On-line Coupling Of Liquid Chromatography Research Articles

Online coupling of liquid chromatography and two-dimensional diffusion ordered spectroscopy for the analysis of oligostyrenes

The aim of this study was to introduce a powerful coupling of Liquid Adsorption Chromatography (LAC) and Diffusion-Ordered Spectroscopy (DOSY) for comprehensive structure analysis. This new hyphenation approach facilitated the simultaneous separation of a polymer mixture and the determination of molar masses within a single 3D experiment. The online coupling of High-Performance Liquid Chromatography (HPLC) and two-dimensional DOSY-NMR will be called 3D-LAC-NMR-DOSY experiment. Our methodology enabled the chromatographic separation of analytes based on their chemical heterogeneity, and provided accurate molar masses of the analytes through 2D-DOSY. This new method was demonstrated on a polystyrene oligomer mixture. In this case, the oligostyrenes could be separated with LAC according to their tacticity and chain length in protonated acetonitrile as eluent and DOSY measurements provided the molar masses of each oligomer. In order to show the power of the 3D-LAC-NMR-DOSY method, the comparison to 2D-DOSY, 3D-DOSY and LAC-NMR was separately evaluated. Furthermore, the recently published solvent-independent molar mass calibration of diffusion coefficients was also successfully applied in our LAC-DOSY studies for molar mass predictions of the oligomers in acetonitrile. The predicted molar masses were in good agreement with the LAC-DOSY measurements and were verified by calibrations of diffusion coefficients and mass spectrometry. Finally, this pioneering 3D technique offers a powerful new tool for advancing structure analysis and enhancing our understanding of complex systems such as oligostyrenes.

The online coupling of liquid chromatography to Fourier transform infrared spectroscopy using a solute-deposition interface: A proof of concept

Hyphenated techniques combining chromatographic and spectroscopic methods are the gold standard to effectively handle the emerging challenges in the analysis of unknown chemical components in mixtures, and in this regard the coupling of liquid chromatography to Fourier transform infrared spectroscopy (LC-FTIR) is no exception. While earlier attempts to couple LC to IR spectroscopy relied almost entirely on offline techniques, clear motivations for implementing online LC-FTIR instrumentation emerged from the need for shorter analysis time, a higher degree of automation and sample throughput, better reproducibility, and reduced contamination. Most recent designs of LC-FTIR concepts have aimed to combine the advantages of both approaches by means of a solvent-elimination interface. The hyphenated instrumentation and method presented in this research are based on a pneumatically assisted LC-FTIR interface, relying on a small-scale self-regulating spray dryer to attain desolvation of the LC eluent stream while retaining the spatial and temporal resolution of the dissolved substrates. Focused deposition of the dried analytes occurs onto a ZnSe disc for continuous transmission mid-IR analysis at a resolution of 4 cm-1. The optimization of the LC-FTIR technique is discussed in terms of interface parameters, limits of detection, and limits of quantification for a pair of furanocoumarin isomers differing in the position (linear or angular type) of the furan ring fused to coumarin. Finally, confident discrimination between the two closely related molecules was attained by matching the experimental FTIR spectra in a dedicated library. The quality match factors obtained were higher than 99% for both molecules. The limit of identification (LOI) was determined experimentally as the minimum amount of substance yielding a library-searchable IR spectrum (affording a quality match factor higher than 90%). Specifically, LOI of 0.6 μg and 1.25 μg was determined for psoralen and angelicin, respectively.

Online HPLC-ESI-HRMS Method for the Analysis and Comparison of Different Dissolved Organic Matter Samples.

Natural dissolved organic matter (DOM) is an ultracomplex mixture that is essential to global carbon cycling but is poorly understood because of its complexity. The most powerful tool for the DOM characterization is high-resolution mass spectrometry (HRMS) generally combined to direct infusion (DI) as sample introduction. Liquid chromatography (LC) represents a compelling alternative to DI; however, state-of-the-art techniques involve only offline LC-HRMS approaches, which have important logistical drawbacks that make DOM analysis more challenging. This study introduces a new method based on online coupling of liquid chromatography to high resolution mass spectrometry, able to overcome the disadvantages of usual approaches. It is characterized by high reproducibility (% Bray-Curtis dissimilarity among replicates ≈ 2.5%), and it reduces transient complexity and contaminant interferences, thus increasing the signal-to-noise ratio (S/N), leading to the identification of an overall larger number of formulas in the mixture. Moreover, the application of an in silico fractionation prior to the statistical analysis allows an easy, flexible, fast, and detailed comparison of DOM samples from a variety of sources with a single chromatographic run.

