Removal of sample background buffering ions and myoglobin enrichment via a pH junction created by discontinuous buffers in capillary electrophoresis

Traditional CE sample stacking is ineffective for samples containing a high concentration of salt and/or buffer. We recently reported the use of a discontinuous buffer system for protein enrichment that was applicable to samples containing millimolar concentrations of salt. In this paper, the technique was investigated for samples containing unwanted buffering ions, including TRIS, MES, and phosphate, which are commonly used in biological sample preparation. Using myoglobin as a model protein, the results demonstrated that background buffering ions can be effectively removed or separated from the enriched protein. The key is to use either the acid or the base of the discontinuous buffers to adjust the pH of the sample, such that the net charge of the unwanted buffering ions is near-zero. The successful isolation and enrichment of myoglobin from up to 100 mM TRIS and 50 mM MES was demonstrated. The enrichment factors remained at approximately 200. Removal of phosphate was more challenging because its net charge was anionic in both the acid and the base of the discontinuous buffers. The enrichment was only achievable up to 30 mM of sodium phosphate, the enrichment factors observed were significantly lower, below 50, and the process was delayed due to the higher ionic strength resulted from phosphate. The migration of phosphate during enrichment was studied using a UV-absorbing analogue, phenyl phosphate. In addition, Simul 5.0 was used to simulate the discontinuous buffers in the absence and presence of TRIS and phosphate. The stimulated TRIS and phosphate concentration profiles were generally in agreement with the experimental results. The simulation also provided a better understanding on the effect of phosphate on the formation of the pH junction.     

Separation of human serum lipoproteins into three major classes by hydroxyapatite chromatography.

The separation of normolipidemic male serum lipoprotein fraction, prepared by ultracentrifugal flotation, was studied on hydroxyapatite columns. Potassium phosphate buffers in the pH range 5.6-7.4 were evaluated as eluents. The three main classes of the lipoproteins (high density, low density and very low density) can be separated on the Tiselius-type hydroxyapatite (Bio-Gel HTP DNA grade) column by elution with 75, 250 and 300 mM potassium phosphate buffer (pH 7.4), respectively.     

Optimization of separation and migration behavior of cephalosporins in capillary zone electrophoresis

The influences of buffer pH, buffer concentration and buffer electrolyte on the migration behavior and separation of 12 cephalosporin antibiotics in capillary zone electrophoresis using three different types of buffer electrolyte, including phosphate, citrate, and 2-(N-morpholino)ethanesulfonate (MES), were investigated. The results indicate that, although buffer pH is a crucial parameter, buffer concentration also plays an important role in the separation of cephalosporins, particularly when cefuroxime and cefazolin, cephalexin and cefaclor, or cefotaxime and cephapirin are present as analytes at the same time. The electrophoretic mobility of cephalosporins and electroosmotic mobility measured in citrate and MES buffers are remarkably different from those measured in phosphate buffer. With citrate buffer, optimum buffer concentration is confined to a small range (35-40 mM), whereas buffer concentrations up to 300 mM can be used with MES buffer. Complete separations of 12 cephalosporins could be satisfactorily achieved with these three buffers under various optimum conditions. However, the separability of 12 cephalosporins with citrate or MES buffer is better than that with phosphate buffer. As a consequence of a greater electrophoretic mobility of cephalosporins than the electroosmotic mobility with citrate buffer at pH below about 5, some cephalosporins are not detectable. The cloudiness of the peak identification and of the magnitudes of the electrophoretic mobility of cefotaxime and cefuroxime reported previously are clarified. In addition, the pKa values of cephradine, cephalexin, cefaclor, and cephapirin attributed to the deprotonation of either an amino group or a pyridinium group are reported, and the migration behavior of these cephalosporins in the pH range studied is quantitatively described.     

