Method of Determination of 9-(Alkylthio)Acridines in Urine by Capillary Liquid Chromatography

Reversed-phase capillary liquid chromatography was applied for quantification of thioderivatives of acridine. Ten 9-(alkylthio)acridines, three containing and seven lacking amino groups, were analyzed on Nucleosil C₁₈ with acetonitrile-water-triethylamine (90.0:9.0:1.0, v/v/v) mixture as the mobile phase. The presence of triethylamine in the eluent greatly improved the peak shape and markedly shortened the analysis time. Calibration curves of all the studied acridine derivatives were measured over a concentration range of 1.0·10^?6 – 1.0·10^?3 mol. L^?1 at two parallel detection wavelengths, 265 and 270 nm. Detection and quantification limits were evaluated for all the derivatives. The detection limits were in the region of μmol. L^?1 for most of the thioacridines. The calibration curves were used for quantification of the derivatives in human urine. No pretreatment of the real biological samples was necessary. The optimized CLC method enabled a good separation of the individual thioacridines from other compounds present in human urine samples and their quantification in a reasonable analysis time.     

Separation and quantification of 9-(alkylthio)acridines by capillary micellar electrokinetic chromatography and capillary liquid chromatography

Various thioacridine derivatives are potential chemotherapeutics against various diseases which are intensively synthesized, characterized, and investigated by many research groups. Efficient, fast, and reliable separation and quantification methods for their analysis are still to be developed. MEKC and capillary LC (CLC) were applied for the separation and quantification of five highly hydrophobic, weakly basic, and structurally similar 9-(alkylthio)acridines. Since the common anionic and cationic surfactants failed to separate the strongly hydrophobic thioacridines by MEKC, sodium cholate was used in an alkaline BGE and successfully employed for their fast separation. In CLC, the weakly basic nature of the thioacridines necessitated use of LiChrosorb RP-select B sorbent as the stationary phase, which combined with a very simple mobile phase methanol/water yielded an efficient chromatographic separation system. Both, the MEKC and CLC optimized separation methods were then applied to quantify the thioacridines within a concentration range of 1.0 x 10(-5)-1.0 x 10(-3) mol/L and the obtained experimental results were critically compared. In practical terms, the MEKC analytical method can quantify the analytes much faster but with a lower reliability while the CLC method performs slower analysis with a higher repeatability of the experimental results.     

Determination of furanic aldehydes in sugarcane bagasse by high‐performance liquid chromatography with pulsed amperometric detection using a modified electrode with nickel nanoparticles

A glassy carbon electrode chemically modified with nickel nanoparticles coupled with reversed‐phase chromatography with pulsed amperometric detection was used for the quantitative analysis of furanic aldehydes in a real sample of sugarcane bagasse hydrolysate. Chromatographic separation was carried out in isocratic conditions (acetonitrile/water, 1:9) with a flow rate of 1.0 mL/min, a detection potential of – 50 mV vs. Pd, and the process was completed within 4 min. The analytical curves presented limits of detection of 4.0 × 10^?7 mol/L and 4.3 × 10^?7 mol/L, limits of quantification of 1.3 × 10^?6 and 1.4 × 10^?6 mol/L, amperometric sensitivities of 2.2 × 10⁶ nA mol/L and 2.7 × 10⁶ nA mol/L for furfural and 5‐hydroxymethylfurfural, respectively. The values obtained in this sample by the standard addition method were 1.54 ± 0.02 g/kg for 5‐hydroxymethylfurfural and 11.5 ± 0.2 g/kg for furfural. The results demonstrate that this new proposed method can be used for the quick detection of furanic aldehydes without the interference of other electroactive species, besides having other remarkable merits that include excellent peak resolution, analytical repeatability, sensitivity, and accuracy.     

