Ionic liquid-based liquid-phase microextraction, a new sample enrichment procedure for liquid chromatography

Room temperature ionic liquids (RTILs) were used as extraction solvent in liquid-phase microextraction (LPME) coupled with liquid chromatography. Using 1-hexyl-3-methylimidazolium hexafluorophosphate ([C6MIM][PF6]) as extraction solvent, some parameters related to LPME of 4-nonylphenol (4-NP) and 4-tert-octylphenol (4-t-OP) were optimized. Although [C6MIM][PF6] can suspend a much larger volume of drop on the needle of the microsyringe than the conventional solvents such as 1-octanol and carbon tetrachloride, the method sensitivity was analyte dependent because of the different partition coefficients and the relatively large viscosity of [C6MIM][PF6]. The proposed procedure has a detection limit and enrichment factor of 0.3 microg l(-1) and 163 for 4-NP, and 0.7 microg l(-1) and 130 for 4-t-OP, respectively. Aqueous samples including tap water, river water, and effluent from sewage treatment plant were analyzed by the proposed method and the recoveries at 10 microg l(-1) spiked level were in the range of 90-113%.     

Optimization of a comprehensive two-dimensional normal-phase and reversed-phase liquid chromatography system

The present investigation is based on the evaluation of the performance of a comprehensive two-dimensional liquid chromatography (LCxLC) system during method optimization. The LCxLC set-up, operated in normal phase (NP) mode (adsorption) in the first dimension (1D) and reversed-phase (RP) mode in the second dimension (2D), is equipped with a 1D microbore silica column and a 2D monolithic C(18) column with a 10-port two position valve as the interface. A photodiode array detector is used after the 2D separation. A possible cause of peak distorsion because of the immiscibility of the mobile phases employed in the two dimensions is resolved. The optimization of the analytical run time and flow rate for both dimensions and the initial gradient in the 2D is carried out with various standard compounds. The potential and versatility of this LCxLC approach is demonstrated through the separation of 11 standard components, most of them allergens. The latter, which are characterized by a scattered distribution on the 2D space plane, underwent separation on both a hydrophobicity and polarity basis.     

Bioanalytical capabilities of micellar liquid chromatography

Micellar liquid chromatography (MLC) offers numerous advantages over other high-performance liquid chromatographic methods in bioanalytical applications, allowing improvements in simultaneous separation of ionic and non-ionic compounds, faster analysis, and higher detection sensitivity and selectivity. In addition, MLC can facilitate the estimation of the hydrophobic character of biologically active molecules in structure—activity relationships.     

Comprehensive two-dimensional liquid chromatography

Having nearly exhausted the possibilities for generating peak capacity through improvements in column technology, chromatographers are increasingly looking to alternative ways of maximising chromatographic separation. In recent years there has been increasing activity in the field of comprehensive multidimensional separations to meet analysis demands. Comprehensive two-dimensional liquid chromatography (LC×LC) approaches offer high peak capacity which leads to significantly improved analytical performance over single-column liquid chromatography. There are several closely related avenues available for achieving an LC×LC separation and this review pays special attention to the different valve-based interfaces that have been used to comprehensively couple the first and second dimension columns in LC×LC systems. A brief discussion of column choices for selected applications and the conditions employed is also presented.     

Phase system selectivity and two-dimensional separations in liquid column chromatography

Correlations between the separation selectivity in aqueous and non-aqueous reversed-phase systems and in normal-phase LC systems were investigated for samples containing different numbers of two repeat structural elements. Such samples are best separated in "orthogonal" two-dimensional chromatographic systems, showing selectivity for one type of the repeat structural element only in the first dimension and for the other structural element only in the second dimension. The number of resolved compounds improves as the degree of orthogonality of the separation systems increases with decreasing correlation between the selectivities for the sample structural distribution in the two dimensions. Orthogonal systems with non-correlated selectivities for each repeat structural element provide the highest number of separated peaks and regular arrangement of the peaks over the two-dimensional retention space according to the individual structural element distribution and the best use of the available peak capacity. Fully orthogonal systems are difficult to find in practice. Partially orthogonal system with correlated selectivities for one structural type distribution, but with one system non-distinguishing the distribution for the other structural element are still useful for the two-dimensional separations. The correlations between the selectivities for repeat regular structural increments were employed to evaluate the suitability of phase systems for two-dimensional HPLC separations. The selectivity correlation in various reversed-phase and normal-phase systems was evaluated for two sample types: (1) Various RP columns show significantly inversely correlated selectivities for acyl lengths and numbers of double bonds distribution, but the differences in the double bond selectivity can be used for practical separations of triacylglycerols with the same equivalent carbon numbers. (2) Synthetic EO-PO block (co)oligomers with two-dimensional distribution of oxyethylene and oxypropylene monomer units were separated according to the two distribution types using on-line two-dimensional reversed-phase-normal-phase LC with a C18 column in the first dimension and an aminopropyl silica column in the second dimension.     

