Anion-exchange Chromatography Method Research Articles

Estimation of Hexenuronic Acids and Kappa Number in Kraft Pulps of Eucalyptus Globulus by Fourier Transform near Infrared Spectroscopy and Multivariate Analysis

Fourier transform near infrared (FT-NIR) spectroscopy was evaluated as an analytical tool for monitoring residual lignin, kappa number and hexenuronic acids (HexA) content in kraft pulps of Eucalyptus globulus. Sets of pulp samples were prepared under different cooking conditions to obtain a wide range of compound concentrations that were characterised by conventional wet chemistry analytical methods. The sample group was also analysed using FT-NIR spectroscopy in order to establish prediction models for the pulp characteristics. Several models were applied to correlate chemical composition in samples with the NIR spectral data by means of PCR or PLS algorithms. Calibration curves were built by using all the spectral data or selected regions. Best calibration models for the quantification of lignin, kappa and HexA were proposed presenting R 2 values of 0.99. Calibration models were used to predict pulp titers of 20 external samples in a validation set. The lignin concentration and kappa number in the range of 1.4–18% and 8–62, respectively, were predicted fairly accurately (standard error of prediction, SEP 1.1% for lignin and 2.9 for kappa). The HexA concentration (range of 5–71 mmol kg−1 pulp) was more difficult to predict and the SEP was 7.0 mmol kg−1 pulp in a model of HexA quantified by an ultraviolet (UV) technique and 6.1 mmol kg−1 pulp in a model of HexA quantified by anion-exchange chromatography (AEC). Even in wet chemical procedures used for HexA determination, there is no good agreement between methods as demonstrated by the UV and AEC methods described in the present work. NIR spectroscopy did provide a rapid estimate of HexA content in kraft pulps prepared in routine cooking experiments.

An anion-exchange high-performance liquid chromatography method coupled to total organic carbon determination for the analysis of water-soluble organic aerosols

The chemical composition of water-soluble organic carbon (WSOC) in atmospheric aerosol particles is largely unexplored, due to the myriad of individual compounds, which has hampered attempts to attain a full characterization at the molecular level. An alternative approach, focusing on the analysis of a few main chemical classes, allowed the quantitative fractionation of WSOC into neutral compounds (NC), mono- and di-acids (MDA) and polyacids (PA) through an anion-exchange liquid chromatographic method. Previous attempts to quantify NC, MDA and PA relied on a low-pressure chromatographic technique using a volatile buffer, followed by total organic carbon (TOC) analysis of the fractions, or alternatively on a faster HPLC-UV method which provided a quantification of the fractions based on empirical relationships between UV signal and TOC concentration. Here, we report an upgraded anion-exchange HPLC technique, allowing direct TOC analysis of the eluted fractions, without any pre-treatment, thus permitting a great simplification of quantitative analysis and preventing sample losses. The new HPLC-TOC methodology, employing completely inorganic eluents shows the same efficiency of the former HPLC-UV method employing organic additives, with the exception of phenolic compounds, which are retained on the column by secondary interactions, and low-molecular weight tricarboxylic acids, which are recovered among MDA. The new anion-exchange liquid chromatographic method can recover a substantial amount (86–100%) of water-soluble organic carbon from atmospheric aerosol extracts, thus allowing a higher retention and fractionation capacity with respect to alternative techniques, like solid phase extraction.

Separation and determination of carbohydrates in drinks by ion chromatography with a self-regenerating suppressor and an evaporative light-scattering detector

Analysis of glucose and other carbohydrates are often performed by use of normal phase HPLC methods with acetonitrile as major eluent coupled with evaporative light-scattering detector (ELSD) or by use of anion-exchange ion chromatography (IC) methods with NaOH as eluent coupled with pulsed amperimetric electrochemical detector. In this work, a novel method for the determination of carbohydrates by IC in conjunction with a self-regenerating suppressor and an ELSD detector was investigated. Three carbohydrates (glucose, fructose, and sucrose) were separated using a KOH eluent generator to avoid the effect of carbon dioxide absorption in the alkaline eluent. Due to the use of the suppressor, non-volatile components were removed and a low salt background (K + ∼0.070 μg/mL) can be obtained so the suppressed eluent could directly go into an ELSD detector without obvious interference of inorganic salts. After examining the changes in retention and resolution, an optimized method was established (for IC: using 32 mM KOH as the eluent at a flow rate of 1 mL/min; for ELSD: operated at 95 °C, 4.0 bar nitrogen with a gas flow rate of 2.0 L/min) and the linearity, reproducibility, and the limit of detection (LOD) for the three carbohydrates were further evaluated. Regression equations revealed acceptable linearity (correlation coefficients = 0.994–0.998) across the working-standard range (100–1000 μg/mL for glucose and sucrose, 150–1000 μg/mL for fructose) and LODs of glucose, fructose, and sucrose were 93, 126, and 90 μg/mL, respectively. This method has successfully been applied to the determination of the three carbohydrates in carbonated cola drinks and fruit juices. The recoveries were between 95 and 113% ( n = 3) for different carbohydrates.

