Comprehensive two-dimensional liquid chromatography in analysis of Lamiaceae herbs: characterisation and quantification of antioxidant phenolic acids

A fast and effective dynamic sonication assisted ethanol extraction method was developed for extracting phenolic acids from basil, oregano, rosemary, sage, spearmint and thyme of the Lamiaceae family. The results were compared with results obtained by conventional solvent extraction techniques. A comprehensive two-dimensional liquid chromatography (LC x LC) system interfaced to electrospray ionisation time-of-flight (TOF) mass spectrometry was then optimised for analysis and quantification of the herb extracts. The optimised LC x LC system employed a combination of C18 and cyano columns. The relative standard deviations for the retention times were better than 0.05% (rosmarinic acid 0.1%) and those for the peak areas 2-14% (2 mg/l, n=3). Limits of detection were 18-90 ng/ml. The LC x LC-MS method was applied to the quantitative analysis of phenolic acids, and the results were compared with those obtained with conventional LC-MS.     

Development and optimization of a system for comprehensive two-dimensional liquid chromatography with UV and mass spectrometric detection for the separation of complex samples by multi-step gradient elution

Comprehensive two-dimensional liquid chromatography (LC x LC) is a powerful tool for the separation of complex biological samples. This technique offers the advantage of simplified automation and greater reproducibility in a shorter analysis time than off-line two-dimensional separation systems. In the present study, an LC x LC system is developed enabling simultaneous UV and MS detection, and which can be easily converted to a conventional reversed-phase LC-UV/MS system. In LC x LC, a 60-min reversed-phase LC separation with a linear solvent gradient in the first dimension is coupled to a second-dimension separation on a mixed-mode cation-exchange/reversed-phase column with a modulation time of 60s. The isocratic separation in the second-dimension column is optimized by the use of a multi-step gradient where the organic and the ionic modifier are varied independently. Intraday (n=3) and interday (n=4) variability of the retention times were evaluated with the complete system and found to be 0.5% and 0.7%, respectively. Good linearity was observed in calibration curves for three different compounds varying in polarity.     

Cavity ring-down spectroscopy for detection in liquid chromatography at UV wavelengths using standard cuvettes in a normal incidence geometry

Liquid chromatography (LC) with cavity ring-down spectroscopy (CRDS) detection, using flow cuvettes (put under normal incidence inside the ring-down cavity), is demonstrated. Fresnel reflections are maintained within the capture range of a stable cavity of 4 cm length. This method circumvents the need for specific Brewster's angles and possible mirror degradation is avoided. The flow cuvettes are commercially available at low cost. At 355 nm (the frequency-tripled output of a Nd:YAG laser), the system surpasses the performance of conventional absorbance detectors; the baseline noise was 1.3 x 10(-5)AU and detection limits (injected concentrations) were between 40 and 80 nM for nitro-polyaromatic hydrocarbons with an extinction coefficient epsilon of 7.3-10.2 x 10(3)M(-1)cm(-1). The system was also tested at 273 nm, but in the deep UV the reflectivity of the currently best available mirrors (R>or=99.91%) is still too low to show a significant improvement as compared to conventional UV-vis detection.     

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction, followed by high-speed liquid chromatography/mass spectrometry, for the determination of a basic drug in human plasma

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction (PPT/SPE) procedure has been investigated. A mixture of acetonitrile and methanol along with formic acid was used to precipitate plasma proteins prior to selectively extracting the basic drug. After vortexing and centrifugation, the supernatants were directly loaded onto an unconditioned Oasis MCX microElution 96-well extraction plate, where the protonated drug was retained on the negatively charged sorbent while interfering neutral lipids, steroids or other endogenous materials were washed away. Normal wash steps were deemed unnecessary and not used before sample elution. The sample extracts were analyzed under both conventional and high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) conditions to examine the feasibility of the PPT/SPE procedure for human plasma sample clean-up. For the conventional LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 50 mm column with gradient elution (k' = 5.5). The mobile phase contained 0.1% formic acid in water and 0.1% formic acid in acetonitrile. For the high-speed LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 10 mm guard column with gradient elution (k' = 2.2, Rt = 0.26 min). The mobile phase contained 0.1% formic acid in water and 0.001% trifluoroacetic acid in acetonitrile. Detection for both conventional and high-speed LC/MS/MS methods was by positive ion electrospray tandem mass spectrometry on a ThermoElectron Finnigan TSQ Quantum Ultra, where enhanced resolution (RP 2000; 0.2 amu) was used for high-speed LC/MS/MS. The standard curve, ranging from 0.5 to 100 ng/mL, was fitted to a 1/x weighted quadratic regression model.This combined PPT/SPE procedure effectively eliminated time-consuming sorbent conditioning and wash steps, which are essential for a conventional mixed-mode SPE procedure, but retained the advantages of both PPT (removal of plasma proteins) and mixed-mode SPE (analyte selectivity). The validation results demonstrated that this PPT/SPE procedure was well suited for both conventional and high-speed LC/MS/MS analyses. In comparison with a conventional mixed-mode SPE procedure, the simplified PPT/SPE process provided comparable sample extract purity. This simple sample clean-up procedure can be applied to other basic compounds with minor modifications of PPT solvents.     

