Highly sensitive indirect photometric detection of cations by capillary electrophoresis with the cationic dye chrysoidine

The cationic dye, chrysoidine, has been used for the first time as a probe for the indirect photometric detection of cations. The dye has been used as a probe at concentrations of 5 mM, which is roughly an order of magnitude higher than for other cationic dyes used previously for the same purpose, in order to minimise electromigrational dispersion. Baseline instability was minimised by a combination of coating the capillary with poly(ethyleneimine), addition of a neutral polymer to the electrolyte, and the application of a small amount (20 mbar) of hydrodynamic pressure during the separation. Separation of a mixture containing alkali metals, alkaline earths, transition metals and lanthanides was achieved by the addition of 2-hydroxyisobutyric and lactic acid as complexing agents. Excellent peak shapes were observed over a wide range of analyte mobilities due to the moderate mobility of the probe. The high absorptivity (26733 l mol(-1) cm(-1)) provided by chrysoidine in comparison with typically used, less absorbing probes, was reflected in limits of detection which were typically less than 0.5 microM. These are amongst the lowest reported using hydrodynamic injection without the use of large volume stacking methods. The use of 2-hydroxyisobutyric and lactic acids as complexing agents at pH values close to their pKa values provided suitable buffering which was highlighted by very good reproducibility of migration time, corrected peak area and peak height.     

邻苯二甲酸为背景吸收电解质的毛细管电泳法测定低分子量肝素中的阴离子

在生产和贮存低分子量肝素的过程中,糖链上的硫酸酯基团会被水解而损失活性,因此肝素样品中常可以检测到游离的硫酸根离子,此外在生产过程中还会引入其他阴离子。为了检测低分子量肝素的质量稳定性,常用离子色谱检测低分子量肝素中游离的阴离子。但是相对分子质量较大的低分子量肝素会污染离子色谱柱和抑制器。为此发展了一种灵敏的毛细管电泳方法用于测定低分子量肝素中游离的SO~(2-)_4、Cl~-、F~-、PO~(3-)_4和OAc~-。不同于常用的背景吸收离子铬酸根,采用邻苯二甲酸根作为背景吸收电解质。与铬酸根相比,邻苯二甲酸根与所有待测阴离子电泳淌度匹配得更好,因此可以获得较窄的峰形。而且邻苯二甲酸根在230 nm检测波长下的摩尔吸光系数(4 754 L/(mol·cm))比铬酸根(254 nm,2 400 L/(mol·cm))高。因此,可以将毛细管电泳检测阴离子的灵敏度提高到与离子色谱法相当的水平。通过验证,该方法在0.002~1 mmol/L的浓度范围内具有较好的线性关系,日内(n=6)和日间(n=3)迁移时间和峰面积的相对标准偏差均小于3%。所测阴离子的检出限(S/N=3)和定量限(S/N=10)分别为0.4~0.8μmol/L和2~4μmol/L。该方法可用于监测低分子量肝素的稳定性。     

Sensitive indirect photometric detection of inorganic and small organic anions by capillary electrophoresis using Orange G as a probe ion

This study addresses the two major problems in the use of dyes as highly absorbing probes for indirect photometric detection in capillary electrophoresis (CE). First, effective electroosmotic flow (EOF) modification or suppression to allow separation and detection of a wide mobility range of analytes is not straightforward when electrolytes containing increased dye concentrations are used. The suppression of EOF to less than + 5x10(-9) m(2)V(-1)s(-1) was achieved with a combination of a poly(ethylenimine) (PEI)-coated capillary and the addition of the neutral polymer hydroxypropylmethylcellulose (HPMC) to the background electrolyte. Second, the deterioration of baselines due to adsorption of the dye probe to the capillary wall is generally a problem. In this work, baseline quality at higher probe concentrations was significantly improved by a rather unusual but highly effective combination of a simultaneous application of a slight overpressure (25 mbar) at the injection end during the separation, and the use of a relatively narrow capillary of 50 microm inner diameter. Both measures would appear to be counterproductive. Optimisation of the probe concentration with regard to signal-to-noise ratio resulted in an electrolyte of 4 mM Orange G, 0.05% HPMC buffered at pH 7.7 by the addition of 10.0 mM histidine isoelectric buffer. Very high separation efficiencies of 128 000-297 000 plates were made possible by the relatively high probe concentration. Combined with excellent detection sensitivity, even with the introduction of hydrodynamic flow and a reduced optical path length, these measures resulted in limits of detection ranging from 0.216 to 0.912 microM with a deuterium lamp light source (248 nm) and from 0.147 to 0.834 microM with a 476 nm blue light-emitting diode (LED) light source. Reproducibility over 30 consecutive runs without changing the electrolyte was excellent, with relative standard deviation (RSD) values of 0.14-0.80% for migration time, 1.27-3.36% for peak area and 0.88-5.12% for peak heights. The optimised electrolyte was used for the analysis of inorganic anions in air filter samples, providing good agreement with results obtained by ion chromatography.     

