Zone Electrophoresis Technique Research Articles

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.

Evaluation of capillary zone electrophoresis for the determination of protein composition in therapeutic immunoglobulins and human albumins

Capillary zone electrophoresis (CZE) provides an alternative means of separating native proteins on the basis of their inherent electrophoretic mobilities. The major advantage of CZE is the quantification by UV detection, circumventing the drawbacks of staining and densitometry in the case of gel electrophoresis methods. The data of this validation study showed that CZE is a reliable assay for the determination of protein composition in therapeutic preparations of human albumin and human polyclonal immunoglobulins. Data obtained by CZE are in line with “historical” data obtained by the compendial method, provided that peak integration is performed without time correction. The focus here was to establish a rapid and reliable test to substitute the current gel based zone electrophoresis techniques for the control of protein composition of human immunoglobulins or albumins in the European Pharmacopoeia. We believe that the more advanced and modern CZE method described here is a very good alternative to the procedures currently described in the relevant monographs.

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.

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.

Electrophoresis, surface charge and surface activity coefficient of ionic adsorbates at interfaces

Historical development of measurements of electrophoretic mobilities of solid particles and proteins using moving boundary techniques have been discussed in brief. Results on micro-electrophoretic methods of mobility measurements of solid particles adsorbed with proteins and other biopolymers have also been elaborated and features of electrophoretic separation of biomolecules using various types of zone electrophoresis techniques have been mentioned. The corrections required in connecting electrophoretic mobilities with electrokinetic or zetapotential using Smoluchowski relation discussed by Henry, Booth, Ghosh and others have been mentioned. The pictures included in the electrical double layers proposed by Helmholtz, Gouy-Chapman and Stern have been discussed. From the adsorption experiments of surfactants at oil-water interfaces, the charge densities (σ) and diffuse double layer potentials (ψ δ ) at a given concentration have been evaluated. The data are observed to fit the Stern layer model with counter-ion binding at the charged surface. ψ δ is found to be significantly higher than zetapotential. From the boundary tension measurement, the surface tension lowering or surface pressure II for soluble monobasic and dibasic acids have been calculated at different values of area A per adsorbed molecule. The electrical pressure II e obtained for these acids in the monolayer does not fit the Gouy model of double layer but using Stern model, the extents of counter-ion binding in the monolayer can be calculated. Using the Langmuir surface balance experiments, II-A curves for insoluble monolayers of different behenate salts have been obtained. At pH 12.0, values of IT e depends upon extents of counter-ion binding, hydration and other factors. A new concept of surface activity coefficient based on Raoult's law, and Hennry's law have been introduced to explain deviation of II from their ideal values.

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.

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.

RAMSES: Applied research on separation methods using space electrophoresis

Eight european industrial companies, the CNRS and University Paul Sabatier and CNES/ Centre National d'Etudes Spatiales collaborate on the SBS (Space Bio Separation) project which aims at demonstrating the possibility of preparing high-purity biomaterials under microgravity conditions. As a first step of SBS, the proposal of a cooperative flight of the RAMSES facility on board Spacelab during the IML-2 mission, scheduled January 1993, has been selected by NASA. RAMSES allows basic and applied research on free flow zone electrophoresis, in order to assess the influence of a low-gravity environment on the purification of biological products. Experiments will be performed by European and American scientists. The facility will be integrated in a Spacelab single rack. Using in situ diagnostics with a U.V. photometer and a cross illuminator, RAMSES investigates a wide variety of transport phenomena to better understand the basic mechanisms which govern electrophoresis method. RAMSES should be a basis for a more complete facility dedicated to the purification of biomaterials, associating various separation methods. This paper will provide an overview of this space facility RAMSES with emphasis on continuous flow zone electrophoresis technique, scientific back-ground, RAMSES experimental program, RAMSES main functions and an overall description of the RAMSES main units.

Isotope effect on electromigration of cations in molten nitrates A comment on the interpretation of some electrophoresis experiments

The zone electrophoresis technique has been used by Wuhl, Lantelme and Chelma to measure electrical mobilities of 22Na and 24Na in some nitrate melts, and by Lantelme and Turq for 6Li and 7Li. Displacements along an asbestos strip were measured, and “external” mobilities, were thus obtained. If, however, we consider “internal” mobilities, e.g. mobilities of the different cations relative to the common anion, some details of the interpretation are changed. Further conclusions can be drawn from a comparison of the isotope effects for strongly diluted ions with those for pure salts. The differences between isotope effects in nitrates and in halides (and sulphates) are due to stronger anion-cation interaction in nitrates than in halides. The isotope effects fit a model where transport is a collective motion with only short displacements at a time.

Electrophoresis of isolated mitochondria

Isolated respiring mitochondria suspended in an isotonic medium migrate to the anode when they are exposed to an electric field. A simple method has been devised to visualize this electrical property of the organelles utilizing a modified zone electrophoresis technique. Uncouplers of oxidative phosphorylation inhibit this anaphoretic property of isolated mitochondria.

