Fixed-wavelength Detector Research Articles

In-capillary probing of quantum dots and fluorescent protein self-assembly and displacement using Förster resonance energy transfer.

Herein, a Förster resonance energy transfer system was designed, which consisted of CdSe/ZnS quantum dots donor and mCherry fluorescent protein acceptor. The quantum dots and the mCherry proteins were conjugated to permit Förster resonance energy transfer. Capillary electrophoresis with fluorescence detection was used for the analyses for the described system. The quantum dots and mCherry were sequentially injected into the capillary, while the real-time fluorescence signal of donor and acceptor was simultaneously monitored by two channels with fixed wavelength detectors. An effective separation of complexes from free donor and acceptor was achieved. Results showed quantum dots and hexahistidine tagged mCherry had high affinity and the assembly was affected by His6 -mCherry/quantum dot molar ratio. The kinetics of the self-assembly was calculated using the Hill equation. The microscopic dissociation constant values for out of- and in-capillary assays were 10.49 and 23.39 μM, respectively. The capillary electrophoresis with fluorescence detection that monitored ligands competition assay further delineated the different binding capacities of histidine containing peptide ligands for binding sites on quantum dots. This work demonstrated a novel approach for the improvement of Förster resonance energy transfer for higher efficiency, increased sensitivity, intuitionistic observation, and low sample requirements of the in-capillary probing system.

Process analysis of fluidized bed granulation

This study assesses the fluidized bed granulation process for the optimization of a model formulation using in-line near-infrared (NIR) spectroscopy for moisture determination. The granulation process was analyzed using an automated granulator and optimization of the verapamil hydrochloride formulation was performed using a mixture design. The NIR setup with a fixed wavelength detector was applied for moisture measurement. Information from other process measurements, temperature difference between process inlet air and granules (T(diff)), and water content of process air (AH), was also analyzed. The application of in-line NIR provided information related to the amount of water throughout the whole granulation process. This information combined with trend charts of T(diff) and AH enabled the analysis of the different process phases. By this means, we can obtain in-line documentation from all the steps of the processing. The choice of the excipient affected the nature of the solid-water interactions; this resulted in varying process times. NIR moisture measurement combined with temperature and humidity measurements provides a tool for the control of water during fluid bed granulation.

Simple and rapid high-performance liquid chromatographic method for the determination of aspartame and its metabolites in foods

A method for the determination of aspartame (N-L-α-aspartyl-L-phenylalanine methyl ester) and its metabolites, applicable on a routine quality assurance basis, is described. Liquid samples (diet Coke, 7-Up, Pepsi, etc.) were injected directly onto a mini-cartridge reversed-phase column on a high-performance liquid chromatographic system, whereas solid samples (Equal, hot chocolate powder, pudding, etc.) were extracted with water. Optimising chromatographic conditions resulted in resolved components of interest within 12 min. The by-products were confirmed by mass spectrometry. Although the method was developed on a two-pump HPLC system fitted with a diode-array detector, it is straightforward and can be transformed to the simplest HPLC configuration. Using a single-piston pump (with damper), a fixed-wavelength detector and a recorder/integrator, the degradation of products can be monitored as they decompose. The results obtained were in harmony with previously reported tedious methods. The method is simple, rapid, quantitative and does not involve complex, hazardous or toxic chemistry.

Determination of ibuprofen and its major metabolites in human urine by high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic assay for free and total ibuprofen and its major metabolites in human urine is described. Urine is acidified, drug and metabolites are extracted into hexane-propanol, back-extracted into sodium bicarbonate, neutralized and chromatographed. Ibufenac (4-isobutylphenylacetic acid) and 2-phenylpropionic acid were employed as internal standards. The extraction efficiencies were 94–100% for all compounds. The two metabolites and their internal standard were separated using an isocratic chromatographic system, followed by an abrupt step gradient to a second eluent for separation of ibuprofen and its internal standard with a total run time of 18 min. Detection was by a fixed-wavelength detector (214 nm). Sample-to-sample and day-to-day reproducibility studies yielded coefficients of variability of less than 9% for all compounds. The sensitivity was sufficient to determine 2.5 μg/ml free ibuprofen in 100 μl urine.

