Capillary Zone Electrophoresis Technique Research Articles

High separation efficiency of gold nanomaterials of different aspect ratio and size using capillary transient isotachophoresis

Two modes of capillary electrophoresis (CE), capillary zone electrophoresis (CZE) and capillary transient isotachophoresis (ctITP), were compared for the detection and separation of spherical gold nanoparticles (AuNPs) and gold nanorods (AuNRs). The development of ctITP using two different leading ions is described. Overall, when compared to traditional capillary zone electrophoresis (CZE), ctITP resulted in improved peak shape and peak efficiency. Specifically, the number of theoretical plates for AuNR samples increased by a factor of 2–2.5 depending on the choice of leading ion. Further, using ctITP two AuNRs differing by aspect ratio were baseline resolved, whereas the same AuNRs could not be separated using CZE or other techniques like single particle inductively coupled plasma mass spectrometry (spICP-MS) and asymmetric flow field-flow fractionation (AF4). The results of this study demonstrate that ctITP is an efficient on-line technique for the improved detection and separation of gold nanomaterials in CE.

The influence of different pH on the electrophoretic behaviour of Saccharomyces cerevisiae modified by calcium ions

The effect of a different pH on Saccharomyces cerevisiae cells modified with calcium ions was investigated by the capillary zone electrophoresis technique. For the identification of the wild strain of S. cerevisiae, the ribosomal nucleic acid sequencing and internal transcribed spacer sequencing as well as spectrometric approach were applied. The potentiometric titration and Fourier transform infrared spectroscopy have shown the occurrence of active functional groups such as carboxyl, amine/hydroxyl, phosphate/hydrogen phosphate groups on the surface of native yeast cells. Moreover, the spectroscopy study in a medium infrared range was carried out to identify the functional groups of yeast cells that participate in calcium ions binding interaction. Furthermore, the microscopic and spectrometric analysis shows that the pH value of the calcium ions solution has a significant effect on the intensity yeast cells clumping. Additionally, the impact of yeast cell clumping on the electrophoretic behaviours was examined. The modification of surface functional groups by calcium ions significantly affected the efficiency of electrophoretic separation. However, these changes did not affect the accuracy of S. cerevisiae identification by MALDI equipment with BioTyper platform. These results form the analytical solution for coupling of electrophoresis and MALDI-TOF MS technique.

Multivariate optimization of a capillary zone electrophoresis assay method for simultaneous quantification of metformin and vildagliptin from a formulation

ABSTRACTA rapid, accurate, precise, and optimized capillary zone electrophoresis assay was established and validated for the simultaneous quantification of metformin and vildagliptin in tablets. The electrophoretic separation was achieved on an untreated bonded silica capillary with a background electrolyte comprising 25 mM of borate buffer at pH 7.5 at 207 nm. The concentration of the buffer and the pH of BGE were optimized using the multivariate optimization method for determining the retention time and peak area. Furthermore, the sample injection time, capillary oven temperature, and applied voltage were optimized. The capillary zone electrophoresis technique was validated for all required parameters as per the International Conference on Harmonization recommendations. The linearity ranged in the concentrations of 5–500 µg/mL and 5–100 µg/mL with the limit of detections of 0.22 µg/mL and 0.40 µg/mL for metformin and vildagliptin, respectively. In addition, the percent relative standard error for repeatability and inter-day precision was within the acceptable range. The mean recoveries determined by the capillary zone electrophoresis method were 99.2% and 100.4% for metformin and vildagliptin, respectively. Finally, the capillary zone electrophoresis process was effectively used for the assays of metformin and vildagliptin in their solid dosage form, and statistical outcomes were in agreement with the outcomes of the previously validated RP-HPLC method.

The sensitive capillary electrophoretic-LIF method for simultaneous determination of curcuminoids in turmeric by enhancing fluorescence intensities of molecules upon inclusion into (2-hydroxypropyl)-β-cyclodextrin.