Retention of ionisable compounds on high-performance liquid chromatography XVIII: pH variation in mobile phases containing formic acid,piperazine, tris,boric acid or carbonate as buffering systems and acetonitrile as organic modifier

In the present work dissociation constants of commonly used buffering species, formic acid, piperazine, tris(hydroxymethyl)–aminomethane, boric acid and carbonate, have been determined for several acetonitrile–water mixtures. From these pK_a values a previous model has been successfully evaluated to estimate pH values in acetonitrile–aqueous buffer mobile phases from the aqueous pH and concentration of the above mentioned buffers up to 60% of acetonitrile, and aqueous buffer concentrations between 0.005 (0.001 mol L⁻¹ for formic acid–formate) and 0.1 mol L⁻¹. The relationships derived for the presently studied buffers, together with those established for previously considered buffering systems, allow a general prediction of the pH variation of the most commonly used HPLC buffers when the composition of the acetonitrile–water mobile phase changes during the chromatographic process, such as in gradient elution. Thus, they are an interesting tool that can be easily implemented in general retention models to predict retention of acid–base analytes and optimize chromatographic separations.     

Salt effects in capillary zone electrophoresis. IV. Resolution versus time and the effect of potassium phosphate and its concentration in the high ionic strength separation of sulphonamides.

The effects of potassium phosphate buffer and its concentration upon the capillary zone electrophoretic separation of 23 sulphonamides and a neutral marker were examined at pH 7. The resolution between the pairs was improved with the increased concentration of the buffer from 65 mM to 174 mM. Nineteen sulphonamides, a hydrolysis product and several unidentified minor components were baseline resolved in both 101 and 138 mM phosphate buffers. In 174 mM buffer all 21 ionised sulphonamides and the other compounds were separated. A simple relationship between the resolution of analyte pairs (Rs) and the square root of the mean analysis time for the pair (square root of tapp) was derived, but few of the pairs displayed this behaviour. For the majority of pairs of compounds, Joule heating appeared to cause a maximum in the Rs versus square root of tapp relationship, while non-ideality and shifts in ionisation with increasing salt concentration appeared dominant in other cases.     

Enhanced selectivity of hydrogel-based molecularly imprinted polymers (HydroMIPs) following buffer conditioning

We have investigated the effect of buffer solution composition and pH during the preparation, washing and re-loading phases within a family of acrylamide-based molecularly imprinted polymers (MIPs) for bovine haemoglobin (BHb), equine myoglobin (EMb) and bovine catalyse (BCat). We investigated water, phosphate buffer saline (PBS), tris(hydroxymethyl)aminomethane (Tris) buffer and succinate buffer. Throughout the study MIP selectivity was highest for acrylamide, followed by N-hydroxymethylacrylamide, and then N-iso-propylacrylamide MIPs. The selectivity of the MIPs when compared with the NIPs decreased depending on the buffer conditions and pH in the order of Tris > PBS > succinate. The Tris buffer provided optimum imprinting conditions at 50 mM and pH 7.4, and MIP selectivities for the imprinting of BHb in polyacrylamide increased from an initial 8:1 to a 128:1 ratio. It was noted that the buffer conditions for the re-loading stage was important for determining MIP selectivity and the buffer conditions for the preparation stage was found to be less critical. We demonstrated that once MIPs are conditioned using Tris or PBS buffers (pH7.4) protein reloading in water should be avoided as negative effects on the MIP's imprinting capability results in low selectivities of 0.8:1. Furthermore, acidifying the pH of the buffer solution below pH 5.9 also has a negative impact on MIP selectivity especially for proteins with high isoelectric points. These buffer conditioning effects have also been successfully demonstrated in terms of MIP efficiency in real biological samples, namely plasma and serum.     

Glutaraldehyde activated eggshell membrane for immobilization of tyrosinase from Amorphophallus companulatus: application in construction of electrochemical biosensor for dopamine

Tyrosinase from a plant source Amorphophallus companulatus was immobilized on eggshell membrane using glutaraldehyde. Among the three different approaches used for immobilization, activation of eggshell membrane by glutaraldehyde followed by enzyme adsorption on activated support could stabilize the enzyme tyrosinase and was found to be effective. K_m and V_max values for dopamine hydrochloride calculated from Lineweaver-Burk plot were 0.67 mM and 0.08 mM min⁻¹, respectively. Studies on effect of pH showed retention of more than 90% activity over a pH range 5.0-6.5. Membrane bound enzyme exhibited consistent activity in the temperature range 20-45 °C. Shelf life of immobilized tyrosinase system was found to be more than 6 months when stored in phosphate buffer at 4 °C. An electrochemical biosensor for dopamine was developed by mounting the tyrosinase immobilized eggshell membrane on the surface of glassy carbon electrode. Dopamine concentrations were determined by the direct reduction of biocatalytically liberated quinone species at −0.19 V versus Ag/AgCl (3 M KCl). Linearity was observed within the range of 50-250 μM with a detection limit of 25 μM.     

