Parallel supercritical fluid chromatography/mass spectrometry system for high-throughput enantioselective optimization and separation

An automated parallel four-column supercritical fluid chromatography (SFC)/MS system to perform high-throughput enantioselective chromatographic method development and optimization is described in this paper. The initial screening was performed in parallel on four chiral SFC columns over several buffer conditions. Optimization of the separation of enantiomers was achieved on a single chiral column. The screening and optimization were accomplished in a fully automated, user-independent manner. Incorporation of column control valves in front of each chiral column allowed the system to switch from parallel four-column screening mode to single-column optimization mode. To facilitate the process, a custom software program, we termed, intelligent parallel optimization for chiral SFC separation (IPOCSS), was developed in-house. The custom software monitored each of the runs in real-time, processed each data set, and by incorporating user-defined criteria (e.g., resolution of the two enantiomer chromatographic peaks), selected the next set of experiments and automatically optimized the enantioseparation. This new approach, combining parallel SFC/MS screening and intelligent software-controlled method optimization, has resulted in a streamlined, high-throughput tool for enantioselective method development, which has been applied successfully to enantioseparations in support of drug discovery.     

Monitoring of veterinary drug residues by a combination of continuous flow techniques and column-switching high-performance liquid chromatography. I. Sulphonamides in egg, meat and milk using post-column derivatization with dimethylaminobenzaldehyde

By using a combination of a low-pressure continuous flow module and a column-switching high-performance liquid chromatographic system, procedures have been developed for the automated residue analysis of sulphonamides and selected other drugs in meat, egg and milk. Aqueous extracts are purified by on-line dialysis and subsequent trace enrichment on a short column containing silica-based or polymer material. After backflush to the analytical column, detection is performed either at 280 nm or, after post-column derivatization with p-dimethylaminobenzaldehyde, at 450 nm. With these procedures the fully automated determination of both polar and apolar sulphonamides and dapsone above 5-20 micrograms/kg is possible. Coefficients of variation are 4-10%, recoveries compared to standards are 85-90%. The methods developed were tested in routine veterinary drug control.     

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 biogenic amines in wine by multidimensional liquid chromatography with online derivatisation

An online multidimensional liquid chromatographic system was developed for online clean-up, derivatisation and separation of biogenic amines in wines. The system consists of a cation-exchange precolumn, a derivatisation coil and an analytical column, and a column-switching valve. The entire system can be easily automated. The method proved to be quantitative and sensitive. Limits of detection were below 0.05 mg l(-1) for all amines and linearity was preserved over the tested range (0.05-15 mg l(-1)). The method was applied to the analysis of red wines of different origin.     

On-line sample cleanup in the liquid chromatographic analysis of pharmaceuticals for citrate content

Matrix interferents are removed from pharmaceutical samples via an on-line, automated column switching process in which the difference in hydrophobicity between the analyte and the interferents allows these species to be isolated in different parts of the chromatographic system. In this case, the interferents are trapped on a cleanup column and are flushed to waste as the analyte undergoes additional separation on an analytical column. The utility of this approach is demonstrated by the quantitation of citrate in pharmaceutical samples by ion suppression, reversed-phase liquid chromatography. The performance of this system is statistically equivalent to that of a manual pretreatment method employing disposable, solid-phase extraction cartridges.     

Efficiency of preparative and process column distribution systems

An analytical method for computing the residence time distribution of the liquid distribution system in chromatography columns is described. The impact of the distributor design on the separation efficiency is predicted as a function of media properties and packed bed dimensions. The efficiency loss due to the distributor when increasing column diameter during scale-up is quantified. It is shown that this loss can be compensated by modulating the local bed height via a moderate inclination of the bed support. It is concluded that the selection of an appropriate distributor design concept with optimised dimensions enables a scale-up of chromatographic separations without any significant loss of chromatographic efficiency due to the distribution system.     