Curcuminoids have received great attention in the past decades due to their health benefit properties. The aim of this study is to develop a very simple, rapid, and sensitive capillary zone electrophoresis technique coupled with a laser induced fluorescence detector (LIF) for the simultaneous determination of three major curcuminoids of turmeric, namely, curcumin, demethoxy curcumin (DMC), and bisdemethoxy curcumin (BDMC). Background electrolyte was selected as borate at pH 9.6 and (2-hydroxypropyl)-β-cyclodextrin (2-HP-β-CD) was added to prevent rapid alkali degradation of curcuminoids in buffer and to increase fluorescence intensities of molecules. With the addition of 2-HP-β-CD to the separation electrolyte, the fluorescence signal intensities of curcuminoids were enhanced considerably by 30, 40, and 54 fold for curcumin, DMC, and BDMC, respectively. The three curcuminoids of turmeric were fully separated and quantified in less than 4.5 min. The repeatability of the peak areas of curcuminoids for intra-day and inter-day experiments was in the satisfactory range of 2.26 and 2.55%, respectively. The LOD and LOQ values for the developed method were equal to or less than 0.081 and 0.270 μg/mL, respectively, for all curcuminoids. The developed method was successfully applied to find curcuminoids amount in turmeric samples and herbal supplements.

A rare haemoglobin-variant produces discrepant results between haemoglobinopathy screening techniques

The validity of a haemoglobinopathy diagnostic technique is dependent on several factors including the sensitivity and specificity of the methodology used. We describe a case of a 69-year-old man who was referred for investigation of a chronic, mild normocytic anaemia. Initial investigations by a private, interstate pathology service reported a fast-migrating haemoglobin-variant of over 40% in Zone 11 using a capillary zone electrophoresis technique (Sebia Capillarys). However, confirmatory testing in a major haemoglobinopathy screening centre failed to identify the presence of a haemoglobin-variant using a high performance liquid chromatography technique (HPLC; Biorad Variant II) in conjunction with agarose gel at acid pH and cellulose acetate at alkaline pH haemoglobin electrophoresis techniques. Extensive investigation of pre-analytic variables and laboratory variables ensured the accuracy of the discrepant results reported by both laboratories. Subsequent molecular sequencing identified the pathognomonic amino acid substitution of Arginine (AGG) for Serine (AGT) at codon 30 of the β haemoglobin gene consistent with haemoglobin Tacoma. With nearly 70% of the laboratories participating in the RCPA 2013 Haemoglobinopathy Program using HPLC techniques, this case suggests that some haemoglobin-variants will remain undiagnosed due to limitations in the sensitivity of the methodology used. The validity of a haemoglobinopathy diagnostic technique is dependent on several factors including the sensitivity and specificity of the methodology used. We describe a case of a 69-year-old man who was referred for investigation of a chronic, mild normocytic anaemia. Initial investigations by a private, interstate pathology service reported a fast-migrating haemoglobin-variant of over 40% in Zone 11 using a capillary zone electrophoresis technique (Sebia Capillarys). However, confirmatory testing in a major haemoglobinopathy screening centre failed to identify the presence of a haemoglobin-variant using a high performance liquid chromatography technique (HPLC; Biorad Variant II) in conjunction with agarose gel at acid pH and cellulose acetate at alkaline pH haemoglobin electrophoresis techniques. Extensive investigation of pre-analytic variables and laboratory variables ensured the accuracy of the discrepant results reported by both laboratories. Subsequent molecular sequencing identified the pathognomonic amino acid substitution of Arginine (AGG) for Serine (AGT) at codon 30 of the β haemoglobin gene consistent with haemoglobin Tacoma. With nearly 70% of the laboratories participating in the RCPA 2013 Haemoglobinopathy Program using HPLC techniques, this case suggests that some haemoglobin-variants will remain undiagnosed due to limitations in the sensitivity of the methodology used.

