sorptive enrichment procedure originally developed for relatively nonpolar analytes was used to preconcentrate polar phenylurea herbicides from aqueous samples. PDMS extraction columns of 5, 10 and 25 cm were used to extract the herbicides from distilled, tap and river water samples. A model that allows prediction of retention and breakthrough volumes is presented. Despite the essentially apolar nature of the PDMS material, it is possible to concentrate sample volumes up to 10 ml on PDMS cartridges without losses of the most polar analyte under investigation, fenuron. For less polar analytes significantly larger sample volumes can be applied. Since standard UV detection does not provide adequate selectivity for river water samples, an electrospray (ES)-MS instrument was used to determine phenylurea herbicides in a water sample from the river Dommel. Methoxuron was present at a level of 80 ng/l. The detection limit of the current set-up, using 10 ml water samples and ES-MS detection is 10 ng/l in river water samples. Strategies for further improvement of the detection limits are identified.

Separation and characterization of oligomers by reversed-phase high-performance liquid chromatography: a study on well-defined oligothiphenes.

Reversed-phase high-performance liquid chromatography (RP-HPLC) was used for the separation of 3-hexylthiophene oligomers in the range of 3 to 30 monomeric units, while systematically varying stationary and mobile phases. A set of different columns was chosen, covering a broad range of silica types, pore sizes and bonding chemistry. Mobile phases of tetrahydrofuran (THF) combined with water, acetonitrile (ACN) or methanol (MeOH) were used. Although differences between columns were small, a higher selectivity correlated with a lower hydrophobicity parameter from the Galushko column test. The model of Jandera, based on the linear solvent strength model of Snyder, was used to describe the retention of the oligomers in gradient mode. This gave information about selectivities on different stationary phases similar to the hydrophobicity parameter. Contrary to the stationary phase, the mobile phase had a major influence on the selectivity. The THF-water combination gave much higher selectivities compared to THF combined with MeOH or ACN. Using the aqueous mobile phase even enabled separation of different isomers. Determination of thermodynamic parameters for the model compounds showed that retention of the different isomers was mainly determined by the orientation of the side chains at both ends of the chain. An additional repeating unit in the middle of the polymer backbone gave a similar contribution to retention, irrespective of the orientation of its side chain. Three model isomers were separated by preparative RP-HPLC and identified by proton nuclear magnetic resonance spectroscopy. The combination of subsequent preparative size-exclusion chromatography, RP-HPLC and matrix-assisted laser desorption ionization time-of-flight mass spectrometry enabled the identification of the two major oligomeric series in the sample as the regioregular product with one bromine end group and, in smaller amounts, a regioirregular product with two bromine end groups. reserved.

Separation and quantification of the linear and cyclic structures of polyamide-6 at the critical point of adsorption.

The linear and cyclic structures of polyamide-6 were separated by liquid chromatography at critical conditions (LCCC) and identified with different mass spectrometric (MS) techniques and quantitated by LCCC with evaporative light-scattering detection (ELSD). Electrospray ionization MS was not suitable to identify the higher cyclic structures. For this purpose, matrix-assisted laser desorption ionization time-of-flight MS performed better and cyclic and linear structures were oligomerically resolved and separately identified in the mass spectrometer. The highest cyclic structure present and detected was the cyclic pentacontamer. It could be demonstrated that cyclic and linear oligomers follow different ionization and fragmentation routes/patterns. Quantification with ELSD of the components separated by LCCC using a universal calibration curve or an iterative procedure was developed. An area correction to account for different peak widths of coeluting components improves precision and accuracy of the calibration curve and improves quantitation accuracy for the samples analyzed. With these corrected values, no molecular mass dependency was observed for the cyclic and linear structures. Under critical conditions, the linear and cyclic structures of polyamide-6 were separated, identified and quantified.

Characterisation of reversed-phase stationary phases for the liquid chromatographic analysis of basic pharmaceuticals by thermodynamic data.

This paper describes the characterisation of reversed-phase liquid chromatography (RPLC) columns using thermodynamic measurements. Retention versus 1/T data were used to construct Van't Hoff plots. The slope of these plots indicates the standard enthalpy of transfer of the analyte from the mobile to the stationary phase. The standard entropy can be calculated from the intercept. Van't Hoff plots were linear for the investigated RPLC columns, meaning that for basic analytes over the temperature range studied no changes in the retention mechanism occurred. Enthalpies and entropies of transfer of basic analytes from the mobile to the stationary phase revealed information about the types of interaction of protonated and neutral compounds with the stationary phases. However, a clear view using the present set of basic compounds on how these thermodynamic data may explain the observed substantial differences in peak symmetry cannot be given. It is considered that addition of N,N-dimethyloctylamine (DMOA) to the eluent will results in a dynamically coating of the stationary phase. Addition of DMOA to the eluent resulted for protonated basic compounds in a reduction of both enthalpy and entropy. In practice, with DMOA in the eluent symmetrical peaks were obtained. It is assumed that this is due to blocking residual silanols and/or ion exclusion effects.