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.     

Comprehensive two-dimensional liquid chromatography coupled with mass spectrometry

In this review, instrumental aspects of comprehensive two-dimensional liquid chromatography coupled with mass spectrometry are presented. The milestones of LC×LC are briefly summarized. Instrument configuration, selection of experimental conditions, the different interfaces used in the system and the current applications of LC×LC–MS systems are described.     

Comprehensive two-dimensional liquid chromatography of polymers

The need for and the emergence of comprehensive two-dimensional liquid chromatographic separations of synthetic polymers are reviewed in this paper. LC×SEC is shown to be a particularly valuable two-dimensional technique in this domain. An improved (symmetrical) configuration based on a single 10-way switching valve is described. The use of LC×SEC to understand and optimize one-dimensional separations is illustrated, as well as the potential of the technique for the separation and characterization of functional polymers and copolymers.     

Column selectivity for two-dimensional liquid chromatography

Selectivity of phase system is of primary concern when designing a 2-D separation, as it affects the 2-D system orthogonality and consequently the peak capacity controlling the number of peaks that can be separated in the available 2-D retention space limited by the time of analysis. Possibilities for characterization of LC phase system selectivity with respect to different polar and nonpolar structural units are compared, with special attention to multidimensional samples with various types of repeat groups, such as homopolymers, (co)polymers, fatty acid esters with various acyl lengths and number and position of double bonds, etc. Possibilities of the 2-D LC separations of these and other sample types, including pharmaceuticals, natural phenolic compounds, biopolymers, etc., using various combinations of separation modes are reviewed. Rules for design of comprehensive 2-D LC x LC systems are discussed, with respect to mobile phase compatibility in the two systems and modulation techniques suppressing band broadening connected with the sample fraction transfer from the first to the second dimension. Pitfalls connected with online connection of normal-phase and RP LC systems and their possible practical solutions are addressed and illustrated by practical examples.     

Fast, comprehensive two-dimensional liquid chromatography

The absolute need to improve the separating power of liquid chromatography, especially for multi-constituent biological samples, is becoming increasingly evident. In response, over the past few years, there has been a great deal of interest in the development of two-dimensional liquid chromatography (2DLC). Just as 1DLC is preferred to 1DGC based on its compatibility with biological materials we believe that ultimately 2DLC will be preferred to the much more highly developed 2DGC for such samples. The huge advantage of 2D chromatographic techniques over 1D methods is inherent in the tremendous potential increase in peak capacity (resolving power). This is especially true of comprehensive 2D chromatography wherein it is possible, under ideal conditions, to obtain a total peak capacity equal to the product of the peak capacities of the first and second dimension separations. However, the very long timescale (typically several hours to tens of hours) of comprehensive 2DLC is clearly its chief drawback. Recent advances in the use of higher temperatures to speed up isocratic and gradient elution liquid chromatography have been used to decrease the time needed to do the second dimension LC separation of 2DLC to about 20s for a full gradient elution run. Thus, fast, high temperature LC is becoming a very promising technique. Peak capacities of over 2000 and rates of peak capacity production of nearly 1 peak/s have been achieved. In consequence, many real samples showing more than 200 peaks with signal to noise ratios of better than 10:1 have been run in total times of under 30 min. This report is not intended to be a comprehensive review of 2DLC, but is deliberately focused on the issues involved in doing fast 2DLC by means of elevating the column temperature; however, many issues of broader applicability will be discussed.     