Development and Optimization of a Capillary Zone Electrophoresis Technique for Simultaneous Determination of Miconazole Nitrate and Hydrocortisone Acetate in a Cream Pharmaceutical Formulation

A simple, fast, inexpensive, and reliable capillary zone electrophoresis (CZE) method for the determination of a mixture of miconazole nitrate (MCZ) and hydrocortisone acetate (HCZ) in a cream formulation has been developed and validated. Optimum conditions were sodium dihydrogen phosphate buffer (50 mM, pH 4) and 30 kV applied voltage in a 85 cm x 75 pm id capillary. Direct UV detection at 230 nm led to adequate sensitivity without interference from the sample excipients. MCZ and HCZ migrated in approximately 165 and 415 s, respectively. The analytical curves had a coefficient of correlation, r, of 0.9999 and 0.9996 for MCZ and HCZ, respectively. The LOD and LOQ were 0.28 and 0.93 microg/mL for MCZ and 0.38 and 1.27 microg/mL for HCZ, respectively. Thus, excellent accuracy and precision were obtained. Recoveries varied from 98 to 102%, and intraday and interday precision, calculated as the RSD, were less than 2.0% for each drug. The proposed CZE method displayed advantageous performance characteristics and can be considered suitable for QC of the MCZ and HCZ cream formulation.

Determination of pKa Values of Some Benzoxazoline Derivatives and the Structure–Activity Relationship

The acid ionization constant (pKa) values of 2-(3H)-benzoxazolinone and its 17 derivatives were determined in buffered solutions by UV–vis spectrophotometry, potentiometry, and capillary zone electrophoresis techniques. The pKa values of the studied compounds are found to be in the range of 9.01 to 7.15. The advantages and limitations of each technique are discussed. The results suggest that the removal of a proton from the molecule occurred on the nitrogen atom of the 2-(3H)-benzoxazolinone ring and the analgesic/anti-inflammatory activities of the benzoxazolinone derivatives decrease when the pKa values of the compounds increase.

Capillary electrophoresis as a tool for the characterization of pentosan nanoparticles

Because capillary zone electrophoresis (CZE) showed higher resolution for highly charged large carbohydrates and complex structures when compared to other chromatographic separation methods, it was chosen for the characterization of nanoparticles (NPs) of pentosan polysulfate (PPS). Thus, using the CZE technique, we developed a reliable, sensitive and rapid protocol that allowed the detection and characterization of PPS NPs. This protocol was able to determine the profile of both the NPs and the species of PPS entrapped into them, and to quantify free and bound PPS showing high reproducibility, acceptable accuracy and a good degree of precision. Moreover, it allowed the evaluation of the size and charge of the NPs. This protocol might be suitable for the characterization of other kinds of NPs also.

Fast determination of chlorogenic acid in tobacco residues using microwave-assisted extraction and capillary zone electrophoresis technique

In this work, for the first time, capillary zone electrophoresis (CZE) technique combined with microwave-assisted extraction (MAE) was developed for the fast quantification of chlorogenic acid (CA) in tobacco residues. CA in tobacco residue samples were extracted by MAE technique, and then analyzed by CZE. As a new sample preparation method for tobacco residues, the MAE procedure is optimized, validated and compared with conventional methods including ultrasonic extraction (USE) and reflux extraction (RE). It is found that MAE gives the best result due to the highest extraction efficiency within shortest extraction time (only 4.0 min). Here, CA is determined by CZE based on the calibration curve of its authentic standard. The method linearity, detection limit, precision and recovery are studied. The results show that the combined MAE and CZE method has a linearity ( R 2 0.991, 0.003–0.5 mg ml −1), a limit of detection (0.003 mg ml −1), a limit of quantification (0.01 mg ml −1), good precision (R.S.D. = 4.28%) and a finer recovery (89.0%). The proposed method was successfully applied to the analysis of CA in tobacco residue samples. The experiment results have demonstrated that the CZE combined with MAE is a convenient, fast, economical and reliable method for the determination of CA in tobacco residues.

Analysis of galactoglucomannans from spruce wood by capillary electrophoresis

The aim of this study was to setup a method for detection and quantification of monosaccharide components in technical galactoglucomannas (T-GGM) from spruce wood using capillary zone electrophoresis (CZE). CZE technique was optimised regarding borate buffer concentrations, EOF modifier application, and system pH. Aqueous solution of T-GGM was chemically hydrolysed by sulphuric acid, in an autoclave. In this way obtained monosaccharides were derivatized with 4-amino benzoic acid ethyl ester via reductive amination using sodium cyanoborohydride. The results of the optimisation procedure showed that the borate buffers at lowest concentrations (100 and 200 mM) with acetonitrile addition as EOF modifier gave the optimal measurement results, as it showed sufficient separation at relatively short migration times. The amounts of single monosaccharide components in the T-GGM samples obtained by the optimised CZE procedure were practically the same in comparison to the results of the well established HPLC-anion exchange chromatography. On the basis of this research, it was concluded that the capillary zone electrophoresis is an efficient analytical procedure for the characterisation of galactoglucomannans derived from softwoods.