Indirect spectrophotometric detection of inorganic anions in ion-exchange capillary electrochromatography

The application of indirect spectrophotometric detection was investigated for a capillary electrochromatographic system in which an anion-exchange stationary phase (in the form of aminated latex particles) was coated onto the wall of a fused-silica capillary. The study has focused on the choice of the type and concentration of the absorbing coion (probe) added to the background electrolyte and the role of this species in manipulating the ion-exchange contributions to the separation with a view to controlling the selectivity of the separation. Common inorganic anions were used as analytes and nitrate, p-toluenesulfonate, nicotinate, and chromate were investigated as probes. It was found that most of these probes produced only a limited range of separation selectivities when their concentration was varied over the practically accessible range. p-Toluenesulfonate provided the greatest variation in selectivity, but peak distortion due to electromigration dispersion was evident for the faster ions. When variation of the separation selectivity - from predominantly electrophoretic in nature to predominantly ion-exchange in nature - was desired, this was best achieved by varying the type of probe rather than its concentration. For example, the nitrate probe provided predominantly electrophoretic separations with good peak shapes and high efficiencies. A comprehensive list of probes, ranked in order of ion-exchange selectivity coefficients determined by ion chromatography, was compiled and this proved to be a useful tool to assist in the selection of a probe for a desired separation selectivity. The limits of detection for the analytes and probes studied ranged from 20-55 micromol for the chromate system to 230-600 micromol for the nicotinate system, with nitrate and p-toluenesulfonate giving intermediate values.     

Brompheniramine as a novel probe for indirect UV detection and its application for the capillary electrophoresis of adamantane drugs

Brompheniramine, an antihistamine drug, was employed as a novel UV probe for capillary electrophoresis with indirect UV detection of adamantane drugs (memantine, amantadine, and rimantadine). The probe possesses high molar absorptivity of 24 × 10³ L/mol cm at 6 mM, which enables the measurement of these nonchromophore analytes without derivatization. The simple background electrolyte (10 mM sodium dihydrogen phosphate (pH 5.0) containing 5 mM brompheniramine and 6 mM β‐cyclodextrin) provided the separation of the analytes in a short time (7.5 min). Under these conditions, brompheniramine had similar mobility to that of the analyte ions resulting in symmetric peaks with minimal electrodispersion. The analytes displace the probe at a one‐to‐one ratio with transfer values close to unity. β‐Cyclodextrin played a role in the resolution of the structurally similar adamantane derivatives. Method validation showed good linearity (r ² > 0.98), precision (%RSD ≤ 3.30), and accuracy (recoveries ranging from 98 to 109%). The proposed method was successfully applied to determine the adamantane content in pharmaceutical products.     

Development of a gas diffusion probe for the rapid measurement of pCO2 in aquatic samples

A capillary electrophoresis (CE) procedure with contactless conductivity detection (C(4)D) has been developed for monitoring of neutral mono- and disaccharides in drinks and foodstuffs. The separation of a mixture of seven neutral saccharides (glucose, fructose, galactose, mannose, ribose, sucrose and lactose) employed a quartz capillary, 5 μm i.d., with an effective length of 18.3 cm, and 75 mM NaOH (pH 12.8) as the background electrolyte (BGE). The limit of detection (LOD) values obtained lied within a range from 0.4 μmol L(-1) for lactose to 0.9 μmol L(-1) for ribose, with a separation time shorter than 140 s. The procedure was successfully applied to determinations of saccharides in fruit juices, Coca-Cola, milk, red and white wines, yoghurts, honey and a foodstuff additive.     