Method development and validation for the separation and determination of omeprazole enantiomers in pharmaceutical preparations by capillary electrophoresis

A new capillary zone electrophoresis (CZE) method for the separation of omeprazole enantiomers has been developed. Methyl-β-cyclodextrin (methyl-β-CD) was chosen as the chiral selector, and several parameters, such as cyclodextrin structure and concentration, buffer concentration, pH, and capillary temperature were investigated in order to optimize separation and run times. Analysis times, shorter than 8 min were found using a background electrolyte solution consisting of 40 mM phosphate buffer adjusted to pH 2.2, 30 mM β-cyclodextrin and 5 mM sodium disulphide, hydrodynamic injection, and 15 kV separation voltage. Detection limits were evaluated on the basis of baseline noise and were established 0.31 mg/l for the omeprazole enantiomers. The proposed method was applied to five pharmaceutical preparations with recoveries between 84 and 104% of the labeled contents.     

An electrochemical on-field sensor system for the detection of compost maturity

A maturity sensor system was developed, based on the combination of three electrically measured parameters, pH, NH₄⁺ concentration, and phosphatase activity in the water extracts of compost samples. One of these parameters, the apparent phosphatase activity in crude test solutions was determined using screen-printed carbon strips (SPCSs) coated with α-naphthyl phosphate (α-NP) in Nafion film. The phosphatase activity was monitored in connection with differential pulse voltammetry (DPV) with an aliquot (30 μL) of the test solution on SPCS. The phosphatase activity sensor was validated using alkaline phosphatase (ALP) in Tris-HCl buffer (pH 8.0) and acid phosphatase (ACP) in citric acid buffer (pH 5.0). The activity of the spiked enzymes in the water extract of the compost sample could be confirmed with the change of corresponding oxidation peak current signal of the product, α-naphthol. The water extracts of compost samples (n = 24) collected in various composting days were applied to our compost maturity sensor system, and the conventional germination tests. Using multiple regression analysis, the germination index (GI) was expressed by the multi-linear regression equation consisting of pH, NH₄⁺ concentration, and the phosphatase activity. The calculated GI from the regression equation had a good correlation with the measured GI of the corresponding composts (r = 0.873). As a result, we have determined an equation for the determination of the compost stability using our portable sensor system rapidly at the composting site.     

Fast electrophoretic separation of sulfur- and selenium-containig amino acid enantiomers with vancomycin as a chiral selector in coated capillaries

Baseline separation of the enantiomers of the negatively charged 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives of cystine, methionine, ethionine, and their seleno analogs can be achieved in 3–5 min with capillary electrophoresis in polyacrylamide coated capillaries and submillimolar concentrations of vancomycin as the chiral selector. In addition to the vancomycin concentration, the separation is affected by the type, concentration and pH of the buffer. Good buffers are more suitable than phosphate buffer. At pH values above the isoelectric point of vancomycin the mobility difference between the enantiomers becomes smaller. This effect is larger than would be expected from the reversal of the vancomycin migration alone.     

Molecularly bonded chitosan prepared as chiral stationary phases in open-tubular capillary electrochromatography: comparison with chitosan nanoparticles bonded to the polyacrylamide phase