Crystalline degradation products of vancomycin as chiral stationary phase in microcolumn liquid chromatography

The ability of crystalline degradation products (CDPs) of vancomycin as a chiral stationary phase was reported in a previous study for enantioselective separation of drugs, amino acids and agrochemical toxins by conventional LC column (250 x 4.6 mm). In this work, the potential of CDP of vancomycin for the enantiomeric separation in micro-LC (200 x 1 mm) has been studied. The obtained separation results are better than in our previous study with conventional LC columns. The enantiomers of D,L-phenylalanine, D,L-alanine, methyldopa, atropine and propranolol were used for this evaluation. Experiments have been carried out in a stainless steel tube that was packed with chiral silica particles of 3 and 12 microm diameters. Also, three different ratios of 3 and 12 microm silica particles were used for packing material of chiral columns and the effect on aspect ratio and resolving powers was compared.     

Comprehensive two-dimensional liquid chromatography applying two parallel columns in the second dimension

The design of a new interface for comprehensive two-dimensional liquid chromatography (LC x LC) is described. To the conventionally used LC x LC system with the loop-type interface consisting of a two-position/ten-port switching valve equipped with two loops, an extra two-position/ten-port switching valve, a detector, a pump and a second column placed in parallel with the column in the second dimension, are added. The features of the interface are that the separation space in the second dimension is significantly enlarged and that the number of fractions transferred from the first to the second dimension can be increased, reducing the risk to lose resolution of the primary dimension. The potential of the system in NPLC x 2RPLC is illustrated with the analysis of a standard mixture and a lemon oil extract. For the lemon oil analysis, the effective peak capacity was increased from 437 using a conventional interface to 1095 with the new interface. RPLC x 2RPLC in combination with reduced modulation times was applied to the analysis of steroids and to the detection of impurities at the 0.05% relative concentration level in a sulfonamide drug sample.     

Recent developments in liquid chromatography--impact on qualitative and quantitative performance

In order to reduce the analysis time and maintain good efficiency in liquid chromatography (LC), several solutions are currently being investigated. The focus of this study was to compare, both qualitatively and quantitatively, the chromatographic performance of a conventional LC with selected approaches, namely monolithic supports, high temperature LC (up to 90 degrees C), and sub-2 microm particles combined with high pressure (up to 1000 bar). This comparison was achieved from a qualitative point of view with a special attention paid to the analysis of time reduction, efficiency improvement, and pressure constraint. For this purpose, the different approaches were discussed using Knox curves and other kinetic plots. It appeared that columns packed with sub-2 microm particles under high-pressure conditions (UPLC) were well adapted and this option represents an attractive alternative to conventional LC; however, the other alternative approaches should not be neglected. The quantitative evaluation of these techniques was performed on the basis of the validation of results of a pharmaceutical formulation (Rapidoca?ne), following SFSTP 2003 guidelines. Fast-LC approaches demonstrated equivalent performance to conventional LC in terms of trueness, precision, and accuracy profile, with a significant time reduction (up to 8x) according to the selected methodology.     

High throughput screening of active pharmaceutical ingredients by UPLC

Ultra performance LC (UPLC) was evaluated as an efficient screening approach to facilitate method development for drug candidates. Three stationary phases were screened: C-18, phenyl, and Shield RP 18 with column dimensions of 150 mm x 2.1 mm, 1.7 microm, which should theoretically generate 35,000 plates or 175% of the typical column plate count of a conventional 250 mm x 4.6 mm, 5 microm particle column. Thirteen different active pharmaceutical ingredients (APIs) were screened using this column set with a standardized mobile-phase gradient. The UPLC method selectivity results were compared to those obtained for these compounds via methods developed through laborious trial and error screening experiments using numerous conventional HPLC mobile and stationary phases. Peak capacity was compared for columns packed with 5 microm particles and columns packed with 1.7 microm particles. The impurities screened by UPLC were confirmed by LC/MS. The results demonstrate that simple, high efficiency UPLC gradients are a feasible and productive alternative to more conventional multiparametric chromatographic screening approaches for many compounds in the early stages of drug development.     