Reversed-phase liquid chromatography on a microchip with sample injector and monolithic silica column

In micro total analysis systems, liquid chromatography (LC) works under pressure-driven flow is the essential analysis component. There were not, however, much works on microchip LC. Here we developed a microchip for reversed-phase LC using porous monolithic silica. The chip consisted of a double T-shaped injector and a approximately 40-cm serpentine separation channel. The octadecyl-modified monolithic silica was prepared in the specified part of the channel on the microchip using sol-gel process. Furthermore, the effect of geometry of turn sections on band dispersion at turns was examined under pressure-driven flow. High separation efficiencies of 15,000-18,000 plates/m for catechins were obtained using the LC chip.     

Multidimensional liquid chromatography for sample characterisation

While LC finds enormously widespread use in almost all areas of chemical science, the technique is limited as a means of identification because compounds do not elute with unique retention times. This limitation spurred the growth of hyphenated instrumental methods of analysis, such as LC-MS/MS, which because of the MS/ MS detection became a method of identification. However, techniques like LC-MS/ MS are specialised and require high initial purchase and running costs, inhibiting the more widespread growth of the technique. In an attempt to increase the separation power of LC, multi-dimensional LC was developed. This expanded the separation space and subsequently has allowed the development of methods with fingerprinting ability due to the lower probability of component overlap. The work in this study illustrates the application of 2-D LC as a means of chemical fingerprinting. We employed a sample base of various low molecular weight oligostyrenes and their diastereomers that represent a population of compounds whose selectivities in a one-dimensional separation are almost unity and hence essentially impossible to separate. Yet in a 2-D domain almost all individual components occupy unique 2-D retention times.     

One-dimensional and two-dimensional liquid chromatography of sulphonated lignins

One- and two-dimensional separation methods have been developed for the analysis of lignosulphonates and sulphonated kraft lignins. The evaluated sulphonated lignins are all used as dispersants in agrochemical formulations, where some give rise to physical instabilities of formulations. It is of interest to identify the properties of the sulphonated lignins that determine the formulation characteristics. Tetrapentylammonium bromide has been used as an ion-pair reagent in a gradient-elution reversed-phase liquid-chromatographic (IP-RPLC) method, as well as in aqueous size-exclusion chromatography (SEC). Clear differences in the size distribution were observed between different batches of sulphonated lignins. The RPLC and SEC methods were combined in a comprehensive two-dimensional liquid chromatography system. The retention times in the two dimensions were highly correlated. Therefore, the full potential of comprehensive two-dimensional liquid chromatography was not yet realized. However, the results did reveal that retention in IP-RPLC was not determined by the degree of sulphonation of similar-size molecules. Rather, molecules were separated according to size and the degree of sulphonation appears to be approximately constant. The information obtained in this study represents a significant step towards meaningful correlations between the requirements of surfactants within an agrochemical formulation and structural parameters, such as the size and the degree of sulphonation of lignin oligomers.     

Electrodialytic sample treatment coupled on-line with high-performance liquid chromatography

Summary An electrodialytic sample treatment method coupled on-line with high-performance liquid chromatography (EDIST-HPLC) is discussed in this paper. The performance of EDIST as a function of the donor-phase (sample solution) flow rate, the voltage applied over the electrodialysis block, and the time of dialysis has been studied using the basic drug ephedrine as a model compound. Enrichment of the analyte by a factor of 10–20 was possible. The determination of human plasma spiked with ephedrine is briefly discussed.     

Phase diagram of a two-dimensional system with anomalous liquid properties

Using Monte Carlo simulation techniques, we calculate the phase diagram for a square-shoulder square-well potential in two dimensions that has been previously shown to exhibit liquid anomalies consistent with a metastable liquid-liquid critical point. We consider the liquid, gas, and five crystal phases, and find that all the melting lines are first order, despite a small range of metastability. One melting line exhibits a temperature maximum, as well as a pressure maximum that implies inverse melting over a small range in pressure.     

Optimization of two-dimensional gradient liquid chromatography separations

An almost orthogonal comprehensive two-dimensional liquid chromatography was developed for the separation of phenolic and flavone natural antioxidants by using combinations of a polyethylene glycol silica micro-column in the first dimension and a porous-shell fused-core C18 column in the second dimension, both in the reversed-phase mode. System orthogonality was improved using parallel gradients of acetonitrile in buffered mobile phase. A new approach was proposed to optimize matching segmented gradient profiles in the two dimensions. An algorithm was developed for automatic corrections of the shifts in retention in the second dimension induced by the parallel two-dimensional gradient operation technique. Using the porous-shell C18 column in the second dimension at elevated temperature (60 degrees C) and high pressure (480 bar) with optimized segmented profiles of the parallel gradients in the two dimensions, the overall separation time for comprehensive LC x LC was reduced to 30 min.     

