Free Zone Capillary Electrophoresis Research Articles

Effect of heat treatment and pH on the thermal, surface, and rheological properties of Lupinus albus protein

AbstractEndosperm from hand‐dissected and‐ dehulled Lupinus albus seeds was milled into meal, sieved through a 40‐mesh screen, and suspended in phosphate buffers (pH 4, 6.8, and 8) at 20% (wt/vol). The suspensions were treated at 75, 90, or 100°C for 1 h. The heat‐treated protein was characterized by SDS‐PAGE, free zone capillary electrophoresis (FZCE), and DSC; and its surface hydrophobicity, surface tension, and rheological properties were examined. The presence of high M.W. aggregates was apparent from SDS‐PAGE and FZCE results. Solubility was lowest at pH 4 and 100°C. DSC analysis was performed on low moisture content samples (3.1%) and 20% (wt/vol) suspensions. DSC analysis at 3.1% moisture content showed a glass transition around 85°C and an exothermic transition at 160°C, whereas the protein suspension showed a more thermally stable protein as indicated by the higher ΔH values. Lupin protein was surface active as demonstrated by its effectiveness in reducing the surface tension of the aqueous phosphate buffer. Surface hydrophobicity of the heat‐treated protein decreased as the treatment temperature increased, which supports the SDS‐PAGE results. The highest level of aggregation was noted at 90°C and pH 6.8 as indicated by low surface hydrophobicity values. Rheological studies showed direct relationships between the shear storage modulus (G′) of the lupin meal suspension and both pH and temperature treatment, although this effect is minimal at the highest temperature (100°C) and pH 6.8.

Separation of aceclofenac and diclofenac in human plasma by free zone capillary electrophoresis using N-methyl- d-glucamine as an effective electrolyte additive

Aceclofenac (A) and diclofenac (D) are effective non-steroidal anti-inflammatory drugs (NSAIDs) derived from the phenylacetic acid with pronounced antirheumatic, anti-inflammatory, analgesic and antipyretic properties. Our work proposes a new, fast-free zone capillary electrophoresis method for the simultaneous determination of aceclofenac and diclofenac in human plasma. The effect of increasing concentrations of N-methyl- d-glucamine organic base on borate run buffer was investigated. A good separation was achieved using a 40 cm × 75 μm uncoated silica capillary, 300 mmol/l sodium borate buffer, 200 mmol/l N-methyl- d-glucamine, pH 8.9, in about 3 min. Moreover, the plasma sample pre-treatment procedure was examined: acidic precipitants such as trichloroacetic acid (TCA), metaphosphoric acid (MPA), perchloric acid (PCA) or 5-sulphosalicylic acid (SSA) cause a total loss of analytes while acetonitrile allows a recovery of 97–98% of both compounds. Its simplicity and rapidity and the low analysis costs demonstrate that our method is a reliable and efficient mean for the comprehensive determination of aceclofenac and diclofenac in human plasma when pharmacokinetics studies are required.

Optimization of ascorbic and uric acid separation in human plasma by free zone capillary electrophoresis ultraviolet detection

In this paper we propose a new fast free zone capillary electrophoresis method for the simultaneous determination of ascorbic acid (AA) and uric acid (UA) in human plasma. We investigated the effect of analytical parameters, such as concentration and pH of borate running buffer, cartridge temperature, and sample treatment, on resolution, migration times, corrected peak areas, and efficiency. A good separation was achieved using a 60.2-cm × 75-μm uncoated silica capillary and 100 mmol/L sodium borate buffer, pH 8, when metaphosphoric acid was employed as protein precipitant, in less than 4 min. These conditions gave a good reproducibility of migration times (CV 0.35 and 0.34%) and peak areas (CV 3.2 and 3.1%) for ascorbate and urate, respectively. The limit of detection was 0.5 mg/L for both analytes when the detection was performed at 254 nm for AA and at 292 nm for UA. We compared the present method with a validated capillary electrophoresis assay by measuring plasma urate and ascorbate in 32 normal subjects and the obtained data were analyzed by the Passing and Bablok regression.