Combination of COFRADIC and high temperature-extended column length conventional liquid chromatography: a very efficient way to tackle complex protein samples, such as serum

The previously reported COmbined FRActional DIagonal Chromatography (COFRA-DIC) methodology, in which a subset of peptides representative for their parent proteins are sorted, is particularly powerful for whole proteome analysis. This peptide-centric technology is built around diagonal chromatography, where peptide separations are crucial. This paper presents high efficiency peptide separations, in which four 250 x 2.1 mm, 5 microm Zorbax 300SB-C18 columns (total length 1 m) were coupled at operating temperatures of 60'C using a dedicated LC oven and conventional LC equipment. The high efficiency separations were combined with the COFRADIC procedure. This extremely powerful combination resulted, for the analysis of serum, in an increase in the uniquely identified peptide sequences by a factor of 2.6, compared to the COFRADIC procedure on a 25 cm column. This is a reflection of the increased peak capacity obtained on the 1 m column, which was calculated to be a factor 2.7 higher than on the 25 cm column. Besides more efficient sorting, less ion suppression was noticed.     

Coaxially bifocal liquid crystal lens with switchable optical aperture

An electrically tunable liquid crystal (LC) lens with dual hole-patterned electrodes is demonstrated. When the LC lens is operated at low voltages, the dual hole-patterned electrodes with different diameters impart the lens with a coaxial bifocal characteristic. At high voltages, the proposed LC lens functions as a conventional lens with a single focal length but with a switchable optical aperture. The demonstrated LC lens is free of disclination lines because of the presence of voltage-assisted high pretilt angles created from the upmost hole-patterned electrode with small diameter.     

Preparation of liquid crystal emulsion and its application performance study

The aim of this study was the formulation composition and characterization of the liquid crystal (LC) emulsion. First, influences of formulation composition on preparation of LC structure were studied, including the aliphatic alcohols with different carbon chain length, the liquid oils with different polarities, the sodium chloride, and the polygons. The results showed that fatty alcohols which is closer to the hydrophobic group of the emulsifier, little liquid oil, and little polyols were conducive to form LC structure. Then, the application performance of LC structure emulsion was studied. The results showed that compared with the conventional structure emulsion, the LC structure emulsion had more excellent moisturizing property, slow release effect, and the promoted penetration effect.     

Single molecule spectroscopy of conjugated polymer chains in an electric field-aligned liquid crystal

Using single molecule polarization spectroscopy, we investigated the alignment of a polymer solute with respect to the liquid crystal (LC) director in an LC device while applying an external electric field. The polymer solute is poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene] (or MEH-PPV), and the LC solvent is 5CB. The electric field induces a change in the LC director orientation from a planar alignment (no electric field) to a perpendicular (homeotropic) alignment with an applied field of 5.5 x 103 V/cm. We find that the polymer chains align with the LC director in both planar and homeotropic alignment when measured in the bulk of the LC solution away from the device interface. Single molecule polarization distributions measured as a function of distance from the LC device interface reveal a continuous change of the MEH-PPV alignment from planar to homeotropic. The observed polarization distributions are modeled using a conventional elastic model that predicts the depth profile of the LC director orientation for the applied electric field. The excellent agreement between experiment and simulations shows that the alignment of MEH-PPV follows the LC director throughout the LC sample. Furthermore, our results suggest that conjugated polymers such as MEH-PPV can be used as sensitive local probes to explore complex (and unknown) structures in anisotropic media.     

Offline LC-GC x GC in combination with rapid-scanning quadrupole mass spectrometry

The present research is focused on the offline combination of normal-phase LC to double-oven GC x GC-quadrupole MS. Initially, a diesel sample was subjected to automated LC x GC in order to define the elution windows of four fractions, viz., saturated hydrocarbons, monocyclic aromatics, dicyclic aromatics, tri- + tetracyclic aromatics; each fraction was collected exploiting the LC system in a further analysis and subjected to large-volume-injection-GC x GC analysis using an apolar-polar column combination. The GC x GC operational conditions were tuned in relation to the specific separation requirements of each heart-cut. The main benefits of what can be defined as offline LC-GC x GC were: (i) the high first-dimension LC selectivity; (ii) the injection of high sample amounts in the GC x GC system, enabling the detection and quantification of a series of low-amount diesel constituents; (iii) improved GC x GC operational conditions for each heart-cut with respect to direct GC x GC.     