Supramolecular separation mechanism of pentafluorophenyl column using ibuprofen and omeprazole as markers: LC‐MS and simulation study

The pentafluorophenyl (PFP) column is emerging as a new advancement in separation science to analyze a wide range of analytes and, thus, its separation mechanism at supramolecular level is significant. We developed a mechanism for the separation of ibuprofen and omeprazole using different combinations (ranging from 50:50 to 60:40) of water–acetonitrile containing 0.1% formic acid as the mobile phase. The column used was Waters Acquity UPLC HSS PFP (75 × 2.1 mm, 1.8 μm). The reverse order of elution was observed in different combinations of the mobile phases. The docking study indicated hydrogen bonding between ibuprofen and PFP stationary phase (binding energy was −11.30 kJ/mol). Separation at PFP stationary phase is controlled by hydrogen bonding along with π–π interactions. This stationary phase may be used to analyze both aromatic and aliphatic analytes. The developed mechanism will be useful to separate various analytes by considering the possible interactions, leading to saving of energy, time and money. In addition, this work will be highly useful in preparative chromatography where separation is the major problem at a large scale. Moreover, the developed LC‐MS‐QTOF method may be used to analyze ibuprofen and omeprazole in an unknown sample owing to the low value of detection limits.     

New potential humic acid stationary phase toward drug components: Development of a chemometric‐assisted RP‐HPLC method for the determination of paracetamol and caffeine in tablet formulations

A new humic acid based stationary phase has been used, for the first time, to achieve the separation and quantification of paracetamol and caffeine in pharmaceutical preparations under reversed‐phase high‐performance liquid chromatography conditions. Central composite design was applied as a powerful tool to optimize the most dominant parameters that influence the resolution of reversed‐phase high‐performance liquid chromatography, that is, mobile phase composition (acetonitrile percentage in water), flow rate, and column temperature. The optimum conditions were obtained as 21.69%, 1.5 mL/min, and 15°C, respectively, with the aid of a second‐order quadratic model and desirability function. Under the optimum conditions, the peaks could be baseline separated within 10 min. For the developed reversed‐phase high‐performance liquid chromatography method, the linearity was investigated in the concentration ranges of 2–160 mg/mL (R² = 0.999) for paracetamol and 2–9.9 mg/mL (R² = 0.991) for caffeine. Mean recoveries for paracetamol and caffeine were 95.90 and 95.68%, respectively. The limits of detection and quantification were 4.1 × 10^‐4 and 1.3 × 10^‐3 mg/mL for paracetamol and 1.6 × 10^‐4 and 5.0 × 10^‐4 mg/mL for caffeine. The results showed that the new humic acid based stationary phase is very suitable for the separation of paracetamol and caffeine in pharmaceutical preparations and, thus it can be used effectively in the pharmaceutical industry.     

Determination of salbutamol by direct chiral reversed‐phase HPLC using teicoplanin as stationary phase and its application to natural water analysis

A direct chiral LC‐UV method was optimized for the determination of salbutamol (SAL) β₂‐agonist in environmental water. Two commercially available columns were evaluated: teicoplanin Chirobiotic‐T? (150 × 2.1 mm i.d., 5 µm) and vancomycin Chirobiotic‐V? (150 × 2.1 mm i.d., 5 µm). Finally, teicoplanin chiral stationary phase was selected for SAL enantiomer resolution. In order to preserve its integrity and maintain the column performance for longer time, the use of additives such as triethylamine (TEA) in the mobile phase was avoided. Experimental design was applied to simultaneously evaluate the influence of several parameters involved in enantiomer separation and to establish the conditions for acceptable resolution and performance in short analysis time. Optimum mobile phase was methanol–20 mM ammonium acetate buffer at pH 4.5 (98:2, v/v). A solid‐phase extraction procedure for sample pre‐concentration and clean‐up allowed the determination of chiral SAL residues in natural water samples spiked at low concentrations in the range 1.0–20 ng mL^?1. Reproducible recoveries, between 77 and 98%, were obtained and matrix effect was negligible. Injection of sample solutions at low elution strength permitted the SAL enantioresolution in the natural water complex matrix with satisfactory sensitivity and precision. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous Determination of Epinephrine, Noradrenaline and Dopamine in Human Serum Samples by High Performance Liquid Chromatography with Chemiluminescence Detection