The chiral selector, chitosan (CS), was attached to the silanized capillary via a silane coupling agent, (3-glycidyloxypropyl)trimethoxysilane (GTS), to form the GTS-CS capillary, and results for this capillary were compared with those of a previous study on the copolymerization of CS with methacrylamide (MAA) (forming the MAA-CS capillary). The GTS-CS capillary did not exhibit enantioselectivity for d/l-tryptophan, whereas the GTS-BSA capillary, which was prepared by replacement of CS with bovine serum albumin (BSA), succeeded in the chiral separation with an Rs = 2.4 in Tris buffer (50 mM, pH 8.5). To increase CS attachment, the CS units were crosslinked by succinic acid, and the resulting GTS-CS-s capillary phase improved the resolution to 1.9. Alternatively, the SiH-CS-s capillary was constructed by CS attachment on the silicon hydride phase via stepwise silanization and hydrosilation reactions and crosslinking by succinic acid, but this approach could only achieve a resolution of 1.4 in Tris buffer (50 mM, pH 9.5). Although the GTS-CS-s and SiH-CS-s capillaries were still inferior to the MAA-CS capillary (Rs = 3.8), the enantioselectivities of the three capillaries were all in the range of 1.4-1.6. For the (±)-catechin sample, the plate heights of the GTS-CS-s and SiH-CS-s capillaries conditioned in pH 8.5 Tris buffer with 60% MeOH modifier were 0.9 cm ((−)-catechin) and 6.0 cm ((+)-catechin)) and 2.9 cm (−) and 3.2 cm (+), respectively, and these heights were comparable to the MAA-CS capillary (2.5 cm (−), 6.0 cm (+)) in pH 6.6 phosphate buffer with 80% MeOH. Finally, a racemate of ibuprofen, a weakly acidic anti-inflammatory drug, was successfully baseline resolved by the GTS-CS-s and SiH-CS-s capillaries in the borate buffers, which were 30 mM at pH 7.5 and 10 mM at pH 8.0, respectively.     

Liquid chromatographic determination of sitagliptin either alone or in ternary mixture with metformin and sitagliptin degradation product

Two reversed-phase liquid chromatographic (RP-LC) methods have been developed for the determination of sitagliptin phosphate monohydrate (STG). The first method comprised the determination of STG alone in bulk and plasma; and in its pharmaceutical preparation. This method was based on isocratic elution of STG using a mobile phase consisting of potassium dihydrogen phosphate buffer pH (7.8)-acetonitrile (70:30, v/v) at a flow rate of 1 mL min⁻¹ with flourometric detection. The flourometric detector was operated at 267 nm for excitation and 575 nm for emission. In the second method, the simultaneous determination of STG and metformin (MET) in the presence of sitagliptin alkaline degradation product (SDP) has been developed. In this method, the ternary mixture of STG, MET and SDP was separated using a mobile phase consisting of potassium dihydrogen phosphate buffer pH (4.6)-acetonitrile-methanol (30:50:20, v/v/v) at a flow rate of 1 mL min⁻¹ with UV detection at 220 nm. Chromatographic separation in the two methods was achieved on a Symmetry^® Waters C18 column (150 mm × 4.6 mm, 5 μm). Linearity, accuracy and precision were found to be acceptable over the concentration ranges of 0.25-200 μg mL⁻¹ for STG with the first method and 5-160 μg mL⁻¹, 25-800 μg mL⁻¹ for STG and MET, respectively with the second method. The optimized methods were validated and proved to be specific, robust and accurate for the quality control of the cited drugs in pharmaceutical preparations.     

Comparison of peak shapes obtained with volatile (mass spectrometry-compatible) buffers and conventional buffers in reversed-phase high-performance liquid chromatography of bases on particulate and monolithic columns

Retention factor, column efficiency and asymmetry factor were recorded for nine basic compounds on a number of RP-HPLC columns using phosphate and a variety of (MS-compatible) volatile mobile phase buffers of acid and neutral pH, in order to assess any effects of the buffer on performance. With formic or acetic acid, some phases gave partial or complete solute exclusion effects (reduced or negative k) compared with results using phosphate buffers at low pH. Despite its possible suppression of mass spectrometer sensitivity, trifluoroacetic acid was useful in enhancing retention times of relatively hydrophilic protonated bases, due to ion-pair effects. Peak shape was relatively poor on some pure silica-based ODS phases at pH 7 compared with results at acid pH. At low pH and at pH 7, ammonium and potassium phosphate gave very similar k, but the former may be preferable due to its volatile cation. Improved peak shapes, attributed to superior silanol masking effects, were obtained with ammonium phosphate at pH 7, but not at acid pH. Ammonium acetate gave acceptable peak shape at pH 7, but due to very limited buffer capacity, poor results were obtained for solutes having a pKa close to the mobile phase pH. Due to its instability, ammonium hydrogen carbonate is not a viable alternative buffer at pH 7.     