Rapid isolation of plutonium in environmental solid samples using sequential injection anion exchange chromatography followed by detection with inductively coupled plasma mass spectrometry

This paper reports an automated analytical method for rapid determination of plutonium isotopes (²³⁹Pu and ²⁴⁰Pu) in environmental solid extracts. Anion exchange chromatographic columns were incorporated in a sequential injection (SI) system to undertake the automated separation of plutonium from matrix and interfering elements. The analytical results most distinctly demonstrated that the crosslinkage of the anion exchanger is a key parameter controlling the separation efficiency. AG 1-×4 type resin was selected as the most suitable sorbent material for analyte separation. Investigation of column size effect upon the separation efficiency revealed that small-sized (2 mL) columns sufficed to handle up to 50 g of environmental soil samples. Under the optimum conditions, chemical yields of plutonium exceeded 90% and the decontamination factors for uranium, thorium and lead ranged from 10³ to 10⁴. The determination of plutonium isotopes in three standard/certified reference materials (IAEA-375 soil, IAEA-135 sediment and NIST-4359 seaweed) and two reference samples (Irish Sea sediment and Danish soil) revealed a good agreement with reference/certified values. The SI column-separation method is straightforward and less labor intensive as compared with batch-wise anion exchange chromatographic procedures. Besides, the automated method features low consumption of ion-exchanger and reagents for column washing and elution, with the consequent decrease in the generation of acidic waste, thus bearing green chemical credentials.     

Development of a relatively cheap and simple automated separation system for a routine separation procedure based on extraction chromatography

A robust analytical method has been developed in our laboratory for the separation of radionuclides by means of extraction chromatography using an automated separation system. The proposed method is both cheap and simple and provides the advantageous, rapid and accurate separation of the element of interest. The automated separation system enables a shorter separation time by maintaining a constant flow rate of solution and by avoiding clogging or bubbling in the chromatographic column. The present separation method was tested with two types of samples (water and urine) using UTEVA-, TRU- and Sr-specific resins for the separation of U, Th, Am, Pu and Sr. The total separation time for one radionuclide ranged from 60 to 100 min with the separation yield ranging from 68 to 98% depending on the elements separated. We used ICP-QMS, multi-low-level counter and alpha spectroscopy to measure the corresponding elements.     

气相色谱专家系统中柱系统的推荐

本文给出气相色谱专家系统中柱系统推荐软件的总体结构和设计及一个运用所研制的软件进行咨询的例子。用户只要回答与样品中组分结构有关的几个关键问题,软件即可对样品可否采用气相色谱法进行判断和对气相色谱柱系统进行推荐。从初步使用和验证来看,本系统是成功的。     

Isolation of Glycolipids from Blood Elements

Abstract

Several chromatographic e.g. HPTLC, HPLC, etc. methods have been published in the literature for the separation, after sufficient pretreatment, of derivatized or non-derivatized glycolipid samples.

Our task is the extraction, isolation and separation of the glycolipids from different blood elements, followed by suitable fractionation methods, giving the lipid classes in sufficient purity and quantity for HPLC, HPTLC and OPTLC measurements and possibly further biochemical use.

We show the differences between the procedures commonly used and that developed in our laboratory.

The advantage of our method, which employs 3 cm long Brownlee Labs HPLC cartridges, is that it can be automated, it gives class fractionation of the lipid samples and as it is hardware compatible with HPLC equipment it can be used directly in a coupled column system for on line separation in the individual class. The development of this column coupling method for the fractionation of a given lipid class from the total lipid extract on an analytical column is under development.     

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.     

An automated food protein isolation approach on preparative scale by two‐dimensional liquid chromatography with active modulation interface

In this work, an automated 2D‐LC approach for protein isolation from egg samples on preparative scale is proposed. The method is based on the use of a C18 guard column installed in a switching valve to focus the proteins coming from the first dimension column, before their elution in the second column. For the first dimension separation, a size‐exclusion column, packed with 3 μm ultrapure silica particles was used. An RP column based on core‐shell technology was used for the second dimension separation. A standard mixture of BSA, β‐lactoglobulin, and glucose oxidase, chosen as a protein model system, was used to optimize the chromatographic separation conditions. The fully automated workflow allowed to isolate, in a single‐chromatographic analysis, a protein amount of 50 μg for each peak fraction, with a total time of 15 min for the first separation and additional 30 min of the second separation for each trapped protein. The final aim was the development of proper analytical tools for protein isolation from foodstuffs to be used for the molecular identification by MS, as well as for biotherapeutic uses, allergy testing, and large‐scale investigations in biological systems.     