A simple, rapid and accurate high performance liquid chromatographic （HPLC） technique coupled with chemiluminescence （CL） detection was developed for the simultaneous determination of epinephrine （E）, noradrenaline （NA） and dopamine （DA）. It was based on the analyte enhancement effect on the CL reaction between luminol and potassium ferricyanide. The effects of various parameters, such as potassium ferricyanide concentration, luminol concentration, pH value and component of the mobile phase on chromatographic behaviors of the analytes （E, NA and DA） were investigated. The separation was carded out on C18 column using the mobile phase of 0.01 mol/L potassium hydrogen phthalate solution and methanol （92 ： 8, V/V）. Under the optimum condi- tions, E, NA and DA showed good linear relationships in the range of 1 × 10^-8 -5 × 10^-6, 5.0× 10^-9 -1.0× 10^-6 and 5.0×10^-9-1.0× 10^-6 g]mL respectively. The detection limits for E, NA and DA were 4.0×10^-9, 1.0× 10^-9 and 8.0 × 10^-10 g/mL. The proposed method has been applied successfully to the analysis of E, NA and DA in human serum samples.     

Analysis of normal and modified nucleosides in urine samples by high‐performance liquid chromatography with different stationary phases

The main aim of the present work was to study the retention behavior and quantification of nine nucleosides with the use of octadecyl, alkylamide, cholesterol and alkyl‐phosphate stationary phases. The influence of organic solvent and buffer concentration on the separation of these compounds was under investigation. The retention factor had the highest values for the octadecyl and cholesterol packing materials. Complete separation of all the studied nucleosides was achieved in case of cholesterol stationary phase. The optimized separation method was applied for the quantification of nucleosides in the urine samples. Calibration plots showed good linearity (R² > 0.999) and the limits of detection were in a range of 0.3–0.5 µg/mL, while the limits of quantitation were >0.9 µg/mL. Accuracy was in the range of 5–11%. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of enoxacin and ofloxacin by capillary electrophoresis with electrochemiluminescence detection in biofluids and drugs and its application to pharmacokinetics

A novel and sensitive method for the simultaneous determination of enoxacin and ofloxacin has been established using capillary electrophoresis (CE) coupled with electrochemiluminescence (ECL) detection based on the ECL enhancement of tri(2,2‐bipyridyl)ruthenium(II). The conditions for sample solvent type, CE separation and ECL detection were investigated systematically. The analytes were well separated and detected within 7 min. The limits of detection (S/N = 3) of enoxacin and ofloxacin are 9.0 × 10^?9 and 1.6 × 10^?8 mol/L, respectively. The precisions (RSD%) of intraday and interday are less than 2.1 and 4.0%, respectively. The limits of quantitation (S/N = 10) of enoxacin and ofloxacin are 3.2 × 10^?7 and 5.4 × 10^?7 mol/L in human urine samples and 4.1 × 10^?7 and 6.9 × 10^?7 mol/L in human serum samples, respectively. The recoveries of enoxacin and ofloxacin at different concentration levels in human urine, serum and eye drop samples are between 94.0 and 106.7%. The proposed method was successfully applied to the determination of the enoxacin and ofloxacin in human urine, serum and eye drop samples and the monitoring of pharmacokinetics of ofloxacin in human body. Copyright © 2009 John Wiley & Sons, Ltd.     

Enantioseparation and impurity determination of the enantiomers of novel phenylethanolamine derivatives by high performance liquid chromatography on amylose stationary phase

Simple and efficient analytical HPLC methods using Chiralpak AS-H as chiral stationary phase were developed for direct enantioseparation of 11 novel phenylethanolamine derivatives. The chromatographic experiments were performed in normal phase mode with n-hexane–ethanol–triethylamine (TEA) as mobile phase. Excellent baseline enantioseparation was obtained for most of compounds. The effects of the concentration of organic modifiers and column temperature were studied for the enantiomeric separation. The mechanism of chiral recognition was discussed based on the relationship between the thermodynamic parameters and structures of compounds. It was found that the enantioseparations were all enthalpy driven, and the tert-butyl groups of compounds had significant influence on the chiral recognition. Trantinterol enantiomers were resolved (R_s = 2.73) within 14 min using n-hexane–ethanol–TEA (98:2:0.1, v/v/v) as mobile phase with a flow rate of 0.8 mL min⁻¹ at 30 °C. The optimized method was validated for linearity, precision, accuracy and stability in solution and proved to be robust. The limits of detection (LOD) and quantification (LOQ) for (+)-trantinterol were 0.15 and 0.46 μg mL⁻¹. The method was applied for enantiomeric impurity determination of (−)-trantinterol bulk samples.     