Aspects of Degradation Kinetics of Azithromycin in Aqueous Solution

The chemical stability of azithromycin (AZM) in aqueous solution has been investigated utilizing a stability-indicating LC assay with ultraviolet detection. The degradation kinetics were studied as functions of pH (4–7.2), buffer composition (phosphate, acetate, and citrate), buffer concentration, ionic strength, drug concentration and temperature. The observed rate obtained by measuring the remaining intact AZM was shown to follow pseudo-first-order kinetics. The maximum stability of AZM occured at an approximate pH 6.3 in 0.05 M potassium phosphate. The observed degradation rate increased with ionic strength, buffer concentration and obeyed the Arrhenius equation over the temperature range investigated (70–100 °C). The apparent energy of activation (E _a) for AZM in solution was found to be 96.8 kJ mol^?1 and by application of the Arrhenius equation the stability at 25 °C (k ₂₅) and 40 °C (k ₄₀) had been predicted. Moreover, the degradation rate of AZM was independent on its initial concentration. Trace metal ions are unlikely to be involved in the degradation of AZM in aqueous solution. The major degradation product of AZM in aqueous solution was isolated and identified by LC–MS–MS and ¹H and ¹³C NMR spectra.     

Extending the applicability of pressurized hot water extraction to compounds exhibiting limited water solubility by pH control: curcumin from the turmeric rhizome

Pressurized hot water extraction (PHWE, also known as subcritical water extraction) is commonly considered to be an environmentally friendly extraction technique that could potentially replace traditional methods that use organic solvents. Unfortunately, the applicability of this technique is often limited by the very low water solubility of the target compounds, even at high temperatures. In this paper, the scope for broadening the applicability of PHWE by adjusting the pH of the water used in the extraction is demonstrated in the extraction of curcumin (which exhibits very limited water solubility) from untreated turmeric (Curcuma longa L.) rhizomes. Although poor extraction yields were obtained, even at high temperatures when using degassed water or neutral phosphate buffer as the extraction medium, yields exceeding those obtained by Soxhlet extraction were achieved using highly acidic pH buffers due to curcumin protonation. The influence of the temperature, pH, and buffer concentration on the extraction yield were investigated in detail by means of a series of designed experiments. Optimized conditions for the extraction of curcumin from turmeric by PHWE were estimated at 197 °C using 62 g/L buffer concentration at pH 1.6. The relationships between these variables were subjected to statistical analysis using response surface methodology.     

Simultaneous determination of malondialdehyde and ofloxacin in plasma using an isocratic high-performance liquid chromatography/fluorescence detection system

This study establishes the applicability of using high-performance liquid chromatography (HPLC) with fluorescence detection for the simultaneous determination of malondialdehyde (MDA) and ofloxacin (OFL). The MDA and OFL were separated through a reverse-phase C18 column (250 mm × 4.6 mm) at a flow rate of 1.0 mL min⁻¹ and then detected using a fluorescence detector (excitation: 532 nm; emission: 553 nm). The separation conditions were optimized by varying the concentration and pH of the phosphate buffer and the percentage of organic solvent; the optimal mobile phase was a mixture of 50 mM phosphate buffer (adjusted to pH 5.8 with potassium hydroxide) and methanol (45:55, v/v). The retention times of MDA and OFL were 3.6 and 5.9 min, respectively, with detection limits (at a signal-to-noise ratio of 3) of 0.015 and 4.0 μM, respectively. This method afforded linear responses between the MDA and OFL concentrations and the HPLC peak areas within the ranges 0.15-2.43 μM and 0.06-1.0 mM, respectively. The precisions of the determinations of MDA and OFL, measured in terms of relative standard deviations, were 1.6-5.0% and 1.9-3.6%, respectively, for intra-day assays and 1.0-4.3% and 0.3-1.8%, respectively, for inter-day assays. The average recoveries of MDA and OFL spiked in plasma were 100.4% and 98.8%, respectively. To the best of our knowledge, this paper describes the first practical analytical approach toward simultaneously monitoring the levels of MDA and OFL in plasma. The OFL-induced oxidative stress measured using this method indicated that OFL treatment did not markedly increase the level of MDA.     

