Liquid Chromatography Detection Research Articles

Capillary liquid chromatography with off-line mid-IR and Raman micro-spectroscopic detection: analysis of chlorinated pesticides at ppb levels

A flow-through microdispenser was used as a solvent elimination interface, allowing vibrational spectroscopic detection in capillary liquid chromatography in addition to standard UV detection. Using a flow-through microdispenser, robust and stable deposition of picoliter-sized droplets on a CaF(2) plate window was achieved. The CaF(2) window was placed on a thermostated sample holder (80 degrees C) mounted on a computerized x, y stage for achieving fast solvent evaporation and enabling recording of the chromatogram as a trace of deposited material. The dried residues that were formed had diameters of a few tens of micrometers and were analysed by mid-IR and Raman micro-spectroscopy. Conditions were optimized for high sensitivity of measurement and maintaining chromatographic resolution during the deposition step. Due to the destruction-free character of Raman and FTIR spectroscopy, these techniques could be applied sequentially to interrogate the same deposits. To test the usefulness of the methodology for environmental analysis, the determination and unambiguous identification of chlorinated pesticides (chlortoluron, diuron, atrazine, and terbuthylazine) in river water was used as an example, obtaining limits of identification of 2 ng analyte on-column and precision of approximately 10% RSD. The application of the developed method to spiked real river samples demonstrated the identification power of the proposed method as, in addition to the four previously studied pesticides, two additional pesticides (simazine and isoproturon) could also be detected and identified.

Simplex-Stochastic Resonance Algorithm Applied to Detection of Weak Chromatographic Peaks

The simplex-stochastic resonance algorithm (SSRA) was applied to amplification and detection of the weak High Performance Liquid Chromatography (HPLC) chromatographic signals of coumatetralyl in plasma. In this algorithm, modified simplex algorithm was embedded to greatly enhance the signal-to-noise ratio of chromatographic signals. The application of SSRA to detect HPLC/UV weak signals of coumatetralyl showed an excellent quantitative relationship between different concentrations and responses. The assay was linear over the concentration range of 3–100 ng/mL, r = 0.9997, and RSD < 10%. The good linearity and precision demonstrated that it is an effective algorithm for detecting weak chromatographic peaks.

Design and implementation of an array of micro-electrochemical detectors for two-dimensional liquid chromatography—Proof of principle

Simultaneous two-dimensional liquid chromatography (2D-LC) is an implementation of two-dimensional liquid chromatography which has the potential to provide very fast, yet highly efficient separations. It is based on the use of time × space and space × space separation systems. The basic principle of this instrument has been validated long ago by the success of two-dimensional thin layer chromatography. The construction of a pressurized wide and flat column (100 mm × 100 mm × 1 mm) operated under an inlet pressure of up to 50 bar was described previously. However, to become a modern analytical method, simultaneous 2D-LC requires the development of detectors suitable for the monitoring of the composition of the eluent of this pressurized planar, wide column. An array of five equidistant micro-electrochemical sensors was built for this purpose and tested. Each sensor is a three-electrode system, with the working electrode being a 25 μm polished platinum micro-electrode. The auxiliary electrode is a thin platinum wire and the reference electrode an Ag/AgCl (3 M sat. KCl) electrode. In this first implementation, proof of principle is demonstrated, but the final instrument will require a much larger array.

Separation and quantification of short-chain coenzyme A in plant tissues by capillary electrophoresis with laser-induced fluorescence detection

