Liquid Chromatography Detection Research Articles

High performance liquid chromatography with mid-infrared detection based on a broadly tunable quantum cascade laser

This work introduces a tunable mid-infrared (mid-IR) external cavity quantum cascade laser (EC-QCL) as a new molecular specific detector in liquid chromatography. An EC-QCL with a maximum tunability of 200 cm(-1) (1030-1230 cm(-1)) was coupled to isocratic high performance liquid chromatography (HPLC) for the separation of sugars with a cation exchange column (counter ion: Ca(2+)) and distilled water as the mobile phase. Transmission measurements in a 165 μm thick flow cell allowed for on-line coupling and independent quantification of glucose, fructose and sucrose in the concentration range from 5 mg mL(-1) to 100 mg mL(-1) in several beverages. The results obtained with the EC-QCL detector were found to be in good agreement with those obtained using a differential refractive index detector as a reference. The standard deviation of the method for the linear calibration was better than 5 mg mL(-1) for all sugars and reached a minimum of 1.9 mg mL(-1), while the DRI detector reached a minimum of 1 mg mL(-1). Besides the quantification of sugars for which a calibration was performed, also chromatographic peaks of other components could be identified on the basis of their IR absorption spectra. This includes taurine, ethanol, and sorbitol.

Refractive index-based detection of gradient elution liquid chromatography using chip-integrated microring resonator arrays.

Refractive index-based sensors offer attractive characteristics as nondestructive and universal detectors for liquid chromatographic separations, but a small dynamic range and sensitivity to minor thermal perturbations limit the utility of commercial RI detectors for many potential applications, especially those requiring the use of gradient elutions. As such, RI detectors find use almost exclusively in sample abundant, isocratic separations when interfaced with high-performance liquid chromatography. Silicon photonic microring resonators are refractive index-sensitive optical devices that feature good sensitivity and tremendous dynamic range. The large dynamic range of microring resonators allows the sensors to function across a wide spectrum of refractive indices, such as that encountered when moving from an aqueous to organic mobile phase during a gradient elution, a key analytical advantage not supported in commercial RI detectors. Microrings are easily configured into sensor arrays, and chip-integrated control microrings enable real-time corrections of thermal drift. Thermal controls allow for analyses at any temperature and, in the absence of rigorous temperature control, obviates extended detector equilibration wait times. Herein, proof of concept isocratic and gradient elution separations were performed using well-characterized model analytes (e.g., caffeine, ibuprofen) in both neat buffer and more complex sample matrices. These experiments demonstrate the ability of microring arrays to perform isocratic and gradient elutions under ambient conditions, avoiding two major limitations of commercial RI-based detectors and maintaining comparable bulk RI sensitivity. Further benefit may be realized in the future through selective surface functionalization to impart degrees of postcolumn (bio)molecular specificity at the detection phase of a separation. The chip-based and microscale nature of microring resonators also make it an attractive potential detection technology that could be integrated within lab-on-a-chip and microfluidic separation devices.

Determination of five sulfonamides in medicated feedingstuffs by liquid chromatography with ultraviolet detection

Abstract A fast and reliable method of liquid chromatography and ultraviolet detection of sulfaguanidine, sulfadiazine, sulfamethazine, sulfamethizole, and sulfamethoxazole in feedingstuffs was described. The method involves THE procedure of preparation of spiked samples, and extraction of sulphonamides from the matrix using a mixture of methanol and acetonitrile, followed by drying the extract and dissolving it in a phosphate buffer. The analysis uses octadecyl (C18) analytical column with UV detection at λ = 260 nm and a gradient programme of mobile phase composition. The analytical procedure has been successfully adopted and validated for quantitative determination of the sulfonamides in feedingstuff samples. Validation included sensitivity, specificity, linearity, repeatability, and intra-laboratory reproducibility. The mean recovery of sulfonamides was 84%, within the working range of 200-2000 mg/kg. Direct, simple sample preparation and HPLC-UV analysis allow the method to be successfully included in the scope of routine analyses. The presented results could be an answer to a need of simple and easy method for sulfonamide determination applicable in medicated feedingstuffs analysis.