Size dependence of SiO2 particles enhanced glucose biosensor

A wide size range of SiO₂ particles were synthesized and were used as enzyme immobilization carriers to fabricate glucose biosensors. The size of the particles was in the range of 17-520 nm. These biosensors could be operated under physiological conditions (0.1 M phosphate buffer, pH 7.2). Particle size could affect the performance of SiO₂ modified glucose biosensors drastically. The smaller particles had higher performance. The smallest SiO₂ modified biosensor could work well in the glucose concentration range of 0.02-10 mM with a correlation coefficient of 0.9993. Its sensitivity was 2.08 μA/mM and the detection limit was 1.5 μM glucose.     

Determination of triazine compounds in ground water samples by micellar electrokinetic capillary chromatography

In this work, a capillary electrophoresis (CE) method for the determination of a group of eleven triazine compounds by micellar electrokinetic capillary chromatography (MEKC) with diode array detection was developed. The eleven herbicides studied were: desethylatrazin-2-hydroxy (DEA), simazine, prometon, atrazine, simetryn, ametryn, propazine, prometryn, trietazine, terbutylazine, and terbutryn The separation of these compounds was optimised as a function of buffer concentration and pH, concentration of sodium dodecyl sulphate (SDS) and voltage applied. To increase the selectivity of the separation and the resolution of the solutes, different organic solvents were tested as buffer additives, obtaining the best results when 1-propanol was used. The optimised buffer (24 mM of sodium borate, 18 mM of disodium hydrogen phosphate, 25 mM of SDS, pH 9.5, and 5% of 1-propanol) provides the best separation in terms of resolution and migration time. This method allowed the determination of these compounds at concentrations of 0.05 μg l⁻¹ in ground water samples pretreated using solid-phase extraction (SPE).     

Concentrating rotaviruses from water samples using monolithic chromatographic supports

Rotaviruses are the leading cause of diarrhoea in infants around the globe and, under certain conditions they can be present in drinking water sources and systems. Ingestion of 10–100 viral particles is enough to cause disease, emphasizing the need for sensitive diagnostic methods. In this study we have optimized the concentration of rotavirus particles using methacrylate monolithic chromatographic supports. Different surface chemistries and mobile phases were tested. A strong anion exchanger and phosphate buffer (pH 7) resulted in the highest recoveries after elution of the bound virus with 1 M NaCl. Using this approach, rotavirus particles spiked in 1 l volumes of tap or river water were efficiently concentrated. The developed concentration method in combination with a real time quantitative polymerase chain reaction assay detected rotavirus concentrations as low as 100 rotavirus particles/ml.     

Investigation of some critical parameters of buffer conditions for the development of quantum dots-based optical sensors

The unique surface-sensitive properties make quantum dots (QDs) great potential in the development of sensors for various analytes. However, quantum dots are not only sensitive to a certain analyte, but also to the surrounding conditions. The controlled response to analyte may be the first step in the designing of functional quantum dots sensors. In this study, taking the quenching effect of benzoquinone (BQ) on CdTe QDs as model, several critical parameters of buffer solution conditions with potential effect on the sensors were investigated. The pH value and the concentration of sodium citrate in the buffer solution critically influenced the quenching effects of BQ. Dozens folds elevation of the quenching extents were observed with the increase of concentrations of H⁺ and sodium citrate, and the quenching mechanisms were also fundamentally different with the changes of the surrounding buffer solutions. The quenching models were proposed and analyzed at different buffer conditions. Taking pH values for example, QDs quenching obeyed the sphere of effective quenching model with the sphere radii of 8.29 nm at pH 8.0, the linear Stern-Volmer equation with Stern-Volmer constant of 2.0 × 10³ mol⁻¹ L at pH 7.0, and the two binding site static quenching model at basic conditions. The elucidation of parameters for assay performance was important in the development of QDs-based optical sensors.