Voltammetric behavior and quantitative determination of ambazone concentrations in urine and in a pharmaceutical formulation

The use of square wave adsorptive stripping voltammetry (SWAdSV) in conjunction with a cyclic renewable silver amalgam film electrode (Hg(Ag)FE) for the analytical determination of ambazone in urine samples and pharmaceutical formulations is described. A single reduction peak in Britton-Robinson buffer at pH 4.0 was detected at about ?1.4 V versus Ag/AgCl. Mechanistic studies have shown that the compound can act as an electrocatalyst. The method was validated. The analytical curve was linear in the concentration range from 1.0×10^?9 to 1.0×10^?7 mol L^?1. The detection and quantification limits were found to be 3.0×10^?10 mol L^?1 and 1.0×10^?9 mol L^?1, respectively. The proposed method was successfully applied to ambazone determination in real samples.      

Determination of amino acid neurotransmitters in rat hippocampi by HPLC‐UV using NBD‐F as a derivative

A simple, rapid and accurate high‐performance liquid chromatography method with ultraviolet–visible detection was developed for the determination of five amino acid neurotransmitters – aspartate, glutamic acid, glycine, taurine and γ‐aminobutyric acid – in rat hippocampi with pre‐column derivatization with 4‐fluoro‐7‐nitrobenzofurazan. Several conditions which influenced derivatization and separation, such as pH, temperature, acetonitrile percentage mobile phase and flow rate, were optimized to obtain a suitable protocol for amino acids quantification in samples. The separation of the five neurotransmitter derivatives was performed on a C₁₈ column using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)–acetonitrile (84:16, v/v) at a flow rate of 1.0 mL/min with the column temperature at 30°C. The detection wavelength was 472 nm. Without gradient elution, the five neurotransmitter derivatives were completely separated within 15 min. The linear relation was good in the range from 0.50 to 500 µmol/L, and the correlation coefficients were ≥0.999. Intra‐day precision was between 1.8 and 3.2%, and inter‐day precision was between 2.4 and 4.7%. The limits of detection (signal‐to‐noise ratio 3) were from 0.02 to 0.15 µmol/L. The established method was used to determine amino acid neurotransmitters in rat hippocampi with satisfactory recoveries varying from 94.9 to 105.2%. Copyright © 2013 John Wiley & Sons, Ltd.     

Capillary electrochromatography-mass spectrometry of cationic surfactants

The CEC-MS of alkyltrimethylammonium (ATMA+) ions with chain lengths ranging from C1-C18 is optimized using an internally tapered column packed with mixed mode reversed phase/strong cation exchange stationary phase. A systematic study of the CEC separation parameters is conducted followed by evaluation of the ESI-MS sheath liquid and spray chamber settings. First, the optimization of CEC separation parameters are performed including the ACN concentration, triethylamine (TEA) content, buffer pH and ammonium acetate concentration. Using 90% v/v ACN with 0.04% v/v TEA as mobile phase, the separation of longer chain C6-C18-TMA+ surfactants could be achieved in 15 min. Lowering the ACN concentration to 70% v/v provided resolution of shorter chain C1, C2-TMA+ from C6-TMA+ although the total analysis time increased to 40 min. Furthermore, variation of both the ACN and TEA content as well as ionic strength has found to significantly influence the retention of longer chain surfactants as compared to shorter chains. The optimum CEC conditions are 70% v/v ACN, 0.04% v/v TEA, pH 3.0 and 15 mM ammonium acetate. Next, the optimization of the ESI-MS sheath liquid composition is conducted comparing methanol to isopropanol followed by the use of experimental design for analysis of spray chamber parameters. Overall, the developed CEC-ESI-MS method allows quantitative and sensitive monitoring of ATMA+ from < or =10 microg/mL down to 10 ng/mL. Utilizing the optimized CEC-ESI-MS protocol, the challenging analysis of commercial sample Arquad S-50 ATMA+ containing cis-trans unsaturated and saturated soyabean fatty acid derivatives is demonstrated.     

Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

Summary A rapid and simple liquid-chromatographic method has been developed for on-line quantification of amphetamine in biological fluids. Untreated samples (20 μL) are injected directly into the chromatographic system and purified on a 20 mm×2.1 mm i.d. pre-column packed with 30 μm Hypersil C₁₈ stationary phase. After clean-up the analyte is transferred to the analytical column (125 mm×4 mm i.d., 5 μm LiChrospher 100 RP₁₈) for derivatization and separation using a mixture of acetonitrile and the derivatization reagent (o-phthaldialdehyde andN-acetyl-L-cysteine) as the mobile phase. The experimental conditions for on-line derivatization and resolution of the amphetamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method described has been applied to the determination of amphetamine in plasma and urine. Good linearity and reproducibility were obtained in the 0.1–10.0 μg mL⁻¹ concentration range, and limits of detection were 25 ng mL⁻¹ and 10 ng mL⁻¹ with UV and fluorescence detection, respectively. The procedure described is very simple and rapid, because no off-line manipulation of the sample is required; the total analysis time is approximately 8 min.     

Separation of the Enantiomers of Thyroxine by HPLC with Chiral Mobile Phases

A high-performance liquid chromatographic (HPLC) method has been developed for separation of the enantiomers of thyroxine (T4). A silica gel column was used in conjunction with a chiral mobile phase (CMP) comprising 35:65 (v/v) acetonitrile–water containing 0.1 mM copper(II) acetate, 0.2 mM L-proline, and 0.5 mM triethylamine (TEA), pH 5.42. The flow rate was 1.0 mL min^?1 and the analysis temperature 40 °C. L-T4 was eluted before D-T4 by mobile phase containing L-proline copper complex. Inversion of the chirality of the mobile phase resulted in reversal of this order of elution. A racemic mobile phase containing DL-proline copper complex resulted in no separation. The enantioseparation phenomenon is discussed. When the method was used to determine the concentration of T4 enantiomers in the serum of a patients with thyroid disease, different concentrations of T4 enantiomers were found in different patents.     

The New Application of Boron Doped Diamond Electrode Modified with Nafion and Lead Films for Simultaneous Voltammetric Determination of Dopamine and Paracetamol

A new voltammetric procedure for the simultaneous determination of dopamine (DA) and paracetamol (PA) using boron doped diamond electrode modified with Nafion and lead films (PbF/Nafion/BDDE) was investigated. The use of this electrode resolved the overlapped voltammetric waves of DA and PA into well‐defined peaks with peak to peak separation of about 320 mV. Under the optimized experimental conditions in differential pulse voltammetric technique, DA and PA gave a linear response over the ranges 2.0×10^?7–1.0×10^?4 mol L^?1*(R²=0.9996) and 5.0×10^?7–1.0×10^?3 mol L^?1 (R²=0.9979), respectively. The detection limits were found to be 5.4×10^?8 mol L^?1 for DA and 1.4×10^?7 mol L^?1 for PA. They are lower, comparable or in some cases a little bit higher than those obtained using other electrochemical sensors. However, the proposed procedure of the sensor preparation is much simpler than procedures described in the literature with a lower detection limit. The proposed procedure was successfully applied to the determination of PA in some commercial pharmaceuticals as well as to the simultaneous determination of DA and PA in human urine, whole blood and serum samples directly without any separation steps.     

Chromatographic resolution, chiroptical characterization and urinary excretion of the enantiomers of sulindac

Summary The chromatographic resolution of the enantiomers of sulindac has been achieved using a Chiralpak AD CSP (10 μm, 250×4.6 mm) with a mobile phase of hexane: ethanol (85∶15 v/v) containing trifluoroacetic acid (0.05% v/v) at a flow rate of 1.0 mL min⁻¹. Under these conditions the enantiomers eluted with separation and resolution factors of 1.43 and 2.46 respectively. Semipreparative isolation of the enantiomers and their characterization by circular dichroism spectroscopy and NMR, in the presence of a chiral shift reagent, indicated that the elution order was (−)-(S)- before (+)-(R)-sulindac. The enantiomeric composition of sulindac in urine following administration of the racemic drug to man was determined by sequential achiral-chiral chromatography. Achiral analysis was carried out using a Spherisorb S5 ODS2 stationary phase (5 μm, 250×4.6 mm) and a mobile phase of aqueous acetic acid (2% v/v; pH 3.5): acetonitrile: THF (50∶48∶2 by volume) at a flow rate of 1.0 mL min⁻¹. The HPLC eluate containing sulindac (retention time 4.9 min) was collected and following workup, the enantiomeric composition of the drug was determined using the CSP. Over the 24 h collection period sulindac was excreted predominantly as theR-enantiomer, but the enantiomeric composition was found to vary markedly with time which is presumably associated with the complex metabolism of the drug.     