Fraction transfer process in on-line comprehensive two-dimensional liquid-phase separations.

Two-dimensional liquid-phase separations have gained increasing attention for their ability to separate complex sample mixtures. Among the experimental setups used, an on-line approach is preferred to reduce the probability of sample contamination, for easier automation and high-sample throughput. The interfacing of the separation techniques in the on-line mode brings additional demands on proper optimization of the two-dimensional system. In this review, the possibilities of the on-line coupling of liquid chromatography and liquid chromatography with capillary electrophoresis in two-dimensional systems are discussed. Special attention is paid to the fraction transfer process, which includes an overview of interfaces and experimental setups applied, the compatibility issues of separation systems, and instrumental parameters. The benefits and drawbacks of using electromigration separations in combination with liquid chromatography are presented as well.

Target Analysis of Polycyclic Aromatic Hydrocarbons (PAHs) in Consumer Products and Total Content of Polycyclic Aromatic Compounds (PACs)

ABSTRACTThe fabrication of consumer products is tightly linked to the usage of polymeric materials. The application of mineral oil products as plasticizers or lubricants in this manufacture is very common and so is the addition of carbon black for coloring and abrasion resistance purposes. Whereas mineral oils often contain highly alkylated aromatic species, alkylation of polycyclic aromatic compounds (PACs) is rather low or even absent in the case of carbon black. Since they are broadly applied in various products, consumers might get exposed to PACs, for instance, via dermal contacts. Here, we characterized several commodities not only via the common way of targeted analysis for their contents on PAHs, but also with regard to its total amounts of unsubstituted and substituted aromatic hydrocarbons. The methods applied to analyze consumer products were as follows: (i) online coupling of liquid chromatography to gas chromatography with flame ionization detection (LC-GC-FID) for quantification of total PAH and mineral oil saturated hydrocarbon (MOSH) contents; (ii) coupling of tandem mass spectrometry to gas chromatography (GC-MS/MS) to quantify occasionally occurring PAHs with high specificity; and (iii) 2D gas chromatography coupled to time-of-flight mass spectrometry (GCxGC-MS) to characterize the complex mixtures concerning analytes’ ring systems and degrees of alkylation. We found PAH contents in the range of parts per million (ppm) in consumer products, while overall amounts of aromatics were in the percent range.

Use of nitrogen to remove solvent from through oven transfer adsorption desorption interface during analysis of polycyclic aromatic hydrocarbons by large volume injection in gas chromatography

The through oven transfer adsorption desorption (TOTAD) interface allows large volume injection (LVI) in gas chromatography and the on-line coupling of liquid chromatography and gas chromatography (LC–GC), enabling the LC step to be carried out in normal as well as in reversed phase. However, large amounts of helium, which is both expensive and scarce, are necessary for solvent elimination. We describe how slight modification of the interface and the operating mode allows nitrogen to be used during the solvent elimination steps. In order to evaluate the performance of the new system, volumes ranging from 20 to 100μL of methanolic solutions of four polycyclic aromatic hydrocarbons (PAHs) were sampled. No significant differences were found in the repeatability and sensitivity of the analyses of standard PAH solutions when using nitrogen or helium. The performance using the proposed modification was similar and equally satisfactory when using nitrogen or helium for solvent elimination in the TOTAD interface. In conclusion, the use of nitrogen will make analyses less expensive.

