Rolf Karlíček

Reversed-phase porous silica rods, an alternative approach to high-performance liquid chromatographic separation using the sequential injection chromatography technique.

A commercially available porous silica rod column was used as a separation tool for the sequential injection analysis (SIA). A porous solid monolithic column showed high performance at a low pressure, allowing sequential injection analysis to be used for the first time for separation in HPLC fashion. In this contribution, we tried to demonstrate a new separation concept with SIA manifold for the simultaneous determination of four different compounds (methylparaben (MP), propylparaben (PP), triamcinolone acetonide (TCA) and internal standard ketoprofen (KP)) in a pharmaceutical triamcinolon cream 0.1% formulation. A Chromolith Flash RP-18e, 25 mm x 4.6 mm column with a 10 mm pre-column (Merck, Germany) and a FIAlab 3000 system (USA) with an 8-port selection valve and 10 ml syringe were used for sequential injection chromatographic separations in our study. The mobile phase used was acetonitrile-methanol-water (35:5:65, v/v/v) + 0.05% nonylamine, pH 2.5, flow rate 0.6 ml min(-1). The analysis time was <6 min. A novel sequential injection chromatography (SIC) technique with UV spectrophotometric detection was optimised and validated.

Automated simultaneous monitoring of nitrate and nitrite in surface water by sequential injection analysis

A fully automated procedure based on Sequential Injection Analysis (SIA) methodology for simultaneous monitoring of nitrate and nitrite in surface water samples is described. Nitrite was determined directly using the Griess diazo-coupling reaction and the formed azo dye was measured at 540 nm in the flow cell of the fibre-optic spectrophotometer. Nitrate zone was passed through a reducing mini-column containing copperised-cadmium. After the reduction of nitrate into nitrite the sample was aspirated by flow reversal to the holding coil, treated with the reagent and finally passed through the flow cell. The calibration curve was linear over the range 0.05-1.00 mg N l(-1) of nitrite and 0.50-50.00 mg N l(-1) of nitrate; correlation coefficients were 0.9993 and 0.9988 for nitrite and nitrate, respectively. Detection limits were 0.015 and 0.10 mg N l(-1) for nitrite and nitrate, respectively. The relative standard deviation (RSD) values (n = 3) were 1.10% and 1.32% for nitrite and nitrate, respectively. The total time of one measuring cycle was 250 s, thus the sample throughput was about 14 h(-1). Nitrate and nitrite were determined in the real samples of surface water, and the results have been compared with those obtained by two other flow methods; flow injection analysis based on the same reactions and isotachophoretic determination used in a routine environmental control laboratory.

Flow-injection spectrophotometric determination of tetracycline antibiotics

Abstract The determination of the tetracycline antibiotics doxycycline, oxytetracycline, rolitetracycline and tetracycline using a flow-injection method with spectrophotometric detection based on their reaction with 4-aminophenazone and hexacyanoferrate(III) is described. The flow-injection system was optimized using a modified simplex method and the results were compared with those from a univariate procedure of optimization. Under the optimized conditions, the determination of the tetracyclines in the ranges 1−20 and 20−250 mg l−1 with a relative standard deviation of ⩽1% for 10 and 100 mg l−1, respectively (n=10), and a sample throughput of 60−70 h−1 was achieved. The method was applied to the determination of tetracyclines in pharmaceutical preparations.

Spectrophotometric determination of cardiac glycosides by flow-injection analysis

Abstract An optimized flow-injection method is described for the determination of various cardiac glycosides (lanatoside A, B and C, digoxin, digitoxin, acetyldigitoxin, ouabain) based on their reaction with picric acid in alkaline media using spectrophotometric detection at 486 nm. The calibration graph is linear for 0.025−0.5 g 1−1 cardiac glucosides (except ouabain) and 0.01–0.2 g 1−1 ouabain, with R.S.D. between 0.62 and 2.16% (n=6) and a sample throughput of 60 h−1. This method was utilized for the determination of digoxin in tablets and lanatoside C and ouabain in injections.