Separation and Quantification of Enkephalin and Vasopressin Related Peptides in Reversed-Phase Capillary Liquid Chromatography

A method for determination of some biologically active penta- and nona-peptides under isocratic conditions in capillary liquid chromatography was developed. Separation system consisting of XTerra C18 stationary phase and mobile phase composed of a mixture of acetonitrile with 0.1% trifluoroacetic acid (TFA) and water with 0.1% TFA in the ratios 75/25 (v/v) and 85/15 (v/v) was suitable not only for a good resolution of enkephalin and vasopressin related peptides, respectively, but it also enabled separation of the respective biopeptides from other constituents of human urine. Calibration curves for the studied peptides were linear in the measured concentration range from 1.00 to 1.57×10^?2 mg mL^?1. The limit of detection and limit of quantification were in the range of units of μg mL^?1 and tens of μg mL^?1, respectively; slightly higher values were obtained for nonapeptides. Determination of certain biologically active peptides in urine can serve in future as a tool for diagnosis of various diseases, e.g. autism.     

Selective Detection of Peptide-Oligonucleotide Heteroconjugates Utilizing Capillary HPLC-ICPMS

A method for the selective detection and quantification of peptide:oligonucleotide heteroconjugates, such as those generated by protein:nucleic acid cross-links, using capillary reversed-phase high performance liquid chromatography (cap-RPHPLC) coupled with inductively coupled plasma mass spectrometry detection (ICPMS) is described. The selective detection of phosphorus as ³¹P⁺, the only natural isotope, in peptide-oligonucleotide heteroconjugates is enabled by the elemental detection capabilities of the ICPMS. Mobile phase conditions that allow separation of heteroconjugates while maintaining ICPMS compatibility were investigated. We found that trifluoroacetic acid (TFA) mobile phases, used in conventional peptide separations, and hexafluoroisopropanol/triethylamine (HFIP/TEA) mobile phases, used in conventional oligonucleotide separations, both are compatible with ICPMS and enable heteroconjugate separation. The TFA-based separations yielded limits of detection (LOD) of ~40 ppb phosphorus, which is nearly seven times lower than the LOD for HFIP/TEA-based separations. Using the TFA mobile phase, 1–2 pmol of a model heteroconjugate were routinely separated and detected by this optimized capLC-ICPMS method.     

Inductively coupled plasma atomic emission spectroscopic determination of rare earth elements in geological samples after preconcentration by countercurrent chromatography—Part II

This paper directly links up with Part I [Spectrochim. Acta 48B, 1365 (1993)] which treats the first application of countercurrent chromatography (CCC) for pre-separation of rare earth elements (REE) in rocks. The rapid and reliable separation and pre-concentration of “light” REE and Y can be achieved using a system of 0.5 mol/l di-2-ethylhexylphosphoric acid (D2EHPA) in n-decane-hydrochloric acid of different concentrations and a planetary centrifuge as a CCC device. However, Tm, Yb and Lu are partially retained in the stationary phase. Comparative data is presented on three other two-phase liquid systems containing trioctylphosphine oxide (TOPO); D2EHPA and TOPO mixtures and diphenyl(dibutylcarbamoylmethylphosphine)oxide (Ph₂-Bu₂) as extractants in terms of their ability for whole REE group complete isolation from the rock constituents. The partial losses of “light” REE (La and Ce) occurred in the system of 0.1 mol/l solution of TOPO in isobutylmethylketone (IBMK) (stationary phase)-1 mol/l NH₄NO₃-6 mol/l HCl aqueous solutions (mobile phase). Complete isolution of the entire REE group can be reached in two systems: 0.3 mol/l D2EHPA + 0.02 ml/l TOPO in the solvents mixture (3:1) of n-decane + IBMK, respectively (stationary phase)-1 mol/l NH₄NO₃-6 mol/l HCl aqueous solution (mobile phase), and 1.0 mol/l Ph₂-Bu₂ solution in chloroform (stationary phase)-3 mol/l HNO₃ aqueous solution (mobile phase). The D2EHPA + TOPO mixture is recommended as more economic and accessible.