Coenzyme A (CoA) is a group of active metabolic compounds that facilitate over 100 chemical reactions in animal and plant cells. It mainly serves as an acyl carrier in many metabolic reactions and initiates the tricarboxylic acid cycle that produces more than 90% of the energy required for life processes. Measurements of short-chain and long-chain CoA compounds in a variety of tissues by using high-performance liquid chromatography (HPLC) and capillary electrophoresis-ultraviolet (CE-UV) detection have been reported, but these techniques do not allow one to simultaneously determine all the possible coexisting CoAs and their derivatives in plant tissues with sufficient sensitivity. In this paper, a method of quantitative determination of 5 short-chain CoAs in plant tissues by using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was developed. Under optimized derivatization and electrophoresis conditions, different CoAs that were derivatized with fluorescein-5-isothiocyanate (FITC) were separated and quantified at the pmole level. A fused silica capillary with a 75 μm (i.d.) × 57 cm was used for the separation and 150 mM borate buffer (pH 9.00) was used as a background electrolyte. The separation was carried out at 25 kV and completed in less than 13 min. The effects of derivatization time, buffer concentrations, and pH values on derivatization efficiency were also systematically investigated. This newly developed CE-LIF method can be used to detect CoAs in both plant and animal tissues.

Re: "Vitamin C Deficiency in a Population of Young Canadian Adults"

In their report that nearly 50% of healthy young Canadian adults have subnormal serum vitamin C concentrations, Cahill et al. (1) cite our observation that 60% of the acute-care patients in a tertiary care Montreal hospital had subnormal plasma vitamin C concentrations (2). More pertinently, we also observed that only 13% of people concurrently attending the hospital's outpatient test center had a subnormal plasma vitamin C concentration (2). The method used by Cahill et al. (1) for handling, storing, and analyzing serum vitamin C is questionable. To prevent vitamin C degradation, whole blood samples must be kept cold at all times, and as soon as the plasma is obtained it must immediately be stabilized by ice-cold deproteinization or flash frozen with ethanol-dry ice with prompt storage at −70°C to −80°C (3, 4). If not stored at −70°C to −80°C, vitamin C will disappear even from deproteinized plasma samples. The serum samples in the reported study were neither deproteinized nor flash frozen prior to storage, and they were stored at only −20°C. The high performance liquid chromatography detection method is not identified or described, precluding an assessment of its reliability.

Procedure for Automated Background Correction in Flow Systems with Infrared Spectroscopic Detection and Changing Liquid-Phase Composition

This paper describes a partial least squares (PLS) based automatic procedure to correct for changes in the spectral contribution of the solvent or solvent mixtures from solute spectra recorded in these solvents. The procedure was developed for successful on-line Fourier transform infrared (FT-IR) detection in gradient high-performance liquid chromatography (HPLC) separations. It requires a reference FT-IR data set containing all possible combinations of the expected variation in solvent composition. Furthermore, a spectral region (A) in these spectra is required where the solvents show absorption but the analytes do not. This is the case for the system ACN:H(2)O, an often-applied solvent mixture in gradient HPLC, in the spectral region from 2300-2400 cm(-1). By focusing on (A) the developed numerical method selects an appropriate background spectrum from the reference data set, which is then subtracted from the analyte spectra. The method was programmed in Matlab and tested on different isocratic and gradient on-line reversed phase liquid chromatography-Fourier transform infrared spectrometry (LC-FT-IR) data sets. This work describes a successful method to perform eluent correction in on-line coupling of FT-IR spectrometry with gradient LC.

Aerosol-Based Detectors for Liquid Chromatography: Do They Have a Role in Bioanalysis?

Aerosol-Based Detectors for Liquid Chromatography: Do They Have a Role in Bioanalysis?

Light emitting diode induced chemiluminescence and its application as a detector for high performance liquid chromatography

Some categories of compounds, including quinones, coumarins, flavins, and xanthene dyes, were found to produce strong chemiluminescence (CL) signals with luminol in sample solution under the irradiation of light emitting diodes (LED) with proper wavelengths. Based on this phenomenon, a compact photochemical reactor was constructed to develop a novel LED induced CL detector for high performance liquid chromatography (HPLC). The effects of related parameters including LED wavelength, luminol concentration, flow rate, pH, and eluents of HPLC were investigated in detail. Under the optimized conditions, the limits of detections (LODs) were in the range of 0.2-80 ng mL(-1). The applications and accuracy of the proposed method were validated by analyzing food samples such as milk powder, beer, candy and beverage with satisfactory results.