Effects of Blockade of Dopamine D2 Receptors on the Activity of the Nitrergic System of the Nucleus Accumbens in the Rat Brain

In vivo intracerebral microdialysis studies with high-performance liquid chromatography and electrochemical detection in Sprague–Dawley rats showed that administration of the dopamine D2 receptor antagonist raclopride (20 and 100 μM) into the nucleus accumbens did not alter the baseline extracellular citrulline level (a co-product of NO synthesis) in the nucleus accumbens. Administration of NMDA (100 μM), an NMDA glutamate receptor agonist, into the nucleus accumbens induced an increase in the extracellular citrulline level in this structure. Administration of 7-nitroindazole (0.5 mM), an inhibitor of the neuronal isoform of NO synthase, into the nucleus accumbens blocked this increase, while administration of raclopride (20 and 100 μM) into this structure decreased the increase. These data suggest that dopamine D2 receptors in the nucleus accumbens are involved in regulation of the extent of activation of neuronal NO synthase induced by local stimulation of NMDA glutamate receptors.

Preparation and Characterization of Molecularly Imprinted Polymer as SPE Sorbent for Melamine Isolation

In this paper, a separation procedure combining molecularly imprinted-solid phase extraction (MI-SPE) was developed for the isolation of melamine. The molecularly imprinted polymer (MIP) was prepared using precipitation polymerization method where melamine as template, 9-vinylcarbazole as functional monomer, ethtylene glycol dimethacrylate as a cross-linker and benzoyl peroxide as initiator. An off-line MI-SPE method followed by ultra-performance liquid chromatography detection of melamine was established. MIP showed a better affinity toward melamine compared to non imprinted polymer (NIP) with a maximum binding capacity of 53.01 mg/g MIP. Based on the correlation coefficients, the kinetic study indicated that the adsorption of melamine by MIP fit a pseudo-second order model. From isotherm study, adsorption of melamine by MIP increased when the concentration of melamine increased and followed a Freundlich isotherm model, which indicates the sorption can be described by multilayer sorption. The interference study proved that MIP has better binding capacity towards melamine if compared to NIP due to specific sites of melamine occurred in MIP particles.

Preparation of a stir bar sorptive extraction coating based on molecularly imprinted polymer and its application in the extraction of dienestrol and hexestrol in complicated samples

A new stir bar sorptive extraction (SBSE) coating based on molecularly imprinted polymer (MIP) with diethylstilbestrol as replaced template molecule was prepared. The influences of the contents of template molecule and monomer in the polymerization mixture on the extraction performance of MIP-SBSE were investigated thoroughly. The MIP was characterized by elemental analysis, scanning electron microscopy and infrared spectroscopy. In order to evaluate the usability of the new coating, the MIP-SBSE was combined with high performance liquid chromatography (HPLC) and diode array detector (DAD) with dienestrol (DS) and hexestrol (HS) as detected solutes. To achieve optimally selective extraction performance for DS and HS, several parameters, including extraction and desorption times, desorption solvent, ionic strength and pH value in sample matrix were investigated. The results showed that under the optimized experimental conditions, the present method has high selectivity and sensitivity. When drying-redissolving procedure was taken during sample preparation, the limits of detection for DS and HS were as low as 0.04 microg/L and 0.14 micorg/L, respectively. Good linearities were obtained for analytes with the correlation coefficients (R2) above 0.99. Finally, the proposed method was successfully applied to the determination of DS and HS in wastewater, honey and cow urine samples. The recoveries of spiked target compounds in real samples ranged from 61.3% to 120%. The developed method is simple, selective, sensitive and applicable for the analysis of trace DS and HS in complicated samples.