Optimization of CDTE Quantum Dots Synthesis Using Capillary Zone Electrophoresis

Water-soluble, thiol protected cadmium telluride CdTe quantum dots (QDs) exhibit size dependent excitation/emission wavelength tunability. The heating period ensuring the growth of CdTe nanocrystals capped with thioglycolic acid (CdTe@TGA) in aqueous solution is critical to reach a required/desirable diameter. Thus, reaction medium was monitored using a capillary zone electrophoresis (CZE) technique in order to select an appropriate heating time. QDs migrate as anionic species in a borate buffer pH 8.5 used as background electrolyte and they were separated according to their size (larger ones have shorter migration times), as confirmed by measuring their maximum wavelength of absorption, with on-line diode array detection. A relationship between electrophoretic mobilities and λmax was tentatively proposed. Efficiency of the purification process of crude QDs using multiple step alcohol precipitation was also confirmed with the developed CZE technique. Keywords: Quantum dots, synthesis, size-selective precipitation, capillary zone electrophoresis, diode array detection, size-dependent migration

Monitoring folding/unfolding transitions of proteins by capillary zone electrophoresis: measurement of deltaG and its variation along the pH scale.

Free-solution capillary zone electrophoresis (CZE) can be used to monitor folding/unfolding transitions of proteins and to construct the classical sigmoidal transition curve describing this isomerization process. By performing a series of CZE experiments along the pH scale (here between pH 2.5 and 6.0) it is possible to measure the parameter [urea]1/2, which represents the concentration of urea at the midpoint of each transition curve, and its dependence from the local pH value. The [urea]1/2 parameter provides an idea of the stability of the protein at a given pH; in the case of cytochrome c, for example, it shows that at and below pH 2 the protein will spontaneously unfold even in the absence of a denaturant. The equation describing the sigmoidal folding/unfolding transition can be used for deriving the term deltaG degrees, which refers to the intrinsic difference in the Gibb's free energy between the (total or partial) denatured state and the reference state, taken usually as the native configuration of a protein. The variation of deltaG degrees between the two extremes of our measurements (pH 2.5 and 6.0) along the stated pH interval has been measured (and theoretically calculated) to be of the order of 7-10 kcal/mol and is here interpreted by assuming that at pH 2.5 and below there is an additionally stretching of the polypeptide coil due to coulombic repulsion, as the unfolded chain looses its zwitterionic character and assumes a pure (or very nearly so) cationic surface. Given the minute amounts of sample required, the fully automated state of the analysis, the rapidity and ease of operation, it is hoped that the CZE technique will become more and more popular in the years to come for monitoring folding/unfolding transitions of proteins.

Determination of tetracycline and streptomycin in mixed fungicide products by capillary zone electrophoresis.

A method of capillary zone electrophoresis (CZE) was used to determine tetracycline and streptomycin content in commercial agriculture products. The results indicated that this method was capable of analyzing the mixed fungicide in formulated products with instrument detection limit (IDL) of 0.50 microg/mL and a method detection limit (MDL) of 0.52 microg/mL for tetracycline, and IDL of 1.00 microg/mL and MDL of 1.22 microg/mL for streptomycin. Precision expressed by relative standard deviation (RSD) ranged from 1.44 to 4.37% of tetracycline and 1.00 to 4.20% of streptomycin. Recoveries were in the region of 98.2-102.5% for tetracycline and 95.3--103.0% for streptomycin. The low detection limit, the low RSD values, and the high percentage of recovery confirmed that the CZE technique is a sensitive and selective method. And the CZE method can analyze both tetracycline and streptomycin at the same time without complicated extraction and further derivative reaction.