Automated determination of disopyramide and N-monodealkyldisopyramide in plasma by reversed-phase liquid chromatography with a column switching system.

A rapid and simple column liquid chromatographic method involving a column switching system for the determination of disopyramide and its N-monodealkyl metabolite (NMD) in plasma is described. The deproteinized plasma is applied to an automated system. Purification and concentration were performed using a precolumn connected to a six-position valve; analytical separation was done on-line using a cyano reversed-phase column with a mobile phase consisting of 10 mmol/l trimethylamine (pH 2.5, adjusted with phosphoric acid)-acetonitrile-tetrahydrofuran (78:20:2, v/v/v). Absorbance was measured at 265 nm, with a minimum detectable amount of disopyramide and NMD of 0.1 micrograms/ml. The method can be applied to drug monitoring and pharmacokinetic studies.     

Turbulent flow chromatography in bioanalysis: a review

With advances in fast chromatography techniques, and highly sensitive and selective detection methods such as tandem mass spectrometry, very high-throughput bioanalytical methods can now be easily developed. The bottleneck of the analytical process then becomes the sample preparation, which it is now realized is crucial to the robust operation of the analytical system, especially for quantitative assays. Turbulent flow liquid chromatography was developed in the late 1990s, and combines 'size exclusion' and traditional stationary phase column chemistry to separate macromolecules, such as proteins, from smaller molecules and analytes of interest in biological fluids. By definition, the process is very rapid, and the instrumentation and software have been developed for fully automated, on-line extraction of neat biological fluids. This work aims to review the chromatographic theory of turbulent flow chromatography and illustrate, using examples from recent literature, the application of this technique to a range of analytes from a number of different biological matrices.     

Determination of melagatran in rabbit plasma by high-performance liquid chromatography with automated column switching

Melagatran is an active thrombin inhibitor showing oral and parenteral bioavailability for antithrombotic therapy. A simple and convenient liquid chromatographic method has been developed and applied to the analysis of melagatran in rabbit plasma. The clean-up and separation of the sample solutions were performed by automated on-line column switching HPLC. The method validation shows the suitability of the column switching liquid chromatographic system for the quantitation of melagatran in biological fluids.     

Development of a novel high volume band compression injector for the analysis of complex samples like toxaphene pesticide

A new type of injector has been developed for gas chromatographic analysis. The injector has high volume and band compression (HVBC) capabilities useful for the analysis of complex samples. The injector consists essentially of a packed liner operated at room temperature while a narrow heated zone is used to axially scan the liner selectively desorbing the compounds of interest. The scanning speed, distance and temperature of the zone are precisely controlled. The liner is connected to an interface which can vent the solvent or any undesirable compounds, and transfer the analytes to an analytical column for separation and quantification. The injector is designed to be compatible with injection volumes from 1 to more than 250microL. At a low sample volume of 1microL, the injector has competitive performances compared to those of the "on-column" and "split/splitless" injectors for the fatty acid methyl esters and toxaphene compounds tested. For higher volumes, the system produces a linear response according to the injected volume. In this explorative study, the maximum volume injected seems to be limited by the saturation of the chromatographic system instead of being defined by the design of the injector. The HVBC injector can also be used to conduct "in situ" pretreatment of the sample before its transfer to the analytical column. For instance, a toxaphene sample was successively fractionated, using the HVBC injector, in six sub-fractions characterized by simpler chromatograms than the chromatogram of the original mixture. Finally, the ability of the HVBC injector to "freeze" the separation in time allowing the analyst to complete the analysis at a later time is also discussed.     

Determination of hexamethylphosphoramide and other highly polar phosphoramides in water samples using reversed-phase liquid chromatography/electrospray ionization time-of-flight mass spectrometry