Chemiluminescence immunochromatographic analysis for the quantitative determination of algal toxins

For the quantitative detection of algal toxin, microcystin, a chemiluminescence immunochromatographic assay method was developed. The developed system consists of four parts, chemiluminescence assay strip (nitrocellulose membrane), horse radish peroxidase labeled microcystin monoclonal antibodies, chemiluminescence substrate (luminol and hydrogen peroxide), and luminometer. The performance of the chemiluminescence immunochromatographic assay system was compared with high performance liquid chromatography (HPLC) detection. The detection limit of chemiluminescence immunochromatographic assay system is several orders of magnitude lower than with HPLC. The chemiluminescence immunochromatography and HPLC results correlated very well with the correlation coefficient (r 2 ) of 0.979.

Electrogenerated chemiluminescence detector based on Ru[formula omitted] immobilized in cation exchange resin for high-performance liquid chromatography: An approach to stable detection

In this work, an electrogenerated chemiluminescence (ECL) detector with improved stability was developed for high-performance liquid chromatography (HPLC) detection of hydrochlorothiazide (HCTZ). The detector was prepared by packing cation exchanged resin particles in a glass tube, followed by inserting Pt wires (working electrode) in this tube and sealing. The leakage of Ru(bpy)32+ from the resin was compensated by adding a small amount of Ru(bpy)32+ in the mobile phase. Factors affected the performance of the proposed ECL detector were investigated. Under the optimal conditions, the ECL intensity has a linear relationship with the concentration of HCTZ in the range of 5.0×10−8gmL−1−2.5×10−5gmL−1 and the detection limit was 2.0×10−8gmL−1 (S/N=3). Application of the detector to the analysis of HCTZ in human serum proved feasible.

Liquid Chromatographic Detection of Permethrin from Filter Paper Wipes of White-tailed Deer

Liquid Chromatographic Detection of Permethrin from Filter Paper Wipes of White-tailed Deer

Determination of Pyrethroids in Environmental Waters Using Magnetic Chitosan Extraction Coupled with High Performance Liquid Chromatography Detection

The magnetic chitosan was prepared by adding chitosan, Fe2+ and Fe3+ into a basic precipitant of NaOH solution. The synthetic magnetic chitosan was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and physical properties. The adsorption characteristics of magnetic chitosan for extracting pyrethroids from water samples were investigated. The analytes were separated by magnetic solid phase extraction and determined by high performance liquid chromatography. The optimum conditions of adsorption experiments were obtained: the amount of sorbent was 80 mg, the extraction time was 20 min, the washing solvent was 50% aqueous methanol, and the eluent was acetonitrile-acetic acid (99: 1, v/v). The obtained linearity of three pyrethroids was in the range of 30–3000 ng L−1. The detection limits of beta-cyfluthrin, cyhalothrin, and cyphenothrin were 7.5, 5.6, and 6.1 ng L−1, respectively. The intra-day and inter-day precisions of three pyrethroids were in the range of 3.6–5.2% and 5.9–8.6%, respectively. In the optimized conditions, three water samples were analyzed, and the recoveries of pyrethroids obtained were 83.2–95.2%.

Development of sample preparation method for eleutheroside B and E analysis in Acanthopanax senticosus by ionic liquids-ultrasound based extraction and high-performance liquid chromatography detection

An ionic liquids-based ultrasonic-assisted extraction (ILUAE) method was successfully developed for extracting eleutheroside B and E from Radix Acanthopanax senticosus. Thirteen 1-alkyl-3-methylimidazolium ionic liquids with different cations and anions were investigated and 1-butyl-3-methylimidazolium bromide ([C4mim]Br) solution was selected as the solvent. The conditions for ILUAE, including the ionic liquid concentration, soaking time, ultrasonic power, ultrasonic time, solid–liquid ratio and number of extraction cycles, were optimized. With the proposed method, the energy consumption time was reduced to 30min, whereas conventional method requires about 4h. The proposed method had good recovery (97.96–103.39%) and reproducibility (RSD, n=5; 3.3% for eleutheroside B, 4.6% for eleutheroside E). ILUAE was an efficient, rapid and simple sample preparation technique that showed high reproducibility and was environmental friendly.