Optimization of ascorbic and uric acid separation in human plasma by free zone capillary electrophoresis ultraviolet detection

Optimization of ascorbic and uric acid separation in human plasma by free zone capillary electrophoresis ultraviolet detection

Wheat Flour Proteins as Affected by Transglutaminase and Glucose Oxidase

ABSTRACTEnzymes are good tool to modify wheat proteins by creating new bonds between the protein chains. In this study, the effect of the addition of glucose oxidase (GO) and transglutaminase (TG) on the wheat flour proteins is presented. The modification of wheat proteins was determined by analyzing the changes in gluten quality, alveograph parameters, and protein modifications. The amount of wet gluten increased with the addition of GO and TG, but the gluten quality was not improved in any case. Regarding the alveograph parameters, the effect of GO was readily evident obtaining wheat dough with higher tenacity and lower extensibility than the control, while TG led to doughs with lower tenacity and that were also less extensible. The protein modifications were characterized by free‐zone capillary electrophoresis (FZCE). FZCE data indicated that TG polymerizes mainly glutenins and, of those, the high molecular weight glutenin subunits were the most affected.

Extraction of phyllanthusols A and B from Phyllanthus acidus and analysis by capillary electrophoresis.

Extracts of roots Phyllanthus acidus were examined by free zone capillary electrophoresis, micellar electrokinetic chromatography (MEKC), and MEKC using the sweeping technique which involves application of a negative potential to the inlet end of the capillary and very much longer than conventional injection times. The latter technique, using a buffer of 50 mM sodium dihydrogen phosphate (pH 2) containing 80 mM sodium dodecylsulphate and 30% methanol was found to allow complete resolution of the active constituents of P. acidus, phyllanthusols A and B, from each other and from other extracted components in under 30 min. Several other components could be detected when hydrodynamic injection times of 500 s were used. The separation, combined with an appropriate extraction procedure and using an internal standard of proguanil, permitted quantification of both phyllanthusols. Calibrations were linear over the range 2-8 micrograms/mL for phyllanthusol A, and 1-4 micrograms/mL for phyllanthusol B. Within-day and day-to-day repeatability RSDs were below 10%, and the precision of extraction RSD was around 14%. The limits of quantification and detection were 0.55 and 0.24 microgram/mL, respectively.

Effect of Aelia spp. and Eurygaster spp. Damage on Wheat Proteins

ABSTRACTThe effect of Aelia spp. and Eurygaster spp. wheat bugs on the protein fractions of different wheat cultivars has been studied by size‐exclusion high‐performance liquid chromatography (SE‐HPLC) and free‐zone capillary electrophoresis (FZCE). Those methods were used to quantify and characterize the extent of protein modification. A decrease in the amount of alcohol‐insoluble polymeric proteins along with an increase in the alcohol‐soluble polymeric proteins and gliadins were observed in damaged wheat. The high molecular weight (HMW) and low molecular weight (LMW) glutenin fractions were barely detected in the incubated damaged wheat from some cultivars, which indicated hydrolysis of those proteins by the bug proteinases. In damaged wheats, both incubated and unincubated, gliadin electrophoregrams revealed the presence of some new peaks with mobilities similar to the ω gliadins. The overall results suggest that the bug proteinases are potent enzymes that appear to be nonspecific because they hydrolyze all gluten proteins.

Use of SDS to Extract Sorghum and Maize Proteins for Free Zone Capillary Electrophoresis (FZCE) Analysis

ABSTRACTTwo different extraction methods for extracting sorghum (Sorghum bicolor L. Moench.) storage proteins for free zone capillary electrophoresis (FZCE) analysis were compared. A traditional solvent based on 60% t‐butanol was compared with a pH 10 borate buffer containing the anionic detergent SDS followed by precipitation of nonkafirins using 60% t‐butanol. FZCE analysis of both types of extracts showed identical patterns, despite the fact that the SDS should have given all proteins equal charge‐to‐mass ratios. This methodology was also successfully applied to maize proteins. The use of t‐butanol to precipitate nonkafirins, combined with electrophoresis at low pH, is thought to have removed the SDS from the storage proteins. The SDS extraction procedure produced more stable extracts for FZCE analysis. These extracts could also be used directly for SDS capillary electrophoresis (SDS‐CE) separations. Kafirins from 15 genotypes were extracted with this procedure and analyzed by FZCE and SDS‐CE. Resolution of the kafirins by FZCE was much higher than the SDS‐CE, demonstrating that the kafirin proteins possessed a high level of charge density variability within a relatively small molecular size distribution. Two distinct groups of α‐kafirins could be seen in the FZCE electropherograms.

