Application of on-line capillary high-performance liquid chromatography-nuclear magnetic resonance spectrometry coupling for the analysis of vitamin A derivatives

The direct on-line coupling between capillary high-performance liquid chromatography (capillary HPLC) and proton high-field nuclear magnetic resonance (NMR) spectrometry has been used to derive structural information about constituents of a mixture of vitamin A derivatives. ¹H NMR spectra were recorded in the stopped-flow and continuous-flow mode within a 180 μm I.D. capillary column mounted in a micro probe on a 600 MHz NMR spectrometer. The resolution of the ¹H NMR spectra obtained in capillary HPLC-NMR coupling experiments is sufficient to determine coupling constants in the order of 1.5 Hz. The detection limit is in the lower nanogram range. A stopped-flow 2D-TOCSY experiment of a 1% solution of vitamin A acetate acquired within 4 h reveals that the acquisition of 2D NMR spectra is possible in the nanoliter detection scale without any loss of structural information.     

Separation of diastereoisomers of podophyllum lignans by micellar electrokinetic chromatography

A technical solution and development of a method for on-line HPLC monitoring of bioreactor processes in a membrane reactor system are presented. Experiences in system design for the continuous coupling of a bioreactor system with capillary by-pass circuits using membrane flow cells and a dual HPLC system are reported. A continuously working integrated sample purification step by ultrafiltration with the membrane cell coupling is established. Using electrical switching valves and separated pumping and eluent systems, the dual HPLC system allows diode array detection as well as measurement of the refractive index. The application of the on-line HPLC monitoring system is demonstrated by measuring the anaerobic H-acid degradation kinetics. H-acid, 1-amino-8-hydroxynaphthalene-3,6-disulfonic acid, is one of the most important coupling components for a variety of direct, mordant, reactive dyes which remains in the process water and the textile dyeing effluents in high concentration.     

Automated determination of the pK a values of 4-hydroxybenzoic acid in cosolvent–water mixtures and related solvent effects using a modified HPLC system

An automated spectrophotometric method based on an HPLC system with a diode array detector was used to determine the pK _a values of compounds with low water solubility in a universal buffer containing acetonitrile as cosolvent. The column of the system was replaced with a capillary connecting the injection system and the diode array detector. Specific solvent effects were corrected for using the dielectric constants of the mixed solvent and pure water. The method was tested using 4-hydroxybenzoic acid and the results were compared with those obtained with a spectrophotometer. Linear regression lines with different slopes were obtained from spectrophotometric measurements of different cosolvent–water mixtures. These effects were shown to depend upon the polarity of the solventwater mixture, and they were explained by the solvatochromic behavior of the 4-hydroxybenzoic acid in the solvent–water mixture.     

高效液相色谱－核磁共振联用技术及其应用

高效液相色谱－核磁共振（ＨＰＬＣ－ＮＭＲ）在线联用技术是同时进行未知混合物的分离和结构鉴定的最好手段之一。详细介绍了在ＨＰＬＣ－ＮＭＲ联用技术中１Ｈ谱的分辨率、检测限和多重溶剂峰抑制的最新进展，并简要评述了其它分离方法与ＮＭＲ联用情况     

高效液相色谱－核磁共振联用技术及其应用

高效液相色谱－核共共振在线联用技术是同时进行未知混合物的分离和结构鉴定的量好手段之一。详细介绍了在ＨＰＬＣ－ＮＭＲ联用技术中Ｈ谱的分辨率，检测限手多重溶剂峰抑制的最新进展，并简要评述了其它分离方法与ＮＭＲ联用情况。     

Mobile phase compensation to improve NMR spectral properties during solvent gradients

A solvent compensation method based on flow injection analysis is used to obtain high quality nuclear magnetic resonance (NMR) spectra during solvent gradients. Using a binary solvent system containing D2O and CD3OD, NMR line broadening and chemical shift changes are observed with a 10% methanol per min solvent composition gradient. However, by creating a second equal but reverse gradient and combining the two solvent gradients before the NMR detector, the composition of solvent reaching the NMR flow cell is kept constant. We demonstrate a system using flow injection analysis of combining solvent gradients and show constant NMR spectral performance as a function of time as the combined flow has a constant solvent composition irrespective of the initial solvent gradient. Using this approach, methods can be developed to measure high quality NMR spectra during on-flow gradient LC-NMR experiments. The ultimate ability of this approach depends on the ability to compensate for the disturbance of the solvent gradient and reverse gradient by a pair of LC columns (the analytical and reverse gradient columns).     