Lower Concentrations of B-Vitamin Subgroups in the Serum and Amniotic Fluid Correlate to Cleft Lip and Palate Appearance in the Offspring of A/WySn Mice

The pathogenesis and prevention of cleft lip and palate (CL/P) have been studied mainly in clinical and animal experiments. A prophylactic poly-B-vitamin substitution during the first months of pregnancy has provided the most encouraging results for the prevention of CL/P recurrence in families at risk. Invitro studies of the palatal organ in an A/WySn mouse model have confirmed the positive influence of B-vitamins on palatal development. The present animal study was performed to analyze different B-vitamin concentrations in the serum and amniotic fluid of A/WySn mice according to the appearance of CL/P in their offspring. Concentrations of different B-vitamins (B1, B2, B3, B5, B6, and folic acid) in serum and amniotic fluid were analyzed by high-performance liquid chromatographic detection. Immunohistochemical staining against thiamin-1 receptor was performed on histologic midface sections of A/WySn fetuses with (n= 12) and without (n= 14) CL/P. Vitamin B5 (P < .001) and folic acid (P < .004) concentrations in the amniotic fluid of dams with CL/P were significantly lower than in dams without CL/P. Serum concentrations of folic acid (P= .5) and B5 (P= .4) showed no difference between the 2 groups. Dams with CL/P had significantly lower thiamine concentrations in serum (P= .01) and amniotic fluid (P < .001). Histologic midface sections presented high thiamin-1 receptor expression in the palatal shelf of fetuses with CL/P. A decreased use or uptake of some B-vitamin subgroups (B1, B5, and folic acid) in amniotic fluid and serum (vitamin B1) was correlated to an increased cleft appearance in A/WySn mice. The high thiamin-1 receptor expression in the palatal tissue of mouse fetuses with CL/P may be caused by adecreased availability of vitamin B1.

Use of potentiometric detection in (ultra) high performance liquid chromatography and modelling with adsorption/desorption binding kinetics

Observation of a potentiometric sensor's response behaviour after injection in flow injection analysis at different concentrations allowed studying “on” and “off” kinetics of the analyte's adsorption/diffusion behaviour. The alkaloid metergoline was mostly used as an example. kon and koff rate constant values were measured, and the association constant Kass, and ΔG values of the analyte–surface interaction were calculated with an adsorption-based model which proved to be fully applicable. kon increased by decreasing the sensor dimensions, while koff was unaffected by miniaturization. Increasing acetonitrile concentrations in the running buffer increased koff, while kon was unaffected. The experimentally determined ΔG values of the analyte–surface interaction showed a linear relation to the response of the sensor, in mV. This knowledge was applied to optimize the potentiometric detection of plant alkaloids in (U)HPLC. Sub-micromolar detection limits were obtained with the potentiometric detector/(U)HPLC combination. This is the first time that the response rates and the response itself can be modelled accurately for coated wire potentiometric sensors, and it is the first application of a potentiometric detector in UPLC.

Microfluidic LC device with orthogonal sample extraction for on-chip MALDI-MS detection.

A microfluidic device that enables on-chip matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) detection for liquid chromatography (LC) separations is described. The device comprises an array of functional elements to carry out LC separations, integrates a novel microchip-MS interface to facilitate the orthogonal transposition of the microfluidic LC channel into an array of reservoirs, and enables sensitive MALDI-MS detection directly from the chip. Essentially, the device provides a snapshot MALDI-MS map of the content of the separation channel present on the chip. The detection of proteins with biomarker potential from MCF10A breast epithelial cell extracts, and detection limits in the low fmol range, are demonstrated. In addition, the design of the novel LC-MALDI-MS chip entices the promotion of a new concept for performing sample separations within the limited time-frame that accompanies the dead-volume of a separation channel.