Determination of egg white lysozyme by on-line coupled capillary isotachophoresis with capillary zone electrophoresis

An on-line coupled capillary isotachophoresis - capillary zone electrophoresis method for the determination of lysozyme in selected food products is described. The optimized electrolyte system consisted of 10 mM NH(4)OH + 20 mM acetic acid (leading electrolyte), 5 mM epsilon -aminocaproic acid +5 mM acetic acid (terminating electrolyte), and 20 mM epsilon -aminocaproic acid +5 mM acetic acid +0.1% m/v hydroxypropylmethylcellulose (background electrolyte). A clear separation of lysozyme from other components of acidic sample extract was achieved within 15 min. Method characteristics, i.e., linearity (0-50 micrograms/mL), accuracy (recovery 96+/-5%), intra-assay (3.8%), quantification limit (1 microgram/ml), and detection limit (0.25 microgram/mL) were determined. Low laboriousness, sufficient sensitivity and low running costs are important attributes of this method. The developed method is suitable for the quantification of the egg content in egg pasta.     

Electrophoretic behavior of adamantane derivatives possessing antiviral activity and their determination by capillary zone electrophoresis with indirect detection

Separation and determination of adamantane derivatives with antiviral activity, namely amantadine (1-adamantan amine), memantine (1-amino-3,5-dimethyl adamantane) and rimantadine (alpha-methyl-1-adamantane methylamine), were examined by capillary zone electrophoresis. After optimization, an indirect detection method using 5 mM 4-methylbenzylamine in ethanol/water solution (1:4) as simultaneously absorbing and buffering background electrolyte with detection at 210 nm was found suitable for determination of the individual compounds (limit of detection was 0.35 mg L(-1) for memantine hydrochloride, S/N = 3). Baseline separation of all the three compounds was reached by addition of alpha- or beta-cyclodextrins to the electrolyte in concentrations of 20 and 2 mM, respectively.     

The utility of infrared spectroscopy for quantitative conformational analysis at a single temperature

The carbonyl stretching vibration of 2-bromocyclohexanone (1) has been measured in a variety of solvents. It is shown that its component intensities are not only dependent on the populations of the axial and equatorial conformers, but are also dependent on the molar absorptivities (epsilon ) which are specific for each conformer in each solvent. In CCl(4), the axial and equatorial conformers have epsilon values of 417 and 818 l mol(-1) x cm(-1), respectively, while in CH(3)CN solution, the values were 664 and 293 l mol(-1) x cm(-1). These results are supported by results of theoretical calculations of frequencies, which gave an intensity of 223.8 kM mol(-1) x(1782 cm(-1)) for the axial and 174.4 kM mol(-1) x (1802 cm(-1)) for the equatorial conformer, indicating that the axial conformer presents a larger molar absorptivity than the equatorial one in the vapor phase. Moreover, the results presented here clearly demonstrate that although infrared spectroscopy at a single temperature can be an important auxiliary technique for conformational analysis, it must not be used to quantify conformational preferences of a molecule if the absorption molar coefficients for each conformer are not known or not amenable to experimental determination.     

Study of enzymatic reaction by electrophoretically mediated microanalysis in a partially filled capillary with indirect or direct detection

Electrophoretically mediated microanalysis (EMMA), in combination with a partial filling technique and indirect or direct detection, is described for the study of enzymes reacting with the high mobility inorganic or organic anions as substrates or products. Part of the capillary is filled with a buffer optimized for the enzymatic reaction, the rest of the capillary with the background electrolyte being optimal for the separation of substrates and products. With haloalkane dehalogenase, chosen as a model enzyme, the enzymatic reaction was performed in a 20 mM glycine buffer (pH 8.6). Because of the wide substrate specificity of this enzyme, utilizing chlorinated as well as brominated substrates and producing either nonabsorbing chloride or absorbing bromide ions, two different background electrolytes and detection approaches were adopted. A 10 mM chromate-0.1 mM cetyltrimethylammonium bromide background electrolyte (pH 9.2) was used in combination with indirect detection and 20 mM beta-alanine-hydrochloric acid (pH 3.5) in combination with direct detection. The Michaelis constant (K(m)) of haloalkane dehalogenase for 1-bromobutane was determined. The K(m) values 0.59 mM estimated by means of indirect detection method and 0.17 mM by means of direct detection method were comparable with the value 0.13 mM estimated previously by gas chromatography.     