Autosomal determination of erythrocyte glucose 6-phosphate dehydrogenase in domestic chickens and ring-necked pheasants

Erythrocyte glucose 6-phosphate dehydrogenase isozymes of domestic chickens, ring-necked pheasants, and their hybrids were studied, using the starch gel zone electrophoresis technique. In domestic chickens G6PD isozymes were represented by two fast-moving bands and an indistinct third band, whereas in ring-necked pheasants a slow-moving broad band which seemed to consist of two closely apposed G6PD isozymes was observed. The F1 hybrids showed three distinct bands combining the characteristic mobility pattern of the two parents, which seemed to indicate that both parental alleles are expressed in F1 hybrids. Since both male and female hybrids exhibited strikingly similar isozyme patterns representing both sire and dam, it was assumed that the genes controlling the production of G6PD in chicken and pheasant red blood cells are located on the autosomes.

Isozymes: Classification, Frequency, and Significance

Publisher Summary Isozymes are defined as multiple forms of enzymes occurring in the same organism and having similar or identical catalytic activities. They are usually demonstrated by the technique of zone electrophoresis followed by in situ histochemical staining for the specific activity. Most isozymes can be classified into primary and secondary types. The primary isozymes are distinctly different molecules, and are produced from different genetic sites. The others result from secondary alteration in structure of a single polypeptide species, and many of these are in vitro artifacts. This chapter focuses on primary isozymes, which are the only ones that can presently be considered to have biological significance. Primary isozymes can usually be found to have differences between or among them, so that they are not biological redundancies. The differences found among isozymes are of three main classes: tissue and cell localization, regulation, and enzyme kinetics.

Three new phenotypic systems in the blood of sheep.

SummaryThree new phenotypic systems detectable in the blood of sheep by means of the technique of zone electrophoresis in starch gels are described.One of these systems, designated EsA (A or arylesterase), involves a broad, smudge‐like zone of esterase which developed rapidly during staining and was observed in the serum of about ¼ of the animals sampled. The 2 phenotypes, referred to as EsA‐positive and EsA‐negative, are inherited as if controlled by a pair of alleles, EsA+ and EsA‐, with EsA+ being dominant to EsA‐. A quick tube test for the diagnosis of the 2 phenotypes is described. It is shown that the 2 phenotypes correspond respectively with the earlier described phenotypes Halon‐high and Halon‐low discovered by Lee in studies of the hydrolytic activity of ovine serum on di‐(2‐chloroethyl) aryl phosphates.A second system, designated CA, involves 2 electrophoretic forms, CA‐F and CA‐S, of erythrocytic carbonic anhydrase observed in 3 phenotypes CA‐F, CA‐FS and CA‐S. The 3 phenotypes are inherited as if controlled by co‐dominant autosomal alleles designated CAF and CAS. Allele CAS was at or near fixation in 6 of the 9 breeds surveyed in this study. This system has its analogue in the earlier described CA system of cattle.The third system, designated X (unknown) protein, involves 3 zones in electrophoresed red cell lysates which were either present together or absent together. The 2 phenotypes, designated X‐positive and X‐negative, are inherited as if controlled by a pair of alleles, X and x, with X being dominant to x. (This nomenclature is tentative and should be changed when the protein involved becomes identified.) The 2 phenotypes were observed in each of the 9 breeds in the population survey.

An agarose-gel zone electrophoresis technique for in-vivo protein binding studies of radio-active isotopes

An agarose-gel zone electrophoresis technique for in-vivo protein binding studies of radio-active isotopes

Electrophoresis of human blood lipoproteins on starch gel

The electrophoretic pattern of human serum lipoproteins after migration in starch gel is different from that obtained by other zone electrophoresis techniques. The most important component which migrates between the slot and slow alpha — 2 globulins may be identified as the component being precipitated by dextrane Sulfate and calcium chloride. The other components have the same mobility as either beta or alpha globulins.

Heterogeneity studies on several proteins by means of zone electrophoresis on starch

The technique of zone electrophoresis on starch was applied to the study of the electrophoretic homogeneity of several proteins. A special point was made of examining some of the classical proteins that had been shown by other authors to possess a low heterogeneity constant in free electrophoresis, namely, crystalline ribonuclease, lysozyme, bovine serum albumin, chymotrypsinogen, and chymotrypsin. Several different preparations of ribonuclease were examined, and all of them showed the presence of two major components as well as at least four minor ones. All the components were active, though their relative proportions varied from preparation to preparation, and they could be correlated with the various components obtained chromatographically by other workers. Chymotrypsin and chymotrypsinogen behaved as homogeneous proteins at acid pH's, but they could be separated into two distinct biologically active components, A and B, by prolonged electrophoresis in a cacodylic buffer at pH 6.6. Electrophoretically homogeneous chymotrypsinogen A was prepared by rerunning the original peak. Lysozyme yielded a single peak at most pH's, but in acetate buffer there appeared two shoulders, indicating the presence of two minor components. In the case of bovine serum albumin, a single symmetrical peak appeared in acid and alkaline buffers, but incipient separation into two peaks was obtained in phosphate and cacodylic buffers between pH 6.6 and 7.0.