Estudo do inseticida carbofurano em solo e sedimento de área de produção de arroz irrigado e controle do gorgulho aquático Oryzophagus oryzae, Taubaté, São Paulo, Brasil

Resíduos do inseticida e nematicida carbofurano e de seu principal metabólito 3-hidroxicarbofurano foram averiguados em tabuleiros de arroz irrigado e em áreas adjacentes para controle do gorgulho aquático da espécie Oryzophagus oryzae Lima, 1936. As amostras foram coletadas no período de 6/9/1999 a 4/5/2000 e analisadas por cromatografia a líquido de alta eficiência (HPLC) utilizando-se detector de fluorescência. Não foram detectados resíduos de carbofurano e de seu metabólito 3-hidroxicarbofurano dentro dos limites de determinação do método.

Nepem-211 ion exchange conductive membrane immobilized tris(2,2´-bipyridyl) ruthenium(II) electrogenerated chemiluminescence flow sensor for high-performance liquid chromatography and its application

We developed a sensitive and robust electrogenerated chemiluminescence (ECL) flow sensor based on Ru(bpy)3(2+) immobilized with a Nepem-211 perfluorinated ion exchange conductance membrane, which has robustness and stability under a wide range of chemical and physical conditions, good electrical conductivity, isotropy and a high exchange capacity for immobilization of Ru(bpy)3(2+). The flow sensor has been used as a post-column detector in high-performance liquid chromatography for determination of erythromycin and clarithromycin in honey and pork, and tricyclic antidepressant drugs in human urine. Under optimal conditions, the linear ranges were 0.03-26 ng/μL and 0.01-1 ng/μL for macrolides and tricyclic antidepressant drugs, respectively. The detection limits were 0.02, 0.01, 0.01, 0.06 and 0.003 ng/μL for erythromycin, clarithromycin, doxepin, amitriptyline and clomipramine, respectively. There is no post-column reagent addition. In addition to the conservation expensive reagents, the experimental setup was simplified. The flow sensor was used for 2 years with high sensitivity and stability.

A New Application of Charged Aerosol Detection in Liquid Chromatography for the Simultaneous Determination of Polar and Less Polar Ginsenosides in Ginseng Products

Conventional liquid chromatographic methods coupled with ultraviolet or evaporative light scattering detection are not sensitive enough to determine both polar and less polar ginsenosides at low concentrations. To establish a liquid chromatography-charged aerosol detection method for the simultaneous determination of polar and less polar ginsenosides in a variety of ginseng products Fourteen polar and less polar ginsenosides were extracted and concentrated by solid phase extraction. These were subsequently baseline-separated on a conventional reversed-phase C18 -column (250 mm × 4.6 mm, 5 µm) with a simple mobile phase consisting of water and acetonitrile. Components were then detected by means of charged aerosol detection. The method developed allowed the simultaneous determination of six polar ginsenosides (Rg1 , Re, Rb1 , Rc, Rb, Rd) and eight less polar ginsenosides (Rg6 , F4 , Rk3 , Rh4 , Rg3 (S), Rg3 (R), Rk1 , Rg5 ) in a single chromatographic run. Further, the method was linear (R(2) > 0.99), accurate (relative recoveries, 90-112%), and precise (intraday RSD < 5.7% and interday RSD < 10.6%) within the concentration range tested. The method sensitivity was measured in terms of the limit of detection, which ranged from 0.5 to 4.0 µg/mL. Concentrations of 14 ginsenosides were determined simultaneously in one homemade red ginseng and 13 commercial ginseng products of different types (liquid and solid samples), and results showed that ginsenoside content varied significantly among the samples tested. The method developed could serve as a useful analytical tool for the quality control of ginseng products.

