Liquid chromatography of cyclodextrins with indirect photometric detection of phenolphthalein inclusion complexes

Summary - and -cyclodextrin (CD) separated by liquid chromatography have been indirectly detected by depression of the background absorbance at a visible wavelength by means of inclusion complexation with phenolphthalein. The background signal was generated by phenolphthalein in an alkaline medium. Octadecyl-bonded poly(vinylalcohol) packings were employed as the stationary phase, which allowed the use of alkaline mobile phases, and eliminated post column mixing of reagents. The detection limits for - and -CD were 0.1–0.5 M at a signal to noise ratio of 3. -CD could not be measured by the present detection principle.     

Analysis of chlortetracycline by high performance liquid chromatography with postcolumn alkaline-induced fluorescence detection.

A high performance liquid chromatographic method for the analysis of chlortetracycline (CTC) using postcolumn fluorescence detection has been developed. After chromatographic separation of CTC on a polystyrene-divinylbenzene copolymer column, a highly fluorescent derivative isochlortetracycline (iso-CTC) was formed postcolumn in an on-line reaction coil with the addition of 25% NaOH (w/v). Chromatographic separation was achieved on a PRP-1 column, 15 cm x 4.6 mm, with 27:73 acetonitrile:0.2% perchloric acid (v/v), at 1.0 mL/min. Fluorescence derivatization was achieved by the on-line addition of 25% NaOH (w/v), at a flow rate of 0.2 mL/min, into the column eluant in a post-column reaction coil. The reaction coil was 9 m of teflon (1/16 in o.d., 0.3 mm i.d.) knitted into a six-sided coil. The fluorescent derivative was detected at lambda ex 355 nm and lambda em > 389 nm. Using this method after a simple sample cleanup, CTC can be detected in milk at 0.04 micrograms/mL, which is comparable to that obtained by microbiological assays. The detection method was linear between 0.02 micrograms/mL and 4 micrograms/mL. Because of the chromatographic separation, the method is more selective than microbiological assays and more sensitive than ultraviolet detection. With the chromatographic system described, the keto tautomeric forms of CTC and 4-epi-CTC are separated in a system which minimizes their formation on-column. In acidic aqueous organic solutions, the keto tautomer of CTC is the only product formed to any significant amount.     

General theory of indirect detection in chromatography

Indirect detection is used in liquid chromatography to detect sample components that more or less lack detectable properties. A detectable component, the probe, is added to the mobile phase and the sample containing the components is injected. Every solute, even those not normally detectable, will give rise to detectable peaks due to the presence of the probe. The theory currently used to describe this situation is different for different adsorption isotherm models and also restrictions have to be imposed on the adsorption isotherm parameters. We present a general theory that describes both the retention times and areas of the detected peaks without making any a priori adsorption isotherm assumptions. Our general theory is compared with the current theory and validated by computer simulations. An alternative quantification method is suggested based on the measurement of the relative areas of the sample components without the requirement of having standard curves of known solutions of the sample components. Using this approach, the new theory is able to predict the solute concentrations in cases when the current theory did not, i.e., when the saturation capacities of the sample components and the probe differed from each other. In addition, a new application is proposed: the estimation of adsorption isotherm parameters.     

Correlation ion chromatography with indirect UV detection

Summary A binary pseudo-random injection of a sample containing low concentrations of common anions into the anion exchange column was used to enhance the signal-to-noise ratio of indirect UV detection of the ions. The detection limits obtained in our experiment (14 ppb, 14 ppb, and 64 ppb for Cl^–, NO ₃ ^– and SO ₄ ^2– respectively) are in good agreement with theoretical predictions. The large amplitude system peaks inherent to single column IC do not generate remarkable ghost peaks, which is indicative of the suitability of the input devices used in our studies.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

Analysis of glyphosate,glufosinate and aminomethylphosphonic acid by capillary electrophoresis with indirect fluorescence detection

A capillary electrophoresis (CE)-indirect fluorescence detection method is described for the simultaneous determination of glufosinate, glyphosate and aminomethylphosphonic acid. The three analytes were separated by CE in 5 min with a 1 mM fluorescein solution at pH 9.5. Fluorescein also functioned as a background fluorophore for the indirect detection of these nonfluorescent species. Linearity of more than two orders of magnitudes was generally obtained. The concentration limits of detection were in the microM range. Precisions of migration times and peak areas were less than 1.7% and 7.4%, respectively. Quantitation of glyphosate and glufosinate in commercial herbicides is demonstrated. In addition, the applicability of the method for the analysis of ground water was examined.     

Indirect fluorimetric detection of proteins via postcolumn mixing with fluorescence probe in size-exclusion chromatography

Summary Proteins were visualized by postcolumn mixing with 2-p-toluidinyl-6-naphthalene sulfonate or 1-anilino-8-naphthalene sulfonate in size-exclusion chromatography. The indirect detection is based on fluorescence enhancement of the fluorescence probe owing to hydrophobic interaction with proteins. Bovine serum albumin gave the highest signal intensity among the proteins examined.     