Highly sensitive determination of lanthanides by capillary electrophoresis with direct visible detection after precapillary complexation with aromatic polyaminocarboxylate and additionally applying dynamic ternary complexation with nitrilotriacetic acid

A newly synthesized aromatic polyaminocarboxylate (NBD-ABEDTA, H(4)L) was applied to precapillary derivatizing capillary electrophoresis as a chelating reagent for lanthanide ions (Ln(3+)). The Ln-L complexes provide both kinetic stability on dissociation due to their methyl-EDTA coordinating structure, and high light absorptivity (epsilon(max) = 2.4 x 10(4) cm(-1) mol(-1) dm(3)) in the visible region at 469 nm thanks to their nitrobenzofurazan moiety. A ligand was employed for capillary zone electrophoresis based on a unique concept: both precapillary and dynamic on-capillary complexation were carried out on one center-metal ion to achieve high resolution. As a ternary complex-formation agent, iminodiacetate (IDA), bound to the mother complex (Ln-L), was added to the carrier buffer solution. The carrier buffer solution of 9.5 mmol.dm(-3) (pH 9.45) borate and 33.5 mmol.dm(-3) IDA, drastically improved the resolution among Ln(3+) ions. Each of the Ln complexes was effectively separated, except for Pr-Sm. Furthermore, the absence of L from the carrier solution, which stabilizes the baseline fluctuation, provided low LOD (typically 4.2 x 10(-7) mol.dm(-3)). This strongly suggests that Ln-L complexes are kinetically stable even with a large excess of IDA. Quite unexpectedly, the order of migration differs from that of the atomic number, inverting at Nd. This is due to the effect of the cavity size of the residual coordination sites on the ternary complexation and the electronic density of Ln(3+).     

Indirect photomeric detection of anions in capillary electrophoresis using dyes as probes and electrolytes buffered with an isoelectric ampholyte

The use of highly absorbing anionic dyes as probes and isoelectric ampholytes as buffers in background electrolytes (BGEs) combined with the use of a light emitting diode (LED) as a light source has been studied for ultrasensitive indirect photometric detection in capillary electrophoresis (CE). Potential dyes and buffers were evaluated based on characteristics relevant to indirect photometric detection principles, such as the electrophoretic mobility of the probe dye, its solubility and adsorption behaviour, and the isoelectric point and buffering capacity of the ampholytic buffer. Two dyes, tartrazine and naphthol yellow S, and histidine as the ampholytic buffer, were selected for detailed investigation. Purification of the probes was vital to avoid anionic impurities interfering with the detection. For the electrolytes containing a purified probe (0.5 mM) and histidine as the isoelectric buffer (p/ 7.7), hydroxypropylmethyl cellulose (approximately 0.05%) was effective in suppression of the electroosmotic flow (EOF). Analytical method performance characteristics were determined. For both probes, experimentally determined mobilities were generally close to literature values, excellent peak shapes and separation efficiencies of up to 298 000 theoretical plates were obtained, and detection limits were generally at the sub-microM level. For the naphthol yellow S-histidine BGE, linearity and reproducibility were also evaluated, with excellent linearity being observed over a range of 5-500 microM, and reproducibility (relative standard deviation, RSD) less than 1% for migration times and 2-8% for normalised peak areas. The approach developed was applied successfully to several real samples including tap water, mineral waters, and beer.     

Determination of trace amount of oxalic acid with zirconium(IV)-(DBS-arsenazo) by spectrophotometry

A novel method is proposed for the determination of trace amount of oxalic acid in the present article. In 1.0M hydrochloric acid medium, oxalic acid can react with the zirconium(IV) in Zr(IV)-(DBS-arsenazo) complex and replaces the DBS-arsenazo to produce a hyperchromic effect at 520 nm. The hyperchromic degree is proportional to the concentration of the oxalic acid added over a defined range. Based on this property, a new method for the spectrophotometric determination of trace oxalic acid was developed. Beer's law is held over the concentration range of 9.0 x 10(-6) to 5.0 x 10(-4)M for oxalic acid with a correlation coefficient of 0.9995. The apparent molar absorptivity of the method is epsilon520 nm = 1.16 x 10(3)L mol(-1)cm(-1) and the detection limit for oxalic acid is 0.815 microg/mL. The developed method was directly applied to the determination of oxalic acid in tomato samples with satisfactory results.     