Membrane-Based Continuous Remover of Trifluoroacetic Acid in Mobile Phase for LC-ESI-MS Analysis of Small Molecules and Proteins

We developed a "continuous" trifluoroacetic acid (TFA) remover based on electrodialysis with bipolar membrane for online coupling of liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS) using TFA containing mobile phase. With the TFA remover as an interface, the TFA anion in the mobile phase was removed based on electrodialysis mechanism, and meanwhile, the anion exchange membrane was self-regenerated by the hydroxide ions produced by the bipolar membrane. So the remover could continuously work without any additional regeneration process. The established LC-TFA remover-MS system has been successfully applied for the qualitative and quantitative analysis of small molecules as well as proteins.

Titanium release in serum of patients with different bone fixation implants and its interaction with serum biomolecules at physiological levels

Increased concentrations of circulating metal-degradation products derived from the use of Ti orthopaedic implants may have deleterious biological effects over the long term. Therefore, there is an increasing need to establish the basal level of Ti in the serum of the population (exposed and non-exposed) with appropriate highly sensitive techniques and strategies. With this aim, we have developed a quantitative strategy for the determination of total Ti concentration in human serum samples by isotope dilution analysis using a double-focussing inductively coupled plasma mass spectrometer. Minimizing sample handling and therefore contamination issues, we obtained detection limits of about 0.05 μg L(-1) Ti working at medium resolution (m/Δm 4000). Such extremely good sensitivity permitted us to establish the range of Ti concentration in serum of 40 control individuals (mean 0.26 μg L(-1)) and also to compare it with the level in exposed patients with different Ti metal implants. On the other hand, Ti transport "in vivo" studies have been enabled by online coupling of liquid chromatography (anion-exchange) separation and double-focussing inductively coupled plasma mass spectrometry for sensitive detection of Ti. The development of a postcolumn isotope dilution strategy permitted quantitative characterization of the Ti-transporting biomolecules in human serum. The results for unspiked serum revealed that 99.8% of the Ti present in this fluid is bound to the protein transferrin, with column recoveries greater than 95%.

Separation Analysis of Polyisoprenes Regarding Microstructure by Online LCCC-NMR and SEC-NMR

It is shown that online coupling of liquid chromatography at critical conditions (LCCC) and 1H NMR spectroscopy can be used to separate and analyze polyisoprenes with respect to their isomeric microstructures. It was possible to separate blends of copolymers consisting predominantly of 3,4- and 1,4-isoprene units by using critical conditions of 1,4-PI. Critical conditions of 1,4-PI were established such that polymers containing predominantly 3,4-PI elute in the size exclusion mode. Furthermore, on-flow NMR detection allows for the complete eluate analysis and the detection of all three isomeric species, such as 1,2-, 1,4-, and 3,4-isoprene, in the eluting fractions. These three individual moieties were correctly quantified in the individual blend components. It was also found that the 3,4-PI samples were random terpolymers of 1,2-, 1,4-, and 3,4-isoprene, whereas the 1,4-PI samples were copolymers of 1,4- and 3,4-isoprene. In addition, SEC-NMR allowed for a fast and precise molar mass calibration and the calculation of the relevant molar mass parameters Mw and Mn.

Coupling of NMR and Liquid Chromatography at Critical Conditions: A New Tool for the Block Length and Microstructure Analysis of Block Copolymers

The online coupling of liquid chromatography at critical conditions (LCCC) and NMR was used for the analysis of block copolymers. Polyisoprene-b-poly(methyl methacrylate) (PI-b-PMMA) copolymers synthesized by living anionic polymerization were separated regarding chemical composition byLCCCusingsinglesolventsasmobilephases.Theanalysis ofthechemically differentLCCCfractionswas conducted by on-flow 1 H NMR. To separate the copolymers from the respective homopolymers, critical conditions of polyisoprene (PI) and poly(methyl methacrylate) (PMMA) were used. Critical conditions were obtained by varying the column temperature. The comprehensive chemical composition and microstructure analysis by 1 H NMR revealed that quantitative information on the PI/PMMA content as well as the microstructure of PMMA (isotactic, atactic, and syndiotactic triads) and PI (1,2-PI, 1,4-PI, 3,4-PI) was obtained.For thefirst time thedetermination ofthetruemolarmasses and truechemicalcompositions ofthe block copolymers was achieved by determining the microstructure distribution.