A probe for the versatile analysis and characterization of N-linked oligosaccharides.

A novel fluorescent probe, 3-(acetylamino)-6-aminoacridine (AA-Ac), has been synthesized and its applicability to the analysis of picomole levels of N-linked glycans investigated. AA-Ac was found to be an excellent derivatization reagent for N-linked glycans, giving at least twice the intensity of fluorescence as its predecessor 2-aminoacridone. AA-Ac-labeled glycans were analyzed by both normal and reversed-phase HPLC. They were also amenable to enzymatic sequencing and analysis by MALDI-TOF mass spectrometry, free zone capillary electrophoresis, and capillary electrophoresis/electrospray ionization mass spectrometry.

Screening for the beta-39 mutation in thalassemia by capillary electrophoresis in free solution in strongly acidic, isoelectric buffers.

A novel method is reported for screening for point mutations in genomic DNA: free-zone capillary electrophoresis in very acidic buffers. This method exploits the charge difference among the four different bases (C, T, A, G) in a pH window between 2.5 and 3.5, where the four titration curves fan out. The method is applied to the detection of the beta-39 missense mutation in the beta-globin gene in thalassemias. A 60-mer fragment straddling the mutation site has been amplified. In an isoelectric buffer (iminodiacetic acid) of pH 3.3, partial resolution between the wild type and mutated strands is obtained. In a pH 3.0 phosphate buffer, baseline resolution is achieved between the two strands in a heterozygous individual. Due to the short size of the amplified fragment, this method can only be applied to routine screening for known mutations because resolution was lost in a fragment 100 bases long.

Acetonitrile as a Buffer Additive for Free Zone Capillary Electrophoresis Separation and Characterization of Maize (Zea mays L.) and Sorghum (Sorghum bicolor L. Moench) Storage Proteins

An improved method for separating and characterizing maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) storage proteins by free zone capillary electrophoresis (FZCE) was developed. Previous electrophoretic methods for analyzing these proteins required high concentrations of urea to maintain protein solubility during separation. To overcome disadvantages of urea, we developed a FZCE method that mimicked reversed-phase high-performance liquid chromatography (RP-HPLC) in that it used high levels of acetonitrile (ACN) at low pH. The optimized FZCE buffer system consisted of 80 mM phosphate-glycine buffer, nominal pH 2.5, containing 60% ACN and a cellulose derivative to dynamically coat capillary walls. Resolution was similar to or higher than that previously achieved by FZCE buffers utilizing 8 M urea as a buffer additive. ACN concentrations of at least 50% were necessary to achieve acceptable separations; this ACN concentration is approximately that necessary to extract these storage proteins. ACN was equally effective as traditional ethanol solvents and 8 M urea for solubilizing maize and sorghum proteins. The ACN-based FZCE buffer system gave high repeatability (<0.3% relative standard deviation, measured over 15 consecutive injections) for migration time. Subclasses of maize and sorghum storage proteins were identified, and genotypes of each cereal were successfully differentiated using ACN-containing buffers. This FZCE method may be applicable for the analysis of other hydrophobic proteins without the use of urea.

Ultrafast capillary electrophoretic analysis of cereal storage proteins and its applications to protein characterization and cultivar differentiation.

Free zone capillary electrophoresis conditions have been improved to allow rapid (2-8 min) separations of grain proteins from several cereals (wheat, oats, rice, barley, and rye) with high resolution and reproducibility. This new method utilized the isoelectric compound iminodiacetic acid (IDA) in conjunction with 20% acetonitrile and 0.05% hydroxypropylmethylcellulose. Cultivars of all cereals tested could be differentiated in 3 min, including wheat, using either prolamin or glutelin protein patterns. Resolution was similar to or higher than that of separations in other acidic buffers. Migration time repeatability was excellent with run-to-run variability <1% RSD, day-to-day <1.4% RSD, and capillary-to-capillary <3.3% RSD. Because larger inner diameter capillaries (50 microm) could be used with this buffer, sensitivity was improved and capillary rinse times could be reduced when compared to smaller capillaries (25 microm i.d.). This also served to reduce total separation time so that the majority of cereal storage protein from several types of cereals could be analyzed with total analysis times of 2-8 min with extremely high resolution and repeatability. This method would allow unattended, high-throughput ( approximately 180-400 samples/24 h) analysis of cereal proteins without the generation of much organic solvent waste as well as automated data analysis and storage.