毛细管区带电泳法测定葡萄籽中儿茶素类化合物

 采用毛细管区带电泳法测定了 10种中国产葡萄籽中的 4个主要儿茶素类化合物 :(+)儿茶素、(- )表儿茶素、(± )表没食子儿茶素、(± )表儿茶素没食子酸酯的含量。在 0 0 2mol/L硼砂和 0 0 0 5mol/L磷酸盐的混合缓冲体系 (pH 10 0 )的背景缓冲液中 ,4个化合物在 10min内取得了令人满意的分离。迁移时间的重现性(RSD)小于 2 % ,峰面积的重现性 (RSD)小于 5 %。在质量浓度为 0 0 0 5g/L～ 0 5 g/L时 ,线性相关系数大于0 995。检测限为 3mg/L～ 10mg/L。该方法简单、快速、准确 ,可作为葡萄籽分析和药用开发过程中分析儿茶素类化合物的有效方法推广使用。     

Identification of inorganic ions in post‐blast explosive residues using portable CE instrumentation and capacitively coupled contactless conductivity detection

Novel CE methods have been developed on portable instrumentation adapted to accommodate a capacitively coupled contactless conductivity detector for the separation and sensitive detection of inorganic anions and cations in post‐blast explosive residues from homemade inorganic explosive devices. The methods presented combine sensitivity and speed of analysis for the wide range of inorganic ions used in this study. Separate methods were employed for the separation of anions and cations. The anion separation method utilised a low conductivity 70 mM Tris/70 mM CHES aqueous electrolyte (pH 8.6) with a 90 cm capillary coated with hexadimethrine bromide to reverse the EOF. Fifteen anions could be baseline separated in 7 min with detection limits in the range 27–240 μg/L. A selection of ten anions deemed most important in this application could be separated in 45 s on a shorter capillary (30.6 cm) using the same electrolyte. The cation separation method was performed on a 73 cm length of fused‐silica capillary using an electrolyte system composed of 10 mM histidine and 50 mM acetic acid, at pH 4.2. The addition of the complexants, 1 mM hydroxyisobutyric acid and 0.7 mM 18‐crown‐6 ether, enhanced selectivity and allowed the separation of eleven inorganic cations in under 7 min with detection limits in the range 31–240 μg/L. The developed methods were successfully field tested on post‐blast residues obtained from the controlled detonation of homemade explosive devices. Results were verified using ion chromatographic analyses of the same samples.     

双波长叠加吸收光谱法测定药物中的酒石酸美托洛尔

建立了快速、准确测定药物中酒石酸美托洛尔的双波长叠加可见吸收光谱法。在pH 4.55的酸性Tris-盐酸介质及586~740 nm波长范围内,偶氮氯膦Ⅲ与酒石酸美托洛尔反应生成具有两个明显正吸收峰的离子缔合物,最大正吸收波长位于614 nm,次大正吸收波长位于664 nm,表观摩尔吸光系数(κ)分别为6.03×10~4L/(mol·cm)(614 nm)和5.37×10~4L/(mol·cm)(664 nm),酒石酸美托洛尔的质量浓度在0.2~8.6 mg/L范围内服从朗伯-比尔定律,检出限为0.13 mg/L(614 nm)和0.15 mg/L(664 nm)。当采用双波长叠加法测定时,其表观摩尔吸光系数(κ)可达1.14×10~5L/(mol·cm),检出限为0.072 mg/L。该文同时探讨了显色反应的适宜条件、共存物质的影响及吸收光谱特征。实验发现,该反应体系的单波长及双波长叠加吸收光谱法的表观摩尔吸光系数可达5.37×10~4~1.14×10~5L/(mol·cm),方法可用于市售药物中酒石酸美托洛尔含量的测定,加标回收率为98.0%~101%,相对标准偏差(n=6)为1.8%~2.3%。     

On-column complexation and simultaneous separation of vanadium(IV) and vanadium(V) by capillary electrophoresis with direct UV detection