Atmospheric Pressure Free Liquid Infrared MALDI Mass Spectrometry: Toward a combined ESI/MALDI-Liquid Chromatography Interface

A new atmospheric pressure (AP)-MALDI-type interface has been developed based on a free liquid (FL) microbeam/microdroplets and a mid-infrared optical parametric oscillator (mid-IR OPO). The device is integrated into a standard on-line nanoESI interface. The generation of molecular ions in the gas phase is believed to be the result of a fast (explosive) laser-induced evaporative dispersion(not desorption) of the microbeam into statistically charged nanodroplets. Only the lowest charge states appear insignificant abundance in this type of experiment. Mass spectra of some common peptides have been acquired in positive ion mode, and the limit-of-detection of this first prototype (liquid microbeam setup) was evaluated to be 17 fmol per second. To improve the duty cycle and to reduce the sample consumption, a droplet-on-demand system was implemented (generating 100 pL droplets).With this setup, about 20 attomole of bradykinin were sufficient to achieve a signal-to-noise ratio better than five.This setup can be operated at flow rates down to 100 nL/min and represents a liquid MALDI alternative to the nanoESI. Our particular interest was the application of the developed ion source for on-line coupling of liquid chromatography with mass spectrometry. The flow rates(>100 microL/min), required for stable operation of the ion source in continuous liquid microbeam mode, matches perfectly the flow rate range of micro HPLC. Therefore, online LC/MS experiments have been realized, employing a microbore C18 reversed-phase column to separate an artificial peptide mixture and tryptic peptides of bovine serum albumin (performing a peptide mass fingerprint). In the latter case, sequence coverage of more than 90%has been achieved.

Development of an on-line UV decomposition system for direct coupling of liquid chromatography to atomic-fluorescence spectrometry for selenium speciation analysis

Speciation analysis of four selenium species (selenite, selenate, selenocystine, and selenomethionine) has been performed by on-line coupling of liquid chromatography (LC), UV decomposition, hydride generation (HG), and atomic-fluorescence spectrometry (AFS). Because only selenite (Se(IV)) can generate hydrides, on-line conversion of organic and inorganic selenium species is discussed. Preliminary study showed that the use of only UV light was not sufficient to reduce selenate, because no absorption is observed for this compound at the main wavelength of the low-pressure mercury lamp (253.7 nm). Thus, new conditions based on addition of a reducing reagent (I-) were developed. Mechanisms of action are proposed to explain selenium species conversions. Because of their compatibility with on-line treatment, phosphate buffers were used for chromatographic separation on an anion exchange column (Hamilton PRP-X100). Detection limits (19-60 pg Se) and repeatability of the technique were close to those obtained by conventional quadrupole ICPMS. Applications to real samples such as water and oysters are presented and emphasize the robustness of the system.

Towards early detection of the hydrolytic degradation of poly(bisphenol A)carbonate by hyphenated liquid chromatography and comprehensive two-dimensional liquid chromatography

The hydrolytic degradation of poly(bisphenol A)carbonate (PC) has been characterized by various liquid chromatography techniques. Size exclusion chromatography (SEC) showed a significant decrease in molecular mass as a result of hydrolytic degradation, while ‘liquid chromatography at critical conditions’ (LC-CC) was very successful for observing differences in functionality due to degradation, i.e. the formation of OH end-groups. To characterize and identify the observed differences semi on-line coupling of liquid chromatography to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Fourier transform infrared spectroscopy (FT-IR) has proved to be very useful. Comprehensive two-dimensional liquid chromatography (2D-LC) was also applied to study the hydrolytic degradation of poly(bisphenol A)carbonate. LC-CC × SEC showed that the formation of poly(bisphenol A)carbonate with OH end-groups occurred over the whole molecular mass range. This information could not be obtained with the separate liquid chromatographic techniques, thereby illustrating the usefulness of 2D-LC. The main degradation processes of poly(bisphenol A)carbonate under the applied hydrolysis conditions turned out to be disappearance of cyclic PC oligomers, chain scission of PC and (subsequent) formation of OH end-groups. FT-IR, SEC and LC-CC have been used to follow the hydrolytic degradation with time. LC-CC proved to be the most promising technique to detect the degradation of poly(bisphenol A)carbonate at an early stage.