Directly coupled CZE-NMR and CEC-NMR spectroscopy for metabolite analysis: paracetamol metabolites in human urine.

Direct coupling of NMR spectroscopic detection with both capillary zone electrophoresis (CZE) and capillary electrochromatography (CEC) was applied to the separation of metabolites of the drug paracetamol in an extract of human urine. Continuous-flow CZE-NMR and CEC-NMR allowed the detection of the major metabolites, the glucuronide and sulfate conjugates of the drug and the endogenous material hippurate. Identification of these substances was achieved by examination of individual rows of the NMR chromatogram and this also gave estimates of the detection limits. For CEC-NMR, spectra were also obtained in the stopped-flow mode including a two-dimensional TOCSY NMR experiment which afforded confirmatory evidence for paracetamol glucuronide. Characterisation of drug metabolites using NMR spectroscopy is therefore possible with nanolitre sample volumes.     

Separation of chlorins and carotenoids in capillary electrophoresis

The present study reports the investigation of capillary electrophoresis (CE) for the separation of the photosynthetic pigments (chlorophyll derivatives as well as carotenoids) together. Various CE methods, such as micellar electrokinetic chromatography, capillary electrokinetic chromatography, and nonaqueous capillary electrophoresis (NACE) are tested, with coated and uncoated capillary columns to evaluate optimal separation conditions using diode array detection. The effect of different type and composition of organic solvents and surfactants on the separation is discussed. Detection limits are found in the range of 1.14-2.45 ppm. According to the system suitability results, the most effective separation is observed using NACE with Aliquat 336 as cationic surfactant in coated capillary and mixture of MeOH-ACN-THF (5:4:1, v/v/v) as solvent. Quantitative evolution is investigated, and recovery percentage values are found to be 96.7-102%.     

On-Line Coupling of Separation Techniques to NMR

The hyphenation of chromatographic separation techniques with NMR spectroscopy is one of the most powerful and time-saving methods for the separation and structural elucidation of unknown compounds and molecular compositions of mixtures. Most of the routinely used NMR flow-cells have detection volumes between 40–180 μL for conventional separations with analytical columns, and the newest designs employ detection volumes in the order of 200 nL for capillary separations. The low flow rates used in capillary chromatography permit the use of deuterated solvents. Unequivocal structural assignment of unknown chromatographic peaks is possible by two-dimensional stopped-flow capillary HPLC-NMR experiments.     

New approaches on gas phase molecular absorption spectrometry detection in gas chromatography

A hyphenated technique for coupling a spectrophotometric detector to a gas chromatograph with a packed column is described. The packed column was connected directly to the flow cell (path length 1 cm) which was placed in a UV-visible molecular absorption spectrophotometer with diode array detector. This detection system was employed for separating and determining benzene, toluene, 1,4-xylene, 1,2-xylene and mesitylene. Molecular absorption spectra of all the separated compounds have also been obtained. A new possibility for coupling capillary gas chromatography and gas phase molecular absorption spectrometry is currently being tested. In this new case, two fibre optics are used and the measurement is performed in the capillary column.     

New approaches on gas phase molecular absorption spectrometry detection in gas chromatography

A hyphenated technique for coupling a spectrophotometric detector to a gas chromatograph with a packed column is described. The packed column was connected directly to the flow cell (path length 1 cm) which was placed in a UV-visible molecular absorption spectrophotometer with diode array detector. This detection system was employed for separating and determining benzene, toluene, 1,4-xylene, 1,2-xylene and mesitylene. Molecular absorption spectra of all the separated compounds have also been obtained. A new possibility for coupling capillary gas chromatography and gas phase molecular absorption spectrometry is currently being tested. In this new case, two fibre optics are used and the measurement is performed in the capillary column.     