The widely used solvent hexamethylphosphoramide (HMPA) and its biological (metabolic) and chemical (abiotic) phosphoramide-based oxidation products may cause adverse health effects through occupational exposure and intake of contaminated groundwater. However, no current methods exist for the separation and the detection of the many polar HMPA oxidation products. Thus, we developed a new RPLC/ESI-TOF-MS method and further investigated the chromatographic performances of two columns (i.e., XTerra Phenyl and XBridge Phenyl). In addition, the impact of (forced) acid hydrolysis for optimized chromatographic performance of the XTerra Phenyl column is investigated. The XTerra Phenyl column showed the best separation of the less polar major metabolic oxidation products pentamethylphosphoramide and hydroxymethyl-pentamethylphosphoramide, however, only after treating the column with formic acid (acid-treated). The XTerra column separated most of the investigated HMPA oxidation products (11 of 16 compounds) in a single chromatographic run. In contrast, the XBridge Phenyl column requires one method for the less polar and another method for the more polar oxidation products. However, this results in an overall better separation performance of the XBridge Phenyl column, especially for the less polar major abiotic oxidation products hydroxymethyl-pentamethylphosphoramide and formyl-pentamethylphosphoramide, as well as for 11 highly polar oxidation products (R(S)>1.5). The RPLC/ESI-TOF-MS method presented and validated in this study is the first analytical method that can be used to separate and detect HMPA (LOD 0.10 μM without preconcentration) and all of its oxidation products.     

Gas chromatographic subsystem for fast on-line concentration profile measurements for advanced distillation column control

An automated gas chromatographic subsystem for the provision of fast and reliable concentration profile data for distillation column control is presented. The subsystem consists of a gas chromatograph, equipment for sample conditioning and a PC/AT compatible computer communicating with the supervisory process computer. A fast separation time is obtained through the use of a liquid chromatographic packing material and a high pressure drop across the column. Preliminary results show separation times below 10 s for the water-methanol-isopropanol system. Peak areas are quantified by use of parameter estimation in the frequency domain, a method that does not demand complete peak separation.     

Two-dimensional supercritical fluid chromatography/mass spectrometry for the enantiomeric analysis and purification of pharmaceutical samples

A new analytical two-dimensional supercritical fluid chromatography/mass spectrometry system (2D SFC/SFC/MS) has been designed and implemented to enhance the efficiency and quality of analytical support in drug discovery. The system consists of a Berger analytical SFC pump and a modifier pump, a Waters ZQ 2000 mass spectrometer, a set of switching valves, and a custom software program. The system integrates achiral and chiral separations into a single run to perform enantiomeric analysis and separation of a racemic compound from a complex mixture without prior clean up. The achiral chromatography in the first dimension separates the racemate from all other impurities, such as un-reacted starting materials and by-products. Mass-triggered fractionation is used to selectively fractionate the targeted racemic compound based on its molecular weight. The purified racemate from the achiral chromatography in the first dimension is then transferred to the chiral column in the second dimension to conduct the enantiomeric separation and analysis. A control software program, we coined SFC2D, was developed and integrated with MassLynx to retrieve acquisition status, current sample information, and real time mass spectrometric data as they are acquired. The SFC2D program also monitors the target ion signal to carry out mass-triggered fractionation by switching the valve to fractionate the desired peak. The 2D SFC/SFC/MS system uses one CO(2) pump and one modifier pump for both first and second dimension chromatographic separations using either gradient or isocratic elution. Similarly, a preparative 2D SFC/SFC/MS system has been constructed by modifying an existing Waters preparative LC/MS system. All components except the back pressure regulator are from the original LC/MS system. Applications of the 2D SFC/SFC/MS methods to the separation and the analysis of racemic pharmaceutical samples in complex mixtures demonstrated that an achiral separation (in first dimension) and a chiral separation (in second dimension) can be successfully combined into a single, streamlined process both in analytical and preparative scale.     

Ion chromatography characterization of polysaccharides in ancient wall paintings

Specific programming of automated HPLC systems allows total on-line qualification, validation and stability monitoring using the concept of deferred standards. Setting up such a process for routine analyses in an automated HPLC system requires specific autosampler programming as well as specific monitoring software. With an autosampler, a double injection procedure is programmed, the first introducing the sample, and the second, a few minutes deferred, the deferred control standard. Two additional compounds are therefore added to the sample before and during the chromatographic process: the intemal standard for sample quantification and the deferred standard for system control. Specific methodologies are described of how to obtain classical quantitative analysis information as well as system qualification validation stability information. Experiments were performed to develop specified methodologies to monitor the quality of quantitative analysis during the life of the column by using the deferred standard concept to probe the effects of column ageing on separation characteristics.