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

High throughput, outstanding certainty in peptide/protein identification, exceptional resolution, and quantitative information are essential pillars in proteome research. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has proven to meet these requirements. Soft ionization techniques, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), have paved the way for the story of success of CE-MS in the analysis of biomolecules and both approaches are subject of discussion in this article. Meanwhile, CE-MS is far away from representing a homogeneous field. Therefore the review will cover a vast area including the coupling of different modes of CE (capillary zone electrophoresis, capillary isoelectric foscusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as on-line preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) applied to compensate for restricted detection sensitivity. Special attention is given to improvements in interfacing, namely addressing nanospray and coaxial sheath liquid design. Peptide mapping, collision-induced dissociation with subsequent tandem MS, and amendments in mass accuracy of instruments improve information validity gained from MS data. With 2-D on-line coupling of liquid chromatography (LC) and CE a further topic will be discussed. A special section is dedicated to recent attempts in establishing CE-ESI-MS in proteomics, in the clinical and diagnostic field, and in the food sector.

Liquid-core waveguide technology for coupling column liquid chromatography and Raman spectroscopy

The on-line coupling of liquid chromatography (LC) and Raman spectroscopy (RS) via an entirely plastic liquid-core waveguide (LCW) was optimized in terms of excitation wavelength of the laser, especially in relation to the fluorescence background, and the length of the LCW. Excitation at 632.8 nm (He–Ne laser) was found to be a good compromise between a wavelength long enough to strongly reduce the fluorescence background and, on the other hand, short enough to avoid (re)-absorption of laser light and Raman signals by H 2O in LCWs of considerable length. This conclusion is supported by a theoretical discussion on the optimization of LCW lengths as function of the excitation wavelength for H 2O and 2H 2O. When using the He–Ne laser the optimum length is ∼50 cm for H 2O; this corresponds to a detection cell volume of 19 μl for an LCW of 220 μm I.D., which is fully compatible with conventional-size LC. The influence of an organic modifier, usually necessary for reversed-phase LC, on the free spectral window was evaluated. The potential applicability of LC–LCW-RS was shown for a mixture of adenosine 5′-monophosphate (AMP), guanosine 5′-monophosphate (GMP) and uridine 5′-monophosphate (UMP), utilizing an aqueous eluent without the addition of a modifier. Improved detectability was achieved by using the stopped-flow mode and applying a large-volume-injection procedure (injection volume: 200 μl). Under these conditions, the limit of identification for AMP, GMP and UMP was in the 0.1–0.5-mg/ml range.

The autoproteolysis of Lactococcus lactis lactocepin III affects its specificity towards beta-casein.

The effect of autoproteolysis of Lactococcus lactis lactocepin III on its specificity towards beta-casein was investigated. beta-Casein degradation was performed by using either an autolysin-defective derivative of L. lactis MG1363 carrying the proteinase genes of L. lactis SK11, which was unable to transport oligopeptides, or autoproteolyzed enzyme purified from L. lactis SK11. Comparison of the peptide pools by high-performance liquid chromatography analysis revealed significant differences. To analyze these differences in more detail, the peptides released by the cell-anchored proteinase were identified by on-line coupling of liquid chromatography to mass spectrometry. More than 100 oligopeptides were released from beta-casein by the cell-anchored proteinase. Analysis of the cleavage sites indicated that the specificity of peptide bond cleavage by the cell-anchored proteinase differed significantly from that of the autoproteolyzed enzyme.

Preliminary study on the effect of miniaturisation and use of volatile mobile phases in LC for the on-line LC–MS analysis of basic pharmaceuticals

To enhance to compatibility of the on-line coupling of liquid chromatography (LC) with mass spectrometry (MS) for the analysis of basic pharmaceuticals, the use of volatile mobile phase systems in combination with miniaturised LC was investigated. Multifactor analysis of variance (MANOVA) was used to evaluate the data obtained for the various variables (modifier, stationary phase, buffer, buffer pH and buffer concentration) on the resolution, peak symmetry and retention of four basic compounds analysed using LC columns with internal diameters (I.D.) of 0.3, 1.0 and 4.6 mm (conventional). Preliminary results obtained with the investigated micro and conventional columns showed similar behaviour with respect to ruggedness. The various investigated variables showed that miniaturisation by simply downscaling dimensions can result in varying selectivity and peak shapes for basic compounds. When comparing volatile mobile phases (containing ammonium acetate or ammonium citrate) and a conventional non-volatile mobile phase (containing sodium phosphate) under pH 3 conditions, similar separation performances were observed. In the present study, ammonium citrate as the buffering salt, a high buffer concentration and methanol as the modifier showed the best peak symmetry.