Quantitative determination of tolazoline in human serum by high performance liquid chromatography.

A micro high performance liquid chromatography assay is reported for the measurement of tolazoline in newborn infants. Pharmacokinetic data are presented for a single infant receiving tolazoline therapy. Tolazoline and the internal standard, clonidine, are extracted from alkaline serum into butylchloride/isopropanol (95/5). The organic layer is then back extracted with 50 mM phosphoric acid. A portion of the phosphoric acid layer is injected onto a 15-cm CN-bonded phase column. A mobile phase consisting of acetonitrile and phosphate buffer (pH 3) is used to elute tolazoline and the internal standard in less than 5 min. The effluent is monitored with a fixed wavelength detector at 214 nm. Using 50 microliters of serum, concentrations as low as 0.25 mg/L can be routinely determined with a coefficient of variation (CV) of 7.2%. However, when a 100-microliters sample is used, and the injection volume increased, a concentration of 0.05 mg/L can be routinely monitored with a CV of 1.2%. Interference from other drugs that are often used concurrently with tolazoline therapy was not observed.

Complete high-performance liquid chromatographic separation of 4-N,N-dimethylaminoazobenzene-4′-thiohydantoin and 4-dimethylaminoazobenzene-4′-sulphonyl chloride amino acids utilizing the same reversed-phase column at room temperature

Reversed-phase high-performance liquid chromatographic methods for the complete separation of all 4-dimethylaminoazobenzene-4′-sulphonyl chloride and 4-N,N-dimethylaminoazobenzene-4′-thiohydantoin amino acids on the same Supelcosil LC-18 column at room temperature are described. The procedures are simple and reproducible, and the systems are easily interconvertible. The use of a fixed-wavelength detector at 436 nm permits amino acid analysis at levels lower than 1 pmol with a stable baseline.

Determination of total pentachlorophenol in the urine of workers. A method incorporating hydrolysis, an internal standard and measurement by liquid chromatography.

Free pentachlorophenol (PCP) represents a small and variable fraction of total PCP excreted in the urine of exposed workers. A method incorporating hydrolysis is essential to be able to relate PCP excretion to absorbed dose. 3,5-Dichloro-2,4,6-tribromophenol (DTP) is added as an internal standard to a urine sample which is then acidified and steam-distilled. The distillate is made alkaline and extracted with methylene chloride to remove interferences. The distillate is then acidified and the phenols extracted into methylene chloride. The extract is evaporated, redissolved in acetonitrile and the PCP is measured using high pressure liquid chromatography (HPLC) using a 15 X 0.46 cm Spherisob-ODS 5 microns column with isocratic elution (H2O:CH3CN:CH3CO2H, 45:55:0.3 v/v). Detection is by fixed wavelength detector at 313 nm and calculation by the method of internal standardization. The biological threshold value used to trigger action to reduce exposure is 7 mg/l (corrected to a specific gravity of 1.024).

Simple, rapid, and highly efficient separation of amino acid phenylthiohydantoins by reversed-phase high-performance liquid chromatography

A rapid and efficient separation of amino acid phenylthiohydantoins by high-performance liquid chromatography has been accomplished by step-gradient elution with use of a mobile phase of acetate-buffered aqueous acetonitrile and an octadecylsilyl stationary phase. Typical analyses are completed in less than 12 min. Peak elution positions in this procedure are highly reproducible (with about 0.2% variance) and allow unambiguous identification of all residues. A procedure for the optimal positioning phenylthiohydantoin-Arg and -His is given. Molar extinction coefficients at 254 nm, which are particularly useful with common fixed wavelength detectors, are given for 25 amino acid phenylthiohydantoins.