An on-column complexation method has been developed for the simultaneous determination of V(IV) and V(V). Vanadium species were chelated with aminopolycarboxylic acids to form anionic complexes which were separated by capillary zone electrophoresis (CZE) with direct UV detection. Ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentacetric acid (DTPA), nitrilotriacetic acid (NTA), and N-2-hydroxyethylethlendiaminetriacetric acid (HEDTA) were investigated as both ligand and running electrolyte. Of the ligands studied the complexes of EDTA with V(IV) and V(V) resulted in the highest selectivity and UV response.The conditions used for on-column complexation and separation, including pH, and electrolyte ligand concentration, were examined to achieve reasonable separation selectivity and detection sensitivity. The optimum separation of the anionic forms of V(IV) and V(V) was obtained by use of CZE with UV detection at 185 nm and an electrolyte containing 5 mmol L(-1) EDTA at pH 4.0. Linear calibration plots were obtained in the concentration range10-300 micro mol L(-1); detection limits were 3 micro mol L(-1) for V(IV) and 1 micro mol L(-1) for V(V). The proposed method was demonstrated for the determination of vanadium in groundwater spiked with V(IV) and V(V).     

Development of a fully buffered molybdate electrolyte for capillary electrophoresis with indirect detection and its use for analysis of anions in Bayer liquor

A new counterion-buffered molybdate electrolyte was developed and optimized for simultaneous quantitative determination of up to eight anions (chloride, sulphate, oxalate, fluoride, formate, malonate, succinate, and acetate) in Bayer liquor by capillary electrophoresis with indirect detection at 214 nm. The separation parameters were optimized in respect to separation of the critical analyte group fluoride-formate-malonate, with the optimal electrolyte prepared from molybdic trioxide containing 5.0 mmol/L MoO3, 1.3 mmol/L cetyltrimethylammonium bromide (CTAB), and buffered with diethanolamine (DEA) to pH 9.2 (ca. 20 mM DEA). Total length of separation capillary was 80 cm, resulting in run time of under 4 min. The method is suitable for a wide concentration range of the analytes (1-50 mg/L) with linear calibration plots (R2 = 0.9983-0.9999). Relative standard deviations were 0.05%-0.07% for migration times and 0.67%-2.04% for peak areas. The detection limits were in the range of 0.17-0.51 mg/L or 2-10 micromol/L (hydrostatic injection of 30 s of 1000 x diluted sample). Due to its good buffering capacity, the electrolyte exhibited an excellent ruggedness and good tolerance to the alkaline samples. Consequently, Bayer liquor samples could be diluted as little as 100 x which allows more sensitive determination of minor components over previous methods. The method was successfully applied to analysis of Bayer liquor samples with recoveries in the range of 95-105%.     

Determination of anthracycline antibiotics doxorubicin and daunorubicin by capillary electrophoresis with UV absorption detection.

Sweeping preconcentration and electrokinetic injection was used for the capillary electrophoretic analysis of trace amounts of biologically active anthracyclines with UV absorption detection. Phosphate buffer (100 mM), pH 2.5, with addition of 40% v/v methanol was used as background electrolyte (BGE). Sodium dodecyl sulfate (150 mM) was added to BGE in the inlet vial as the sweeping agent. The system enables effective separation of anthracyclines as well as cleanup from matrix impurities. Sweeping preconcentration of sample provides an excellent detection limit (1 x 10(-9) mol L(-1)). The method was applied for the determination of therapeutic levels of doxorubicin in real plasma samples.     

Determination of hesperidin and synephrine in Pericarpium Citri Reticulatae by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection (CE-ED) has been developed for the determination of hesperidin (HP) and synephrine (SP) in the Chinese traditional herbal drug, Pericarpium Citri Reticulatae, the dried rind of the ripe fruits of Citrus reticulata Blanco (mandarin orange). The effects of some important factors such as the acidity and concentration of running buffer, separation voltage, and detection potential were investigated to determine the optimum conditions. The working electrode was a 300 microm diameter carbon disc electrode positioned opposite the outlet of the capillary. Both analytes could be well separated within 5 min in a 40 cm long capillary at a separation voltage of 12 kV in 50 mmol L(-1) borate buffer (pH 9.0). Excellent linearity was observed for the dependence of peak current on analyte concentration in the range from 2.5 x 10(-6) to 1.0 x 10(-3) mol L(-1) for SP and from 5.0 x 10(-6) to 1.0 x 10(-3) mol L(-1) for HP. The detection limits (S/N=3) for SP and HP were 4.96 x 10(-7) mol L(-1) and 6.54 x 10(-7) mol L(-1), respectively. This method has been successfully applied for the analysis of real samples, with satisfactory results.