Integrated pulsed amperometric detection of glufosinate, bialaphos and glyphosate at gold electrodes in anion-exchange chromatography.

A rapid and practical method for direct detection of the herbicides (glufosinate, bialaphos and glyphosate) in anion-exchange chromatography has been developed with integrated pulsed amperometric detection (IPAD). The electrochemical behavior of these herbicides showed catalytic currents based on the oxidation of amines in their structures. Waveform in IPAD was similar to that for amino acids, which exhibited adsorption/desorption catalytic features at gold electrode surface in alkaline solution. Under optimized conditions, detection limits (signal-to-noise ratio of 3) for glufosinate, bialaphos and glyphosate were 20, 65 and 50 ng ml(-1), respectively, with correlation coefficients of 0.995, 0.997 and 0.996 over concentration ranges of 0.1-45, 0.3-32 and 0.1-50 microg ml(-1), respectively. The relative standard deviations (n=5) were 1.7-3.0%. The present method was successfully applied to the determination of glyphosate in urine and serum.     

Determination of vitamin K1 in medical foods by liquid chromatography with postcolumn reduction and fluorometric detection

A liquid chromatographic (LC) method is described for the determination of vitamin K1 in medical foods. The sample is enzymatically digested with lipase and alpha-amylase and extracted with 1% sodium bicarbonate solution-isopropanol (1 + 1). After C18 solid-phase extraction, vitamin K1 is separated by nonaqueous reversed-phase LC, converted to the hydroquinone by postcolumn zinc reduction, and quantitated by fluorescence detection. The limit of detection is 8 pg (3 sigma), and the limit of quantitation is 27 pg (10 sigma) on column. Linear response ranged from 0.1 to 1.0 ng vitamin K1 (r= 0.9999). The mean recovery (n = 38) for all spiking levels was 101.6 +/- 2.85%. Analysis of Standard Reference Material 1846, Infant Formula, gave a mean value of 0.95 +/- 0.088 mg vitamin K/kg (K or K1?) (n = 31) with a coefficient of variation of 9.26.     

Optimization of single-column anion chromatography with indirect ultraviolet photometric and fluorimetric detection

Optimization of single-column anion chromatography with indirect photometric and indirect fluorimetric detection is described. A simplex algorithm is used for signal maximization with eluent concentration, pH and flow rates as dependent variables. Absolute detection limits obtained for chloride, nitrite, nitrate and sulphate by indirect photometric detection were 1.0, 1.2, 1.4, and 2.0 ng, respectively. The use of single-beam indirect fluorimetric detection of these anions is also demonstrated. The method was applied to the detection of anions arising from gaseous acids or atmospheric aerosols.     

Determination of glyphosate by ion chromatography

An ion chromatography system for the determination of glyphosate was described. Ion chromatograph was carried out by suppressed conductivity detection (DX-100). The eluent contained 9 mmol l⁻¹ Na₂CO₃ and 4 mmol l⁻¹ NaOH. The detection limit was 0.042 μg ml⁻¹ (S/N=3). The relative standard deviation was 1.99% and the correlation coefficient of the calibration curve for area was 0.9995. The linear range was 0.042100 μg ml⁻¹. Common inorganic ion and organic acids did not interfere. The recovery was 96.4103.2%. The method was simple, rapid, reliable and inexpensive.     

Evaluation of peroxyoxalate chemiluminescence postcolumn detection of fluphenazine in urine and blood plasma using high performance liquid chromatography

Peroxyoxalate chemiluminescence may be used for sensitive postcolumn detection of phenothiazine analytes separated by high performance liquid chromatography with appropriate optimization of measurement conditions such as solvent, pH and oxalate ester. Detectability of fluorescent analytes by chemical excitation varies greatly, but analytes with low oxidation potentials are generally more readily detected at low levels, as demonstrated for phenothiazines, an important class of fluorescent drugs. Some improvement in detection limits is observed for fluphenazine when chemiluminescence detection is compared to conventional fluorescence detection. Because of the specificity of chemical excitation, fewer interferences from fluorescent impurities in a urine matrix are observed.     

Determination of organic acids, amino acids and saccharides by high-performance liquid chromatography and a postcolumn enzyme reactor with amperometric detection.

A technique for the determination of organic acids, amino acids and sugars is described. The compounds of interest are separated by high-performance liquid chromatography (HPLC) and converted on-line by immobilized enzymes. The enzymes employed are covalently bound to a synthetic carrier. Hydrogen peroxide, which is produced in the reaction with oxidases, makes possible the application of an electrochemical detector. This arrangement combines the separation efficiency of HPLC, the substrate specificity of enzymes and the high sensitivity of electrochemical detection. The enzymes act according to known reaction mechanisms, but coupling with HPLC leads to a promising extension in the field of biosensors. The simple pretreatment of the samples (often a dilution step is sufficient) allows a rapid analysis of foodstuffs and biological or clinical extracts. The examples presented demonstrate the very high sensitivity of the method with detection limits in the nano- to picomolar range and a wide field of application.     