Liquid Chromatography, Chemical Oxidation, and Online Carbon Isotope Dilution Mass Spectrometry as a Universal Quantification System for Nonvolatile Organic Compounds

A procedure for the universal detection and quantification of polar organic compounds separated by liquid chromatography (LC) based on postcolumn carbon isotope dilution mass spectrometry (IDMS) was developed. The eluent from the LC column is mixed online with a continuous flow of (13)C-enriched sodium bicarbonate, and the sodium persulfate oxidation reaction in acidic media is employed to achieve isotope equilibration. All carbon-containing compounds eluting from the column are oxidized to (12)CO(2) and (13)CO(2), respectively, and the carbon dioxide is separated from the aqueous phase using a gas-permeable membrane. The gaseous carbon dioxide is then carried to the mass spectrometer for isotope ratio measurements. Different water-soluble organic compounds were evaluated using a flow injection configuration to assess the efficiency of the oxidation process. Most water-soluble organic compounds tested showed quantitative oxidation. However, chemical structures involving conjugated C═N double bounds and guanidinium-like structures were found to be resistant to the oxidation and were further studied. For this purpose, (13)C(1)-labeled creatine (with the isotopic label in the guanidinium group) was employed as model compound. Specific conditions for the quantitative oxidation of these compounds required lower flow rates and the addition of metallic catalysts. This novel approach was tested as a universal detection and quantification system for LC. A simple standard mixture of four amino acids was separated under 100% aqueous conditions and quantified without the need for specific standards with good accuracy and precision using potassium hydrogen phthalate as internal standard. The main field of application of the developed method is for the purity assessment of organic standards with direct traceability to the International System of Units (SI).

Evaluation of a low cell constant conductance detector for detection of charged species in high-performance liquid chromatography

The present study evaluates high performance liquid chromatography (HPLC) detection based on a commercial conductance detector with a low cell constant of 0.005 cm−1 and a volume of 2 μL and a photodiode array UV-vis detector typically used in HPLC. When detecting a static NaCl solution, the conductance detector yields a limit of detection (LOD, 3 × noise) for NaCl of 39 parts per trillion. When flowing methanol through both conductance and UV-vis detectors and injecting benzoic acid/methanol, the signal-to-noise (S/N) ratio of the conductance detector is 8-fold higher than that of the UV-vis at its optimal wavelength. Using an HPLC with a C-18 column, flowing a 75 : 25 water : methanol solution, and using an acetylsalicylic acid (ASA)/water solution, the conductance detector yielded an ∼18-fold higher S/N ratio. It was found that HPLC system noise reduces the S/N ratio of the conductance detectors. The conductance detector detected non-chromophoric species generated by atmospheric CO2 as well as by decomposition of ASA. Conductance chromatograms yielded ASA peak heights and areas that varied linearly with the ASA concentration from 0.5 ppm to 10 ppm with linear correlation coefficients exceeding 0.999. In view of the sensitivity of conductance detection, its potential application as a sensitive tool for cleaning assessments for pharmaceutical equipment was confirmed by dispersing 50 μg of ASA on a 2′′ × 2′′ stainless steel sheet, swabbing the surface, dissolving the collected material in water and injecting the solution into the HPLC. The results in this study demonstrate that low cell constant conductance detection can be remarkably sensitive to ionized/charged species and thereby has potential to serve as an analytical tool for this important class of molecules.

Monolithic molecularly imprinted column-high performance liquid chromatography for enrichment and determination of trace cytokinins in plant samples

A method based on monolithic molecularly imprinted polymer enrichment combining with high performance liquid chromatography (mMIP-HPLC) detection was developed for the selective determination of trace cytokinins (CTKs) in plant samples. Monolithic molecularly imprinted polymer (mMIP) column was prepared in stainless steel tube by using kinetin as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, and toluene-dodecanol as the porogenic solvents. Compared with non-imprinted polymer (NIP) monolith, the prepared mMIP exhibited selective separation ability, good reproducibility and reusability, and high extraction efficiency in the separation and enrichment of the four CTKs. Under the optimized experimental conditions, mean recoveries were 91.9%, 80.0%, 87.5% and 50.2% for kinetin (K), kinetin glucoside (KR) , trans-zeatin (tZ) and meta-topolin (mT), respectively, with the corresponding RSDs less than 11.8%. The proposed mMIP-HPLC method was successfully applied in the separation and determination of the four cytokinins in different plant samples