High-Pressure Liquid Chromatography Assay for Dane Salt Potassium (-)-N-(1-Methoxycarbonylpropene-2yl)-p-hydroxyphenylglycine

Abstract A rapid, ion-pair, high-pressure liquid chromatographic method for analysis of Dane Salt Potassium (-)-N-(1-Methoxycarbonylpropene-2-yl)-p-Hydroxy-phenylglycine was developed. Tetrabutylammonium hydroxide was used as a counter-ion in the mobile phase. A fixed wavelength detector (λ = 280 nm) and a μ-Bondapak C-18 column were employed. The percent relative range of the method (precision) was 0.6% (n = 3).

Continous-flow scanning of selected high-performance liquid chromatography peak components by microprocessor control : Application to analysis of extracts from human lymphocytes

Continous-flow wavelength scanning of compounds separated by high-performance liquid chromatography is achieved through the use of fixed and variable-wavelength micro ultraviolet detectors connected in series but separated by a low-pressure three-way valve. Activation of the valve allows entrapment of selected peaks in the variable-wavelength detector without interfering with the response of the fixed-wavelength detector which is utilized for peak quantitation. A microprocessor program is employed to maintain control and accuracy during the scanning sequence. Good correlation was found between ultraviolet spectra of standards obtained on a conventional spectrometer and those on separated peaks. This system allows the identification and quantification of picomole amounts of peaks separated during one analysis of a biological sample.

High-performance Liquid Chromatographic Assay of Tolbutamide and Carboxytolbutamide in Human Plasma

A high-performance liquid chromatographic method was developed for the simultaneous measurement of tolbutamide and its major metabolite, carboxytolbutamide, in plasma. The assay involves the ether extraction of 1 ml of plasma, using chlorpropamide as an internal standard. The extract is dried, the residue is taken up in acetonitrile, and 5 μl is injected into a reversed-phase column. The mobile phase consisted of 35% acetonitrile and 65% 0.05 M phosphoric acid buffer (pH 3.9). A fixed-wavelength detector was set at 254 nm. The sensitivity limits for the tolbutamide and carboxytolbutamide assay were 2 and 0.1 μ/ml, respectively. The ratio of carboxytolbutamide to tolbutamide in plasma obtained from a subject given a 500-mg tolbutamide tablet was ˜1:20.

Stability-Indicating High-Pressure Liquid Chromatographic Assay for L-Lysine

Abstract A high-pressure liquid chromatographic method for the assay of L-Lysine is described. L-lysine was derivatized by reacting with 2,4-dinitrofluorobenzene. A fixed wavelength detector (254 nm) and a LiChrosorb RP-18, 10 μm, Hibar-II column from MCB Manufacturing Chemists, Inc., was employed. The method is simple, precise, accurate and stability-indicating.

Quantitative Determination of Guaifenesin, Phenylprolamine Hydrochloride Sodium Benzoate Δ Codeine Phosphate in Cough Syrups by High-Pressure Liquid Chromatography

Abstract A rapid Paired-ion high-pressure liquid chromatographic method for the quantitative determination of guaifenesin, phenylpropanolamine hydrochloride, sodium benzoate and codeine phosphate in cough syrups was developed. Heptane sulfonic acid was used as a counter-ion in the mobile phase. A fixed wavelength detector (λ = 254 nm) and a μ-Bondapak phenyl column was employed. Total analysis time for all the active ingredients was 22 minutes.

Rapid High-Performance Liquid Chromatographic Determination of Bleomycin A2 in Plasma

A rapid and specific method for the determination of bleomycin A2 is described. A 50-μl aliquot of 20% trichloroacetic acid was added to 200μl of plasma. The sample was vortexed and centrifuged, and 50μl of the clear supernate was injected into a liquid chromatograph equipped with a microparticulate reversed-phase column and a fixed wavelength detector. Elution was carried out using methanol–acetonitrile–0.0085M heptanesulfonic acid–acetic acid. A linear calibration curve was found in the 0.05–5–μg/ml range with an estimated precision of±6% (CV). Preliminary pharmacokinetic data in the rabbit also are reported.