Effect of processing on ochratoxin A content in dried beans

Dried pink beans naturally contaminated with ochratoxin A (OTA) and dried carioca beans artificially contaminated with OTA by inoculation with Aspergillus ochraceus (ATCC 22947) were tested for ochratoxin A levels as follows: dried beans were washed with water for 2, 60 or 120 min, soaked in water for 60, 120 min or 10 h, and cooked for 60 or 120 min. At each step, test water and beans were separated. Test water, raw beans and cooked beans were analyzed for OTA. The amount of OTA partitioned into water and in residual beans was determined by methanol–sodium bicarbonate extraction, buffer dilution, immunoaffinity column cleanup, liquid chromatographic separation and fluorescence detection. The results demonstrated that the distribution of OTA in processing water and beans depends on the method of preparation. All treatments (washing, soaking and cooking) when applied individually reduced the amounts of OTA retained in bean flour and whole beans. Higher amounts of OTA remained in whole beans than in bean flour after removing the processing water. The combination of the three treatments eliminated about 50% of the toxin from whole beans. This study provides evidence that discarding the washing, soaking and cooking water leads to a significant reduction in OTA contamination in dried beans.

Revision of Analytical Conditions for Determining Ligand Molecules Specific to Soft Metal Ions Using Dequenching of Copper(I)−Bathocuproine Disulfonate as a Detection System

Reoptimization of analytical conditions was performed for a high-performance liquid chromatographic (HPLC) detection system for Cu(I) chelators based on the dequenching of Cu(I)-bathocuproine disulfonate complexes that occurs in the presence of Cu(I) chelators. The revision corrects for emission and excitation wavelengths that were in fact second-order light of the actual optimal wavelengths and for the composition of the postcolumn solution. These revisions resulted in an order of magnitude decrease in detection limits of phytochelatins, a class of cysteine-rich, heavy metal-binding peptides. The revised technique is capable of phytochelatin quantitation at femtomole quantities.

Lipid analysis via HPLC with a charged aerosol detector

AbstractMost lipid extracts are a mixture of saturated and unsaturated molecules. Therefore, the most successful high performance liquid chromatography (HPLC) detectors for the quantitative analysis of lipids have involved the use of “universal” or “mass” detectors such as flame ionization detectors and evaporative light scattering detectors. Recently a new type of HPLC “universal” detector, a charged aerosol detector (CAD), was developed and is now commercially available. This detection method involves nebulizing the HPLC column effluent, evaporating the solvents, charging the aerosol particles and measuring the current from the charged aerosol flux. During the approximately four years that the charged aerosol detector has been commercially available, several publications have described HPLC‐CAD methods for lipid analysis. The most common lipids can be quantitatively analyzed via HPLC‐CAD except for some volatile lipids such as common fatty acid methyl esters and short chain free fatty acids (&lt;C16). The major results of these publications will be summarized in this report.

Liquid-Phase and Evanescent-Wave Cavity Ring-Down Spectroscopy in Analytical Chemistry

Due to its simplicity, versatility, and straightforward interpretation into absolute concentrations, molecular absorbance detection is widely used in liquid-phase analytical chemistry. Because this method is inherently less sensitive than zero-background techniques such as fluorescence detection, alternative, more sensitive measurement principles are being explored. This review discusses one of these: cavity ring-down spectroscopy (CRDS). Advantages of this technique include its long measurement pathlength and its insensitivity to light-source-intensity fluctuations. CRDS is already a well-established technique in the gas phase, so we focus on two new modes: liquid-phase CRDS and evanescent-wave (EW)-CRDS. Applications of liquid-phase CRDS in analytical chemistry focus on improving the sensitivity of absorbance detection in liquid chromatography. Currently, EW-CRDS is still in early stages: It is used to study basic interactions between molecules and silica surfaces. However, in the future this method may be used to develop, for instance, biosensors with high specificity.