Ionization behaviour and tautomerism-dependent lipophilicity of pyridine-2(1H)-one cardiotonic agents

The acid–base properties of pyridine-2(1H)-one derivatives, analogues of the cardiotonic agent milrinone, were studied by capillary zone electrophoresis (CZE). Electrophoretic mobility and pH data were fitted to equilibrium expressions and apparent dissociation constants (pKa) calculated by non-linear regression. Compared with the ultraviolet (UV) spectrophotometric method and potentiometric titrations, the CZE technique showed advantages, such as rapidity and applicability to compounds that are sparingly soluble in water. Based on the pKa values, intramolecular electronic interactions were assessed. The lipophilicity of a number of derivatives was also examined, by determining their n-octanol/water distribution coefficients over a wide pH range, and found to be significantly affected by 2-pyridone/2-hydroxypyridine tautomerism. As revealed by a comparison between experimental and calculated log P values, electron withdrawing substituents, especially at the C(6) position of 2-pyridone, favour the less polar hydroxypyridine tautomers both in water and octanol. Our results indicate that the positive inotropism of milrinone-related compounds could be explained taking ionization and tautomerism into account.

Serum Proteins by Capillary Zone Electrophoresis: Approaches to the Definition of Reference Values

The Paragon CZE 2000 (Beckman Analytical, Milan, Italy) is an automatic dedicated capillary zone electrophoresis (CZE) system, producing a five-zone serum protein pattern with quantitative estimation of the zones. With the view of substituting this instrument for two previously used serum protein electrophoresis techniques, we planned to produce reference values for the "new" systems leading to compatible interpretation of the results. High resolution cellulose acetate electrophoresis with visual inspection and descriptive reporting (HR-CAE) and five-zone cellulose acetate electrophoresis with densitometry (CAE-D) were the previously used techniques. Serum samples (n = 167) giving "normal pattern" with HR-CAE were assayed with the CZE system, and the results were statistically assessed to yield 0.95 reference intervals. One thousand normal and pathological serum samples were then assayed with the CAE-D and the CZE techniques, and the regression equations of the CAE-D values over the CZE values for the five zones were used to transform the CAE-D reference limits into the CZE reference limits. The two sets of reference values thereby produced were in good agreement with each other and also with reference values previously reported for the CZE system. Thus, reference values for the CZE techniques permit interpretation of results coherent with the previously used techniques and reporting modes.

Serum proteins by capillary zone electrophoresis: approaches to the definition of reference values.

The Paragon CZE 2000 (Beckman Analytical, Milan, Italy) is an automatic dedicated capillary zone electrophoresis (CZE) system, producing a five-zone serum protein pattern with quantitative estimation of the zones. With the view of substituting this instrument for two previously used serum protein electrophoresis techniques, we planned to produce reference values for the "new" systems leading to compatible interpretation of the results. High resolution cellulose acetate electrophoresis with visual inspection and descriptive reporting (HR-CAE) and five-zone cellulose acetate electrophoresis with densitometry (CAE-D) were the previously used techniques. Serum samples (n = 167) giving "normal pattern" with HR-CAE were assayed with the CZE system, and the results were statistically assessed to yield 0.95 reference intervals. One thousand normal and pathological serum samples were then assayed with the CAE-D and the CZE techniques, and the regression equations of the CAE-D values over the CZE values for the five zones were used to transform the CAE-D reference limits into the CZE reference limits. The two sets of reference values thereby produced were in good agreement with each other and also with reference values previously reported for the CZE system. Thus, reference values for the CZE techniques permit interpretation of results coherent with the previously used techniques and reporting modes.

Comparison between HPLC and Capillary Electrophoresis for the Separation and Identification of the Platination Products of Oligonucleotides withcis-[Pt(NH3)2(H2O)2]2+and [Pt(NH3)3(H2O)]2+