Automated two-dimensional HPLC residue procedure for glyphosate on cereals and vegetables with postcolumn fluoregenic labelling

Summary A procedure for glyphosate in cereals and vegetables is described in which a 250-μl extract in water is injected on an AX/Corasil Bondapack pre-column. After washing with water the retarded glyphosate is forward eluted on an lonosphere A anion-exchange column with the buffered eluent. The pre-column is backflushed and regenerated with acetic acid. Recovery experiments with barley, wheat and rye (tolerance 5–20 mg/kg) at the 1 mg/kg level showed recoveries of 80% (C.V. = 10%). For potatoes, strawberries and asparagus, with a tolerance of 0.05 mg/kg, experiments at the 1 mg/kg level indicated promising results suitable for working at residu levels with these products.     

Determination of streptomycin residue in cucumber and Chinese cabbage by high-performance liquid chromatography with postcolumn derivatization and fluorometric detection

A sensitive and accurate method was developed for the determination of streptomycin using HPLC followed by postcolumn derivatization and fluorometric detection. The analyte was extracted, using aqueous solution from cucumber and Chinese cabbage, by a two-step SPE procedure. The extraction, cleanup, and chromatography conditions were optimized, and the performance of the analysis method was evaluated. The conditions of chromatography were as follows: the separation was performed on a C18 column; the isocratic mobile phase consisted of acetonitrile and a mixed solution containing 10 mM sodium 1,2-naphthoquinone-4-sulfonate and 0.4 mM sodium 1-heptanesulfonate (25+75, v/v); and the flow rate was 1 mL/min. The fluorescence detector was set at an excitation wavelength of 263 nm and an emission wavelength of 435 nm. The calibration curve was linear over the range of 50-2000 ng/mL, with a correlation coefficient of 0.9995. The LOD and LOQ were 10 and 30 ng/g, respectively, in both cucumber and Chinese cabbage. The method was validated for selectivity, linearity, precision, and accuracy. The intraday and interday precision and accuracy were within 10%. The mean recoveries from spiked samples were more than 75%, with RSD lower than 10%.     

Coupled-column liquid chromatography combined with postcolumn photochemical derivatization and fluorescence detection for the determination of herbicides in groundwater

This study examines the application of coupled-column LC-photochemically induced fluorimetry-fluorescence detection (LC-LC-PIF-FD), demonstrating its potential for the quantitative and selective detection of six herbicides, including propanil and the phenylureas monuron, monolinuron, chlorotoluron, diuron and neburon in groundwater samples. An AQUASIL C18 50 x 4.6 mm(2) id column coupled to an AQUASIL C18 150 x 4.6 mm(2) id column for analyte clean-up and determination were used, respectively. A simple SPE with Cl8 cartridges was carried out, yielding average recoveries between 80 and 112% (n = 6) with RSDs between 0.5 and 9%. The LODs ranged from 0.0083 to 0.0833 microg/L in the groundwater samples.     

Direct determination of glyphosate using hydrophilic interaction chromatography with coulometric detection at copper microelectrode

A simple, rapid, and low-cost coulometric method for direct detection of glyphosate using hydrophilic interaction chromatography is presented. The principle of detection is based on the enhancement of the anodic current of copper microelectrode in the presence of complexing agents, such as glyphosate, with the formation of a soluble Cu(II) complex. Under optimized conditions, the limit of detection (S/R = 3) for glyphosate was 0.1 mg L⁻¹ (0.59 μM) without any preconcentration method. The calibration curve has been found linear in all concentration range tested (from limit of detection to 34 mg L⁻¹) with an excellent correlation coefficient (0.9999). The present method was successfully applied for the determination of glyphosate in fruit juices without any kind of extraction, clean-up, or preconcentration step, with recoveries of 92 and 90% for apple and grape juice, respectively.     

Rapid determination of bisphosphonates by ion chromatography with indirect UV detection

Rapid methods for etidronate, clodronate, pamidronate, and alendronate assays are presented. The methods are based on ion chromatography with indirect UV detection, which avoids the need for chemical derivatization procedures. Each compound is analyzed on an individual basis. There is no need for having separation among these analytes because the aim of the proposed methods is to analyze each compound separately either in bulk material or pharmaceuticals. Phenosphere (150 x 2.0-mm, 5 microm) and Sphereclone (250 x 2.0-mm, 5 microm) anion exchange columns were employed with sodium citrate (20mM) as the mobile phase. The methods are simple, rapid (analysis time of 5 min for etidronate and clodronate and 7 min for alendronate and pamidronate), and demonstrate precision (relative standard deviation was lower than 2.0% in all concentrations), accuracy, and specificity. Calibration curves are linear with r(2) > 0.99 over the concentration range of 50 to 400 microg/mL for etidronate and clodronate, and of 100 to 500 microg/mL for pamidronate and alendronate. Furthermore, they employed silica-based columns, which are cheaper than polymeric columns frequently used in previous reported methods.