Comparison of separation power of ultra performance liquid chromatography and comprehensive two-dimensional liquid chromatography in the separation of phenolic compounds in beverages

In this study, 1-D and 2-D liquid chromatographic systems, namely, conventional HPLC, UPLC, HPLC x HPLC and HPLC x UPLC systems were developed and evaluated for the separation of phenolic acids in wine and juices. In the LC x LC studies, the first dimension separation was based on RPLC and the second dimension was performed with ion-pair chromatography. Three different columns, namely two short columns packed with either 2.5 or 1.7 microm particles and a monolithic column, were tested for the fast second dimension separation. The best results were obtained when the monolithic column was applied for the second dimension separation. The peak capacities for comprehensive 2-D systems varied from 330 to 616.     

Peak capacity optimization in comprehensive two dimensional liquid chromatography: a practical approach

In this work we develop a practical approach to optimization in comprehensive two dimensional liquid chromatography (LC x LC) which incorporates the important under-sampling correction and is based on the previously developed gradient implementation of the Poppe approach to optimizing peak capacity. The Poppe method allows the determination of the column length, flow rate as well as initial and final eluent compositions that maximize the peak capacity at a given gradient time. It was assumed that gradient elution is applied in both dimensions and that various practical constraints are imposed on both the initial and final mobile phase composition in the first dimension separation. It was convenient to consider four different classes of solute sets differing in their retention properties. The major finding of this study is that the under-sampling effect is very important and causes some unexpected results including the important counter-intuitive observation that under certain conditions the optimum effective LC x LC peak capacity is obtained when the first dimension is deliberately run under sub-optimal conditions. In addition, we found that the optimum sampling rate in this study is rather slower than reported in previous studies and that it increases with longer first dimension gradient times.     

Comparison between capillary electrophoresis and liquid chromatography for the determination of diclofenac sodium in a pharmaceutical tablet

Two novel analytical methodologies using capillary electrophoresis (CE) and liquid chromatography (LC) were developed and compared for the determination of diclofenac sodium in commercial and simulated tablet formulations. The CE analysis was performed in a bare fused-silica capillary with 75 microm id and total length of 50 cm (28 cm to the detector) with a buffer solution of 20 mM sodium tetraborate, pH 9.23. The applied voltage was 20 kV, and acetaminophen was used as the internal standard (IS). The LC analysis was performed with a LiChrospher 100 RP-18 (5 microm) column and a mobile phase of methanol-diluted glacial acetic acid (0.3 parts in 2500; 75 + 25) at a flow rate of 0.9 mL/min with propylparaben as the IS. In both analyses, detection was by ultraviolet absorption at 276 nm. Under optimized conditions, the CE migration times for the diclofenac sodium standard and acetaminophen (IS) were 2.07 and 1.59 min, respectively, and the LC retention times for the diclofenac sodium standard and propylparaben (IS) were 3.98 and 2.26 min, respectively. The resolution and efficiency for CE were 14.2 and 1.6 x 10(5) plates/m, respectively, and for LC, 5.0 and 8.6 x 10(3) plates/m, respectively. Calibration curves of peak area versus concentration gave correlation coefficients of 0.9992 for CE and 0.9994 for LC. The limits of detection and quantitation were 8.40 and 25.46 microg/mL, respectively, for CE and 4.60 and 13.93 microg/mL, respectively, for LC. Coefficients of variation were 1.68 and 0.37% for CE and LC, respectively. Average recoveries obtained with CE and LC were 103.12+/-0.90 and 99.59+/-0.21%, respectively. Although both methodologies were shown to be suitable for the determination of diclofenac sodium in tablets, performing in a similar manner with regard to several aspects (linearity, recovery, and specificity), CE provided faster analysis and better column efficiency, whereas LC provided superior repeatability and sensitivity.     

Evaluation of the coupling between ultra performance liquid chromatography and evaporative light scattering detector for selected phytochemical applications

Fast analysis in LC can be performed with sub-2 microm particles at very high pressures (up to 1000 bar) known as ultra performance LC (UPLC). With this configuration, it is possible to obtain fast and/or highly efficient separations compared to conventional LC. For the analysis of compounds without chromophores, the evaporative light scattering detector (ELSD) is an attractive alternative because of its quasi-universality, versatility, low-cost and good sensitivity. The UPLC-ELSD was investigated in terms of sensitivity and apparent efficiency, with a conventional ELSD instrument, for two types of commercially available nebulisers, using different mobile phase flow rates and column ids. Results were finally compared with the UPLC-UV configuration. Three applications with phytochemical compounds were selected to highlight the potential of this approach (i.e. the isocratic separations of artemisinin and its derivatives, of calystegines and the gradient separation of several tropane alkaloids). Depending on the used column length, baseline separations were obtained in 3-10 min, with an average apparent efficiency ranging from 7000 to 30,000 plates.     