Separation of selected peptides by capillary electroendoosmotic chromatography using 3 μm reversed-phase bonded silica and mixed-mode phases

The retention behaviour and selectivity of selected basic, neutral and acidic peptides have been studied by capillary electroendoosmotic chromatography (CEC) with Hypersil C8, C18, Hypersil mixed-mode, and Spherisorb C18/SCX columns, 250 (335) mm×100 μm, packed with 3 μm particles, and eluted with mobile phases composed of acetonitrile–triethylamine–phosphoric acid (TEAP) at pH 3.0 using a Hewlett-Packard Model HP3DCE capillary electrophoresis system. The selected peptides were desmopressin (D), two analogues (A and B) of desmopressin, oxytocin (O) and carbetocin (C). The peptides eluted either before or after the electroendoosmotic flow (EOF) marker, depending on the concentration of acetonitrile used and the buffer ionic strength. The retention and selectivity of these peptides under CEC conditions were compared to their behaviour in free zone capillary electrophoresis (CZE), where the separation mode was based on the electrophoretic migration of the analytes due to their charge and Stokes radius properties. In addition, their retention behaviour in RP-HPLC was also examined. As a result, it can be concluded that the elution process of this group of synthetic peptides in CEC with a TEAP buffer at pH 3.0 is mediated by a combination of both electrophoretic migration processes and retention mechanisms involving hydrophobic as well as silanophilic interactions. This CEC method when operated with these 3 μm reversed-phase and mixed-mode sorbents with peptides is thus a hybrid of two well-known analytical methods, namely CZE and RP-HPLC. However, the retention behaviour and selectivity of the selected peptides differs significantly in the CEC mode compared to the RP-HPLC or CZE modes. Therefore this CEC method with these peptides represents an orthogonal analytical separation procedure that is complimentary to both of these alternative techniques.

Separation and characterization of barley (Hordeum vulgare L.) hordeins by free zone capillary electrophoresis.

Extraction conditions, separation conditions, and capillary rinsing protocols were optimized for the separation of barley hordeins by free zone capillary electrophoresis. Stable hordein extracts were obtained with a single 5 min extraction after the albumins and globulins were removed. Hordeins had to be reduced for optimal resolution. Optimum separation conditions for hordein separations were 100 mM phosphate-glycine buffer containing 20% acetonitrile and 0.05% hydroxypropylmethylcellulose. The addition of zwitterionic sulfobetaine detergents containing hydrocarbon tails of eight and ten carbons slightly improved the resolution of the separations, but not enough to warrant their use on a routine basis. The migration positions of the hordein subclasses were determined by two- dimensional reversed-phase high-performance liquid chromatography x free zone capillary electrophoresis mapping. The hordein subclasses formed clusters similar to those of wheat gliadins. Separation-to-separation repeatability was good, with migration time relative standard deviations < 1% for a 15-run period. For routine discrimination of cultivars, a 2 min post-separation rinse with 500 mM acetic acid was necessary to prevent protein build-up on the capillary walls. An example of successfully differentiating barley cultivars using this technique is shown.

Simple and rapid analytical method for carbapenems using capillary zone electrophoresis

A simple and rapid analytical method for carbapenems using high-performance capillary electrophoresis is described. All therapeutic carbapenem injections in Japan (imipenem, panipenem and meropenem) and four other β-lactams (piperacillin, cefotiam, cefotaxime, latamoxef) were separated and determined with good repeatability in about 10 min using simple free zone capillary electrophoresis. The electrophoresis buffer was 100 m M phosphate buffer of pH 8.0, and a fused-silica capillary of 25 μm I.D. and 47 cm length was adopted. The present method was successfully applied to monitor the degradation of carbapenems under various conditions (at various temperatures or in coexistence with other drugs prescribed in the case of methicillin-resistant Staphylococcus aureus).