Fluorescence enhancement of Er 3+ ion by Glibenclamide: A practical probe

Glibenclamide, 5-chloro-N-[2-[4-(cyclohexylcarbamoyl–sulfamoyl) phenyl]ethyl]-2-methoxy-benzamide, is an important anti-diabetic drug that can enhance the erbium (Er) intrinsic fluorescence intensity. Based on that fact, Glibenclamide can be monitored using simple and sensitive fluorimetric probes. For this research, an interest arose in using lanthanides as probing biochemical reactions, and to study the interactions between Ca 2+ and biologically important molecules. Methodologies employed are based on the unique fluorescence properties of lanthanides due to their unfilled 4f n electronic orbital, and formation of a complex between Er 3+ and Glibenclamide. The proposed method was found to be simple, accurate and precise, thus allowing it to be used as a detector for high performance liquid chromatography in the future.

Indirect Detection of Ethylene Glycol Oligomers Using a Contactless Conductivity Detector in Capillary Liquid Chromatography

Ethylene glycol oligomers were visualized by indirect conductimetric detection based on dilution of the mobile phase due to the analytes. A high electrical conductivity background was maintained by the addition of 5 mM sodium nitrate in the mobile phase, and the analytes were visualized by decreases in the background when they eluted. A capacitively coupled contactless conductivity detector was convenient to monitor effluents from the microcolumn with minimum extra-column band broadening. The signals as negative peaks were linear to the concentration of the analytes, and a concentration detection limit of 0.025% was achieved for tetraethylene glycol at S/N=3, corresponding to the mass detection limit of 38 ng for 0.15 microl injection. The logarithm of the retention factor of ethylene glycol oligomers was linear to the degree of polymerization (DP) as well as to the acetonitrile composition in the mobile phase. These situations allowed us to estimate the DP of eluted ethylene glycol oligomers by using a few oligomers with known DP. The dynamic reserve, defined as the ratio of the background to its noise level achieved under the present conditions, was 2.3 x 10(5) which was much larger than that achieved by UV absorption detection. The present method was applied to profile ethylene glycol oligomers contained in commercially available PEG reagents.

Determination of Triazine Herbicides in Aqueous Samples Using Solidification of a Floating Drop for Liquid-Phase Microextraction with Liquid Chromatography

A rapid, simple, and efficient liquid-phase microextraction (LPME) method coupled with high-performance liquid chromatography and ultraviolet detection for the analysis of triazine herbicides was developed in this study. Under the optimum conditions, the enrichment factors and extraction recoveries were 33.0–72.6 and 11.2–23.2%, respectively. The detection limits (LODs) were in the range of 0.03–0.10 µg L−1. The relative standard deviations for the determination of the triazine herbicides at μg L−1 levels varied in the range 2.05–8.15%. The method was successfully applied in the determination of the triazine herbicides in aqueous samples with satisfactory results.

Determination of Low-Molecular-Mass Aldehydes in Water Samples by Liquid Chromatography and Chemiluminescence Detection Using Continuous in situ Derivatization/Preconcentration with Dansylhydrazine

A simple, expeditious, and sensitive method has been developed for the determination of low-molecular-mass aldehydes in water samples by liquid chromatography and peroxyoxalate–chemiluminescence detection. The method is based on continuous solid-phase extraction with in situ derivatization/preconcentration of the aldehydes using dansylhydrazine, which was first adsorbed on an RP–C18 mini-column. For 10 mL of aqueous sample, the limits of detection (LOD) for C1 to C4 aldehydes were 20–30 ng L−1, except for formaldehyde, which had an LOD of 400 ng L−1. Application was illustrated by the determination of these aldehydes in water samples; the interday precision was always less than ca. 7%, and relative recoveries were more than 96%.