High-temperature two-dimensional liquid chromatography of ethylene-vinylacetate copolymers

Temperature rising elution fractionation hyphenated to size exclusion chromatography (TREF × SEC) is a routine technique to determine the chemical heterogeneity of semicrystalline olefin copolymers. Its applicability is limited to well crystallizing samples. High-temperature two-dimensional liquid chromatography, HT 2D-LC, where the chromatographic separation by HPLC is hyphenated to SEC (HPLC × SEC) holds the promise to separate such materials irrespective of their crystallizability. A model blend consisting of ethylene-vinyl acetate (EVA) copolymers covering a broad range of chemical composition distribution including amorphous and semicrystalline copolymers and a polyethylene standard was separated by HT 2D-LC at 140 °C. Both axes of the contour plot, i.e. the compositional axis from the HPLC and the molar mass axis from the SEC separation were calibrated for the first time. Therefore, a new approach to determine the void and dwell volume of the developed HT 2D-LC instrument was applied. The results from the HT 2D-LC separation are compared to those from a cross-fractionation (TREF × SEC) experiment.     

A micro trapping system coupled with a high performance liquid chromatography procedure for methylamine determination in both tissue and cigarette smoke

Both endogenous and exogenous methylamine have been found to be involved in many human disorders. The quantitative assessment of methylamine has drawn considerable interest in recent years. Although there have been many papers about the determination of methylamine, only a few of them involved cigarette smoke or mammalian tissue analysis. The major hurdles of the determination of methylamine are the collection of methylamine from samples and the differentiation of methylamine from the background compounds, e.g., biogenic amines. We have solved this problem using a micro trapping system coupled with an HPLC procedure. The interference from other biogenic amines has been avoided. The high selectivity of this method was achieved using four techniques: distillation, trapping, HPLC separation and selective detection. The chromatograms of both mouse tissues and cigarette smoke are simple, with only a few peaks. The method is easy and efficient and it has been validated and applied to the determination of methylamine in tissues of normal CD 1 mice and cigarette smoke. The methylamine contents were determined to be approximately 268.3 ng g⁻¹ in the liver, 429.5 ng g⁻¹ in the kidney and 547.4 ng g⁻¹ in the brain respectively. The methylamine in the cigarette smoke was approximately 213 ng to 413 ng per cigarette. These results in tissues and in cigarette smoke were found to be consistent with the data in the previous literature. To the best of our knowledge, this is the first report on a method suitable for methylamine analysis in both mammalian tissue and cigarette smoke.     

Automation of sample pretreatment for liquid chromatography

The purpose of this review is to discuss the strategic problems of automating sample preparation (SP) for high performance liquid chromatography (HPLC). There is a general feeling that SP is the bottleneck of many HPLC procedures. Despite numerous reports of successful automation of SP, there are still many laboratories using manual or semiautomated SP procedures. This calls for a reevaluation of the present situation.     

Fully automated micro- and nanoscale one- or two-dimensional high-performance liquid chromatography system for liquid chromatography-mass spectrometry compatible with non-volatile salts for ion exchange chromatography

A one- or two-dimensional high performance liquid chromatography system for electrospray ionization mass spectrometers has been developed that is optimized for ion exchange and reversed phase separations. A unique and simple valve configuration permits the use of a variety of non-volatile salts; ammonium sulfate was used in an example of strong cation exchange separations. The system was designed and evaluated for both micro- and nanoflow chromatography. The peptide detection limit was approximately 100 fmol for micro- and 20 fmol for nanoflow, demonstrating the concentration and mass sensitivity improvements expected with nanoelectrospray ionization. The 1D/2D-HPLC MS system is fully automated for routine peptide analyses, compatible with direct injection of proteolytic digests, and exhibits chromatographic reproducibility and sensitivity. Software permits operator selection of either a 1D or 2D configuration with corresponding system parameters as required for individual samples. The hardware elements and resulting performance are described in this paper.