Chiral separation of aromatic amino acids by capillary electrophoresis

3-Carboxyphenyl-substituted amino acids co-occurring with various types of other aromatic amino acids in plants have been investigated with respect to the chirality at the α-carbon atom. A free zone capillary electrophoresis (FZCE) method using cyclodextrin as chiral selector has been developed and found to be suitable for this purpose. Phenylalanine, tyrosine, tryptophan, and all of the 3-carboxyphenyl-substituted amino acids ( m-carboxytyrosine, m-carboxyphenylalanine, m-carboxyphenylglycine and m-carboxy- p-hydroxyphenylglycine) separated well into their enantiomers and from other naturally occurring amino acids using the developed FZCE method. Identification of the separated enantiomers has been confirmed by use of authentic reference compounds, by spiking and use of stereospecific aromatic amino acid decarboxylase, l- and d-amino acid oxidases. The influence of various parameters on the separation efficiency has been investigated and optimized. Theoretical plate numbers of up to 460 000 N·m −1 have been obtained, and resolutions between d- and l-forms in the range from 0.70 to 1.26 (SD<0.10, n=10) of the investigated compounds have been found. The method has been found to be efficient for the determination of the naturally occurring amounts of d- and l-forms of the four meta-carboxy substituted amino acids, with the possibility of the racemisation of these compounds, and as a highly specific and efficient technique for use in amino acid oxidase and decarboxylase assay.

Separation of siderophores by capillary electrophoresis

Capillary electrophoresis (CE) was applied as a fast method of siderophore separation. Siderophores are iron binding and regulating cell products, which facilitate iron transport into cells. A fast and efficient method of siderophore analysis is important for better understanding of the iron pathways in a sea environment or marine organisms. The best results of CE analysis were obtained using free zone CE in 25 m M phosphate buffer at basic pH using a constant voltage of 20 kV. Under these conditions it was possible to detect the presence of siderophores in seawater.

Preparation of linear polyacrylamide-coated capillaries: Study of the polymerization process and its effect on capillary electrophoresis performance

The effect of different parameters controlling the characteristics of linear polyacrylamide coatings deposited on the inner wall of fused-silica capillaries and their influence on capillary electrophoresis (CE) performance of these coated columns is investigated. To carry out this study, a reproducible procedure to obtain capillaries with similar extent of modification of the surface silanols with 7-oct-1-enyltrimethoxisilane was first approached. Next the polymer attachment to the silica wall, via covalent linkage to the silyl reagent grafted onto the silica, was investigated. In this way, by using columns with a similar silylation extent, differences in CE performance observed among capillaries coated under diverse conditions could be assigned to the characteristics of the polyacrylamide layer. It is demonstrated that the characteristics and reproducibility of these polymeric coatings depend on the adequate control of both the temperature of polymerization and the degassing of the polymerizing dissolutions used. More interestingly, it is also demonstrated that the quantities of monomer (acrylamide), initiator (ammonium persulfate) and activator ( N, N, N′, N′-tetramethylethylenediamine), and the ratio among them used in the preparation of the coating polymer have a large influence on the performance of CE columns. The optimum conditions for preparing the polyacrylamide coatings are discussed. The applicability of these linear polyacrylamide-coated capillaries to the separation of basic and acidic proteins in free zone CE is demonstrated. Besides, the use of these coated columns in capillary gel electrophoresis for the separation of DNA fragments is shown.

Conformation of Systemin, a Polypeptide Activator of Proteinase Inhibitor Synthesis in Plants

An 18-amino acid polypeptide systemin was synthesized by the solid-phase method and its conformation was studied by circular dichroism spectroscopy. The peptide in solution is a mixture of random coil structure with β-sheet and β-turn motifs as has been previously suggested with NMR spectra. Free zone capillary electrophoresis analysis proved purity and chemical stability of systemin at different pH.

Quantitative Determination of Cocaine in Illicit Powders by Free Zone Capillary Electrophoresis

Abstract A free zone capillary electrophoresis method was developed for the quantitation of cocaine in illicit powders. The method uses a 75 mM sodium phosphate-sodium borate pH 8.5 buffered system and separates the most common cocaine adulterants and impurities in eight minutes. Linearity, accuracy and reproducibility studies are presented, as well as comparisons with quantitative results obtained by gas liquid chromatography and high pressure liquid chromatography methods.