Study of an electroosmotic pump for liquid delivery and its application in capillary column liquid chromatography

A packed-bed electroosmotic pump (EOP) was constructed and evaluated. The EOP consisted of three capillary columns packed in parallel, a gas-releasing device, Pt electrodes and a high-voltage power supply. The EOP could generate output pressure above 5.0 MPa and constant flow rate in the range of nl/min to a few microl/min for pure water, pure methanol, 2 mM potassium dihydrogenphosphate buffer, the buffer-methanol mixture and the pure water-methanol mixture at applied potentials less than 20 kV. The composition of solvent before/after pumping was quantitatively determined by using a gas chromatograph equipped with both flame ionization detector and thermal conductivity detector. It was found that there were no apparent changes in composition and relative concentrations after pumping process for a methanol-ethanol-acetonitrile mixture and a methanol-water mixture. Theoretical aspect of the EOP was discussed in detail. An capillary HPLC system consisting of the EOP, an injection valve, a 15 cm x 320 microm i.d., 5 microm Spherigel C18 stainless steel analytical column, and an on-column UV detector was connected to evaluate the performance of the EOP. A comparative study was also carried out with a mechanical capillary HPLC pump on the same system. The results demonstrated that the reproducibility of flow rate and the pulsation-free flow property of the EOP are superior to that of mechanical pump in capillary HPLC application.     

Microfluidic liquid-liquid extraction system based on stopped-flow technique and liquid core waveguide capillary

In this work, a miniaturized liquid-liquid extraction system under stopped-flow manipulation mode with spectrometric detection was developed. A Teflon AF liquid-core waveguide (LCW) capillary was used to serve as both extraction channel for organic solvent flow and adsorption detection flow cell. Gravity induced hydrostatic pressure was used to drive the organic and aqueous phases through the extraction channels. During extraction process, a stable organic and aqueous phase interface was formed at the outlet of the capillary, through which the analyte in the flowing aqueous stream was extracted into the stationary organic solvent in capillary. The absorbance of the analyte extracted into the organic solvent was measured in situ by a spectrometric detection system with light emitting diode (LED) as light source and photodiode as absorbance detector. The performance of the system was demonstrated in the determination of sodium dodecyl sulfate (SDS) extracted as an ion pair with methylene blue into chloroform. The precision of the measured absorbance for a 5 mg L⁻¹ SDS standard was 6.1% R.S.D. (n = 5). A linear response range of 1-10 mg L⁻¹ SDS was obtained with 5 min extraction period. The limit of detection (LOD) for SDS based on three times standard deviation of the blank response was 0.25 mg L⁻¹.     

Direct on-line hyphenation of capillary liquid chromatography to nuclear magnetic resonance spectroscopy: practical aspects and application to drug metabolite identification

In combining the high peak concentrations of capillary liquid chromatography (CapLC) with the high mass sensitivity of micro scale nuclear magnetic resonance (NMR) the hyphenation of CapLC to micro NMR offers a substantial gain in overall sensitivity. This paper deals with our experiences gained using a commercial CapLC-NMR system which has very recently become available. The limits of detection (SNR > 3) for a test compound of a molecular weight of M 318 were found to be approximately 100 ng (0.35 nmol) within an hour acquisition time and approximately 25 ng over night (85 pmol). Practical aspects such as the feasibility of stopped-flow experiments and sample handling issues are discussed in detail and first possible drug metabolite applications to hepatocyte incubations and direct analysis of plasma samples are presented.     

Hollow fiber-based liquid-liquid-liquid microextraction followed by flow injection analysis using column-less HPLC for the determination of phenazopyridine in plasma and urine

Hollow fiber-based liquid-liquid-liquid microextraction (HF-LLLME) followed by flow injection analysis and diode array detection (FIA-DAD) was applied as a simple and sensitive quantitative method for the determination of phenazopyridine in urine and plasma samples. Flow injection system included a conventional HPLC system (without a chromatographic column) and a diode array detector. The extraction of phenazopyridine was carried out using diphenyl ether as the organic phase for filling the pores of the hollow fiber wall, and 0.1 M H(2)SO(4) solution as acceptor phase in the lumen of the fiber. The factors affecting the HF-LLLME and flow injection analysis including type of organic solvent, pH of donor phase, extraction temperature, extraction time, stirring rate, and pH of mobile phase were investigated and the optimal extraction conditions were established. With the consumption of 5 mL of sample solution, the enrichment factor was about 230. The limit of detection was 0.5 μg/L with inter- and intra-day precision being (RSD%) 6.9 and 4.9, respectively. Excellent linearity was found between 5 and 200 μg/L.     