Postcolumn Iodine–Azide Reaction as a Detection System in HPLC for the Determination of Methylthiouracil in Urine

This article describes a system for determining methylthiouracil in urine based on the sensitizing induction of methylthiouracil on the reaction between iodine and azide ions and combined techniques of high-performance liquid chromatography and simple postcolumn detection. The optimal conditions for iodine–azide reaction and high-performance liquid chromatographic separation were determined. The reproducibility, linearity, and recovery were evaluated under the optimal conditions. The methylthiouracil standards added to normal urine show that the response of the detector, set at 350 nm (corresponding to unreacted iodine in the postcolumn iodine–azide reaction), is linear within the concentration range of 0.4–5.0 nmol mL−1 urine. The relative standard deviation values for precision and recovery within the calibration range varied from 0.9 to 4.8% and from 94 to 105%, respectively. Limits of detection (LOD) and quantitation (LOQ) were 0.3 and 0.4 nmol mL−1 urine, respectively.

Reverse-Phase High-Performance Liquid-Chromotographic DAD Method for Simultaneous Determination of Five Isoflavones in Millettia nitida var. hirsutissima

A reverse-phase high-performance liquid-chromatographic photodiode array detection (DAD) method has been developed and validated for the simultaneous determination of five isoflavones in Millettia nitida var. hirsutissima: hirsutissimiside B, sphaerobioside, lanceolarin, formononetin, and afromosin. The method utilized a C18 column (4 µm, 250 × 4.6 mm) with an acetonitrile and aqueous H3PO4 gradient and ultraviolet detection at 259 nm. The recovery of the method is 94.1–105.9%, and linearity (r > 0.9996) is obtained for all the isoflavones.

Vinblastine and doxorubicin administration to pregnant mice affects brain development and behaviour in the offspring

Background Administration of chemotherapy during the fetal phase of pregnancy may put late-developing organs like the central nervous system at risk. Methods Transplacental transfer of doxorubicin and vinblastine was measured in C57/BJ mice by high-performance liquid chromatographic detection of the drugs in maternal and fetal plasma, 90 min after intravenous injection. Further, doxorubicin, vinblastine or saline were administered to pregnant C57/6J mouse dams on gestational day 17.5. Effects on brain morphology of the offspring were examined at 24 h p.i. (immediate phase) and at 4–5 months p.i. (residual phase), using light- and electron microscopy. At the age of 3 months, offspring performed a behavioural test battery addressing neuromotor performance, exploration and anxiety, and learning and memory. Results Fetal plasma levels of doxorubicin and vinblastine reached respectively 5.0 ± 0.2% and 13.9 ± 2.4% of the maternal plasma levels. In the immediate phase, pathological examination revealed endothelial and perivascular parenchymal damage to the neocortical subventricular zone and a less constant thickening of the leptomeninx, in some cases also cortical lamination defects were noted. Brain histology was within normal limits in the mice of the residual phase group. Behavioural testing revealed subtle differences between drug-exposed and control mice. Grip strength was reduced in drug-exposed mice, but other tests for motor performance were normal. Several exploratory measures were altered, and there were some indications of increased anxiety in the drug-exposed mice. In the passive avoidance task, step-through latency was shorter in the drug-exposed mice, but their normal performance in the Morris water maze indicated that this was probably not due to impaired memory. Conclusion The current preclinical data reveal subtle changes in behaviour and transiently also in brain morphology in the mice that were prenatally exposed to vinblastine or doxorubicin.

Carbon fiber/epoxy composite ring–disk electrode: Fabrication, characterization and application to electrochemical detection in capillary high performance liquid chromatography

Carbon fiber/epoxy composite materials, which are manufactured using the pultrusion process, are commercially available in various shapes and sizes at very low cost. Here we demonstrate the application of such a material as an electrochemical detector in a flow system. Cyclic voltammetry shows that the material’s electrochemical behavior resembles that of glassy carbon. Using tube and rod composites, we successfully fabricated a ring–disk electrode with a 20 μm gap between the ring and the disk. The narrow gap is favorable for mass transfer in the generator–collector experiment. This composite ring–disk electrode is assembled in a thin-layer radial-flow cell and used as an electrochemical detector. The disk electrode, placed directly opposite to the flow inlet, is operated as a generator electrode with the ring electrode being a collector. The high collection efficiency on the ring electrode (0.8 for a chemically reversible species) enhances the detection selectivity.