In-tube solid-phase microextraction-capillary liquid chromatography as a solution for the screening analysis of organophosphorus pesticides in untreated environmental water samples

This paper describes a method for the selective screening of organophosphorus pesticides in water. In-tube solid-phase microextraction (SPME) in an open capillary column coupled to capillary liquid chromatography (LC) with UV detection has been used to effect preconcentration, separation and detection of the analytes in the same assembly. For in-tube SPME two capillary columns of the same length and different internal diameters and coating thicknesses have been tested and compared, a 30 cm x 0.25 mm I.D., 0.25 micro m thickness coating column, and a 30 cm x 0.1 mm I.D., 0.1 micro m of coating thickness column. In both columns the coating was 95% dimethylpolysiloxane (PDMS)-5% diphenylpolysiloxane. The proposed methodology provided limits of detections (LODs) for the tested organophosphorus pesticides in the 0.1-10 micro g/L range, whereas the direct injection of the samples onto the capillary LC system provided LODs in the 50-1000 micro g/L range. The sensitivity of the proposed in-tube SPME-capillary LC method is adequate to monitorize the analyte levels in drinking water. Several triazines, polycyclic aromatic hydrocarbons (PAHs), nonylphenol, organochloride pesticides or polybrominated diphenyl ethers (PBDEs) have been evaluated as possible interferents. The reliability of the described method is demonstrated by analysing different real water samples.     

Comprehensive two-dimensional liquid chromatography for the characterization of functional acrylate polymers

Comprehensive two-dimensional liquid chromatography-size-exclusion chromatography (LC x SEC) was investigated as a tool for the characterization of functional poly(methyl methacrylate) (PMMA) polymers. Ultraviolet-absorbance and evaporative light-scattering detection (ELSD) were used. A simple method to quantify ELSD data is presented. Each data point from the ELSD chromatogram can be converted into a mass concentration using experimental calibration curves. The qualitative and quantitative information obtained on two representative samples is used to demonstrate the applicability of LC x SEC for determining the mutually dependent molar-mass distributions (MMD) and functionality-type distributions (FTD) of functional polymers. The influence of the molar mass on the retention behavior in LC was investigated using LC x SEC for hydroxyl-functional PMMA polymers. The critical conditions, at which retention is--by definition--independent of molar mass, were not exactly the same for PMMA series with different end-groups. Our observations are in close agreement with theoretical curves reported in the literature. However, for practical applications of LC x SEC it is not strictly necessary to work at the exact critical solvent composition. Near-critical conditions are often sufficient to determine the mutually dependent distributions (MMD and FTD) of functional polymers.     

Implementations of two-dimensional liquid chromatography

Today scientists must deal with complex samples that either cannot be adequately separated using one-dimensional chromatography or that require an inordinate amount of time for separation. For these cases we need two-dimensional chromatography because it takes far less time to generate a peak capacity n(c) twice in a row than to generate a peak capacity n(c)(2) once. Liquid chromatography has been carried out successfully on thin layers of adsorbents and along tubes filled with various adsorbents. The first type of separation sorts out the sample components in a physical separation space that is the layer of packing material. The analysis time is the same for all the components of the sample while their migration distance increases with decreasing retention. The resolution between two components having a certain separation factor (alpha) increases with increasing migration distance, i.e., from the strongly to the weakly retained compounds. In the second type of separation, the sample components are eluted from the column and separated in the time space, their migration distances are all the same while their retention times increase from the unretained to the strongly retained compounds. Separation efficiency varies little with retention, as long as the components are eluted from the column. We call these two types of separation the chromatographic separations in space (LC(x)) and the chromatographic separations in time (LC(t)), respectively. In principle, there are four ways to combine these two modes and do two-dimensional chromatographic separations, LC(t)xLC(t), LC(x)xLC(t), LC(t)xLC(x), and LC(x)xLC(x). We review, discuss and compare the potential performance of these combinations, their advantages, drawbacks, problems, perspectives and results. Currently, column-based combinations (LC(t)xLC(t)) are the most actively pursued. We suggest that the combination LC(x)xLC(t) shows exceptional promise because it permits the simultaneous second-dimension separations of all the fractions separated in the first-dimension, thus providing remarkable time saving.