Packed capillary HPLC: An attractive separation technique for small organic molecules

The feasibility of using packed capillary HPLC for the analysis of small organic molecules has been demonstrated by three examples: the separation of glucose and sucrose telomers, the separation of the different components of a solvent extraction reagent, and the separation of mono-, di-, and trioleins, all of which have been achieved with packed capillary columns of 0.32 mm inner diameter and eluent flow rates of 3 microliters per minute. Application to the analysis of the solvent extraction reagents has shown that this technique can be used as a quantitative tool in just the same way as any conventional HPLC method. The feasibility of a direct packed capillary HPLC – mass spectrometry interface has been demonstrated for the glucose telomer separation; direct coupling is only possible because of the microliter flow rates employed by the technique.     

Determination of polycyclic aromatic compounds by high-performance liquid chromatography with simultaneous mass spectrometry and ultraviolet diode array detection

A major problem in the determination of polycyclic aromatic compounds (PACs) in environmental samples is the extreme complexity of the extracts, even after extensive fractionation. The combination of high-performance liquid chromatography (HPLC) with simultaneous mass spectrometry (MS) and ultraviolet diode array detection (DAD) is a powerful tool for the identification and quantitation of such species with a high degree of confidence. HPLC allows the selective separation of a wide variety of PACs, including thermally labile and high molecular weight compounds. Electron ionization MS with the moving belt interface provides high sensitivity and selectivity, as well as structural information such as molecular weight, functional groups, and elemental composition. The diode array detector helps to differentiate isomeric structures and confirm compound identity.     

Liquid chromatography-nuclear magnetic resonance spectroscopy.

A general overview of the experimental set-up for performing analytical-scale and nanoliter-scale liquid chromatography-1H nuclear magnetic resonance spectroscopy (LC-1H-NMR) experiments is given. The high power of combining LC with 1H-NMR spectroscopy is demonstrated by two examples, where NMR acquisition was performed either in the continuous-flow mode on the analytical scale or in the stopped-flow mode on the nanoliter scale. Current developments employing the on-line coupling of capillary as well as supercritical fluid separation methods with 1H-NMR spectroscopy together with LC-13C-NMR spectroscopy are discussed.     

Chemometric analysis of high performance liquid chromatography-diode array detection-electrospray mass spectrometry of 2- and 3-hydroxypyridine

Triply coupled high performance liquid chromatography using diode array detection and positive ion electrospray mass spectrometry of 2- and 3-hydroxypyridine is presented. Considerations of the physical method for coupling the two detectors, the influence of pH on retention times, the cone voltage of the mass spectrometer and the linear concentration ranges are described. Data from both detectors are aligned and interpolated. The analyte mass spectra are reduced to 20 significant masses. Principal components plots on the raw, normalised and standardised data, derivatives to determine composition 1 regions, deconvolution and procrustes analysis to compare data from both detectors are discussed. Common trends in both mass spectral and diode array chromatograms are interpreted. This paper represents a new approach to common processing of chromatographic data from two detectors.     

Assessment for the light-induced cis-trans isomerization of rhapontigenin and its glucoside rhaponticin by capillary electrophoresis and spectrometric methods

The light-induced cis-trans isomerization of rhapontigenin (RHA) and its glucoside rhaponticin (RHA-Glc) were evaluated under ultraviolet (UV) light irradiation. A simple and rapid capillary electrophoresis method was developed for the kinetic study of four stilbenes (both cis and trans form of RHA and RHA-Glc). These analyses were achieved by using β-cyclodextrin (β-CD) modified capillary zone electrophoresis with diode array detector (CZE-DAD). The method provided reliable separations with a short analysis time of 3 min. The purity of individual compound was checked by UV spectral comparisons with known standards, and further confirmed by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy. Furthermore, the UV absorbance and the molar absoptivity (ε) values were determined by UV-vis spectrophotometer to be 36824 L mol(-1) cm(-1) at λ(max) 324.5 nm for trans-RHA and 43894 L mol(-1) cm(-1) at λ(max) 325 nm for trans-RHA-Glc in methanol/water mixture solution (50%, v:v), respectively. CZE, UV-vis and NMR spectroscopy studies provided similar conclusions by considering the influence of irradiation time and the influence of irradiation wavelength.