Enzyme amplification as detection tool in continuous-flow systems: II. On-line coupling of liquid chromatography to enzyme-amplified biochemical detection after pre-column derivatization with biotin

Enzyme-amplified biochemical detection (EA-BCD) was used as a post-column detection technique, coupled on-line with high-performance liquid chromatography (HPLC). The enzyme detection system was developed to detect biotin or biotin containing compounds. Biotinylation is widely used to label analytes of interest ranging from small molecules to proteins and DNA. Naphthalene aldehyde and anthracene aldehyde were used as model compounds. Both compounds were biotinylated off-line with biotin aminocaproic hydrazide (BACH). On-column biotinylation was performed by preconcentration of anthracene aldehyde on copper phthalocyanine. After biotinylation, samples were introduced to the HPLC system. Enzyme-labeled streptavidin, which possesses high affinity to biotin, was added post-column to the HPLC effluent. Excess of enzyme-labeled affinity protein was removed by means of an immobilized biotin column. After separation of free and bound fraction, substrate was added, which was converted to a fluorescent product by the enzyme label. Using alkaline phosphatase as an enzyme label, a mass detection limit after on-column preconcentration and biotinylation of 250 fmol was achieved.

On-line coupling of liquid chromatography with thin-layer chromatography

On-line coupling of high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) represents a very promising two-dimensional technique because it allows the combination of two completely different separation principles. This is especially so for the combination of reversed-phase HPLC for the first separation and HPTLC with automated multiple development on a normal-phase thin-layer plate for the second separation. The system consists of a microbore HPLC syringe pump and a modified TLC autosampler (CAMAG ATS3-C) which is controlled by new software for a personal computer running under MS Windows. The new dedicated software enables the analyst to spray the effluent from the HPLC column in fractions onto the plate in a freely definable manner. In order to address different analytical problems, six transfer modes are made available. A complete profiling analysis can be carried out with the whole effluent from the liquid chromatographic column being transferred in fractions to the TLC plates. On the other hand, the program supports typical target compound analyses with multiple switching activated according to individual elution times. Typical applications are pesticide residue analysis in foodstuffs or environmental samples. The new dedicated software is expected to fill the gap considered to be the main obstacle for the widespread application of on-line coupling of HPLC with TLC.

Determination of l-lysine N-acetylcysteinate and its mono- and dimeric related compounds by liquid chromatography–mass spectrometry

Methods for the simultaneous determination of N-acetylcysteine, l-lysine, l-cysteine, l-cystine, N, N′-diacetylcystine and N, S-diacetylcysteine in aqueous solutions were developed. The method is based on the on-line coupling of liquid chromatography (LC) to tandem mass spectrometry (MS–MS) with an atmospheric pressure chemical ionisation (APCI) interface. The optimal LC phase system consisted of a C 18 stationary phase and an acidic solution of formic acid in acetonitrile as the mobile phase, while d, l-phenylalanine was selected as an internal reference compound. The best transitions for MS–MS detection were selected after optimisation of the APCI ionisation yield in full scan. The optimisation was performed by loop injection. By coupling LC to MS–MS in the APCI+ (positive ion mode) as well as in the multiple reaction monitoring mode, satisfactory results were obtained with a mixture of the seven compounds, each at a concentration level of 1 μg/ml. For a further improvement of the detection limit of the dimeric compounds, electrospray ionisation was then investigated. By use of this interface a quantitation limit down to 10 −8 M (3 ng/ml) could be achieved for both dimers. The developed method provides improved selectivity based on the combination of chromatography and mass spectrometry thus facilitating identification.