Sequential flow-injection spectrofluorimetric determination of coumarins using a double-injection single-line system

A simple and rapid flow-injection (FI) technique with spectrofluorimetric detection is presented for the sequential determination of two model compounds: 6-methoxy-7-hydroxycoumarin (scopoletine) and 7-hydroxycoumarin (umbelliferone) without prior separation. A double-injection valve with a single-line flow-injection system is used for the simultaneous injection of a sample and a phosphate buffer solution in aqueous 50% (vv) ethanol in order to measure the fluorescence signal of the analytes successively at pH ∗ 6.0 and 11.0. The optimum excitation and emission wavelengths are 350 nm and ≥ 418 nm, respectively. Using a calculation method based on differential fluorimetry it is possible to determine ≥ 1 μmol l−1 of scopoletine in the presence of ≥ 9-fold molar excess of umbelliferone or 1 μmol l−1 of umbelliferone in the presence of ≥ 23-fold molar excess of scopoletine. This technique can be used for sequential determination of various other fluorescent compounds (exhibiting pH-dependent excitation spectra) in two-component mixtures; it allows for a quick interchange of reagents within a single-line FIA system and the reagent consumption is decreased substantially.

Determination of rhodamine 123 by sequential injection technique for pharmacokinetic studies in the rat placenta

The sequential injection (SIA) technique was applied in pharmacokinetic studies of the transporter-mediated passage of a model substrate, rhodamine 123 (Rho123), through the dually perfused rat term placenta. The method described was used for real-time monitoring of Rho123 concentrations in both the maternal and fetal compartments. Determination was processed by fluorescence detection (lambda(ex)=480 nm, lambda(em)580 nm); calibration curve was linear over the range 0.01-50 mumoll(-1) (r=0.99965), detection limit was 10 nmoll(-1) (3sigma) and RSD2% (10 readings). Transport of Rho123 was scanned under various conditions (ATP-synthesis inhibition) and several inhibitors of P-glycoprotein transporter were tested (e.g. quinidine). The advantages of the modern SIA method-an automated analytical tool providing both fast and precise analysis-were successfully demonstrated for examination of transport profiles to investigate the effect of P-glycoprotein on the placental transfer of Rho123.

Spectrophotometric flow-injection determination of urea in body fluids by using an immobilized urease reactor

Abstract A flow system involving a packed-bed enzyme reactor (volume 180 μl) with urease immobilized covalently on poly(glycidyl methacrylate)-coated porous glass is used for determining urea in blood serum and urine. Enzymatically produced ammonia is converted to an indophenolate dye (by oxidative coupling with hypochlorite and sodium salicylate), which is detected spectrophotometrically at 700 nm. The calibration graph is rectilinear for 25–500 μM urea when injecting samples (75 μl) diluted 1:50 for serum or 1:1000 for urine at a frequency of 60 h −1 ; the relative standard deviation is 1.1% for ten injections of 300 μM urea. The immobilized urease is stabilized by the addition of disodium EDTA, sodium azide and 2-mercaptoethanol to a 0.2 M phosphate buffer (pH 6.9) used as the carrier stream, which serves also as a preservative for longterm storage of the urease reactor packing at 4°C.

SIMPLE LABORATORY-MADE AUTOMATED SEQUENTIAL INJECTION ANALYSIS (SIA) DEVICE. I. DESIGN AND TESTING OF THE HARDWARE COMPONENT

The ever increasing demand for analyses in clinical, agricultural, pharmaceutical, industrial, and process analytical control has led to the development of Sequential Injection Analysis (SIA) (1). Use of a flow pattern, rather than constant monotonous flow, used in Flow Injection Analysis, requires synchronisation of sample zone injection with the start of each flow cycle. Therefore, a system configuration must be conceived which will allow sample zone injection, reagent addition, mixing, measurement, and ejection of reacted mixture by a combination of forward and reversed flow steps. These steps may have different lengths, durations, and speeds, provided that they always follow the same pattern. A typical SIA set-up (Fig. 1) utilises a multiport selection valve (MSV) coupled with a pump (PP) which generates the main pulsation-less flow with variable direction and speed. A bi-directional syringe pump complies well with INSTRUMENTATION SCIENCE & TECHNOLOGY, 30(1), 13–20 (2002)

SIMPLE LABORATORY-MADE AUTOMATED SEQUENTIAL INJECTION ANALYSIS (SIA) DEVICE. II. SIA OPERATIONAL SOFTWARE BASED ON LABVIEW® PROGRAMMING LANGUAGE

ABSTRACT The paper presented deals with the development of an automated computer-aided sequential injection analysis (SIA) system and its application to laboratory automation. In Part I, hardware and technical parameters of the SIA system were described. Part II introduces original lab-developed operational software (FaFSIA), enabling one to control the cited SIA system and to process experimental data, including their presentation. The software, based on graphic programming language LabVIEW, is described in detail.

UNIDIRECTIONAL TRANSFER OF D-XYLOSE ACROSS THE RAT PLACENTA

1. The transplacental transfer Of D‐Xylose was investigated in the present study.