HPLC and capillary electrophoresis (CE) were compared in order to separate the platinum adducts formed upon reaction of the double-stranded 18-mer d(AACGGTTAACCGTTAATT)2I with the two complexescis-[Pt(NH3)2(H2O)2]2+1 and [Pt(NH3)3(H2O)]2+2 and of the single-stranded octamer d(CTGGCTCA) II with complex 2. The GG sequence in both oligonucleotides is the only site of platination giving monoadducts on either of the two guanines or the diadduct which is theN7,N7chelate of the Pt(NH3)22+moiety. The HPLC separation of the adducts was performed with a reverse-phase technique similar to that previously used for platinated oligonucleotides up to octamers. The adducts were identified by enzymatic digestion followed by mass spectrometry characterization of the digests collected at the outlet of the column. The CE separation of the platination products of I with 2 and 1 was performed using respectively a simple capillary zone electrophoresis technique and a micellar electrokinetic capillary chromatography technique. For the more difficult separation of the platination products of II with 2, a capillary gel electrophoresis technique was required. In contrast to HPLC, CE gives better resolution the longer the oligonucleotide. Moreover, the separations are run with higher efficiency and time saving. However, with the present machines, CE precludes the recovery of sufficient amounts of material required by product identification.

Determination of the Alkaloids in Coptis-Evodia Herb Couple by Capillary Electrophoresis

A method combining the techniques of micellar electrokinetic capillary chromatography (MECC) and capillary zone electrophoresis (CZE) has been developed to assay seventeen alkaloids in coptis-evodia herb couple. The MECC method, based on SDS, was applied to analyze three indolequinazoline alkaloids and six quinolone alkaloids in evodia within 30 minutes, and a CZE technique was used to determine eight quaternary alkaloids (dehydroevodiamine in evodia, and seven protoberberine alkaloids in coptis) within 25 minutes. The recovery efficiencies were 96.68–103.19% in MECC and 99.65–103.28% in CZE, with a relative standard deviation of 1.67–4.00% for MECC and 2.33–4.38% for CZE. The calibration curves exhibited good linearity over one order of magnitude of concentration, and their minimum detectable concentrations were approximately 15.78 to 47.33 μ/mL using a 0.75 μ inner diameter column. Contents of the seventeen alkaloids in a methanol-water crude extract of coptis-evodia herb couple could easily be determined by this method.

Determination of the alkaloids in Evodiae fructus by capillary electrophoresis

A total of ten Evodia alkaloids was separated successfully by micellar electrokinetic capillary chromatography (MECC) and capillary zone electrophoresis (CZE). The MECC method based on SDS was applied to analyze three indolequinazoline alkaloids (evodiamine, rutaecarpine and carboxyevodiamine) and six quinolone alkaloids (1-methyl-2-nonyl-4(1 H)-quinolone, 1-methyl-2-[(Z)-6-undecenyl]-4(1 H)-quinolone, 1-methyl-2-undecyl-4(1 H)-quinolone, evocarpine, 1-methyl-2-[(6Z,9Z)-6,9-pentadecadienyl]-4(1 H)-quinolone and dihydroevodiamine) in 15 min and CZE technique was used to determine the dehydroevodiamine in 5 min. Linearity over one order of magnitude of concentration was generally obtained and limits of detection for the alkaloids were in the range of 35–47 μg/ml. The relative standard deviations were less than 4% ( n=6). Contents of Evodiae alkaloids in a methanol-water extract of Evodia fructus sampled could easily be determined by this method. The effects of pH, surfactant concentration and organic modifier concentration of the carrier on the migration behaviour of the solutes are discussed.

Capillary zone electrophoresis to study the humic fraction in organic soils and its relationship with radiocaesium mobility

Humic acids and associated radionuclides were extracted from several soil samples with a significant organic matter content, such as peaty soils and forest soil layers. Extractions were made using two alkaline solutions (Na4P2O7 0.1 mol·1−1 and NaOH 0.1 mol·1−1 under N2). The humic acid content in the extract was determined by the capillary zone electrophoresis technique (CZE) and associated radiocaseium was determined by gamma spectrometry. After analizing a large number of samples and studying the relationship between humic acid and organic matter content it was possible to conclude that the CZE technique may be a good alternative to classical techniques in humic acid determinations, with NaOH extractions leading to higher results than Na4P2O7. Furthermore, the quantification of the radiocaseium desorbed by applying different extractant reagents (NaOH, Na4P2O7, NH4AcO and CaCl2) showed that there may be some organic matter-radionuclide interations, other than those originated by humic acids. which may govern radionuclide retention in soils with a high content of organic matter.