Iodide analysis by anion-exchange chromatography and pulsed amperometric detection in surface water and adsorbable organic iodide

An anion-exchange chromatography method in combination with pulsed amperometric detection (PAD) was developed for the analysis of dissolved iodide in surface water and in absorption solutions obtained from adsorbable organic iodide (AOI) determination. The development of the amperometric waveform for a selective detection using a silver-working electrode together with the optimization of the injection volume and digital signal smoothing is described in detail. This combination of excellent selectivity, exhibits a detection limit of 0.02 μg/L, without any need of sample treatment other than micro-filtration. The results of AOI determination of the method described in this article are compared with results obtained with a different ion chromatography approach applying UV detection.

Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography

Adeno-associated virus (AAV) empty capsids typically co-purify with genome containing AAV2 vectors purified by column chromatography. This study describes a method to remove empty capsids from genome containing vector particles by anion exchange chromatography. The separation is based on the slightly less anionic character of empty particles compared to vectors. Detailed methods to achieve AAV2 vector purification and particle separation using cation exchange resin POROS 50HS followed by anion exchange resin Q-Sepharose xl are described. Chromatographic separation of AAV2 particles was achieved using gradients based on sodium acetate and ammonium acetate, and was optimal at pH 8.5. Efficient removal of particle surface nucleic acid impurities was found to be important to achieve good particle separation. In a large scale experiment performed using partially purified vector containing a mixture of 1.56 × 10 14 vg and 2.52 × 10 15 empty capsids as a starting material, the optimized anion exchange chromatography method resulted in a vector peak of 1.15 × 10 14 vg containing 0.25 × 10 14 empty capsids, corresponding to 74% vector yield and 86-fold reduction in empty capsids in the vector product.

Recent Developments in Proteoglycan Purification and Analysis

Proteoglycans are ubiquitous biomolecules in the body located in the extracellular matrix, on the cell surface and also within the cells. They contain at least one glycosaminoglycan (GAG) chain covalently attached to a core protein and may also present N- or O-linked glycans. The high structural diversity and distribution relate to the various biological functions of proteoglycans. In recent years, new members have enlarged the proteoglycan family and advances in molecular biology and glycobiology contributed to elucidate more of the biological functions of proteoglycans. In order to study the structure of a proteoglycan molecule and relate it to its function (or dysfunction), its isolation and purification from cell culture or tissue extracts is necessary. Next to the widely used anion exchange chromatographic methods, techniques based on lectin affinity chromatography have created new possibilities to increase the degree of purity. Introduction of electrospray ionization (ESI) and matrix-assisted laser desorption-ionization (MALDI) sources, together with tandem mass spectrometry (MS/MS or MSn), mark a further important step towards the structural analysis of glycosaminoglycans. The aim of this review is to present the most recent advances in proteoglycan purification and analysis.

Ion chromatographic determination of perchlorate in foods by on-line enrichment and suppressed conductivity detection.

Systemic uptake of perchlorate anion, a rocket fuel component and potential thyroid function disruptor, by leafy vegetables and other crops grown in contaminated waters is a public health concern. A column-switching anion-exchange chromatographic method with suppressed conductivity detection, described in this paper, achieved a 3-6 microg/kg method limit of quantitation in analysis of the wet weight edible portion of cantaloupe, carrots, lettuce, and spinach samples with field-incurred perchlorate. A test portion was blended with dilute nitric acid, and the extract was filtered under vacuum. A portion of the measured filtrate was acidified to pH approximately 2 by addition of cation-exchange resin, 4 mL was passed through a graphitized carbon cleanup column, and an aliquot of a collected fraction was pushed through a short precolumn for anion extraction, enrichment, and injection onto the analytical column. Statistical comparison with determination by tandem mass spectrometry-ion chromatography analysis of untreated filtrate revealed that the difference between means was not significant at the 95% confidence level (P value > or = 0.12) for crops tested. In addition, the method was applied to cooked vegetables processed as baby food.

Comparison of micro-enzymatic and high performance anion exchange chromatography methods for the analysis of glucose in human serum

In this study we have performed a comparison of the most used spectroscopic glucose-oxidase assay for routine clinical monitoring of serum glucose, with the high-performance anion- exchange chromatographic method using pulsed amperometric detection (HPAEC-PAD). The linearity, repeatability and internal reproducibility of the chromatographic method were determined. The correlation by linear regression and difference plot was analyzed. It is known that the enzymatic method leads to unreliable results when glucose must be determined in samples containing high concentration of bilirubin or lipids. On the contrary, the accuracy of the HPAEC-PAD method with these complex matrix sera resulted similar to normal samples showing no interferences. Another advantage was the minimal amount of human serum needed: one microliter was largely sufficient to perform the whole analysis. This fact would make the HPAEC-PAD method applicable when samples of preterm infants or new born babies of low weight must be analyzed.

Purification of transferrin from Cohn supernatant I using ion‐exchange chromatography

The present paper describes an anion-exchange chromatography method to separate iron-free apo-Tf (apo-transferrin) from albumin and IgG in Cohn supernatant I. The method uses DEAE-fast flow Sepharose chromatography along with optimized protein concentration (5%, w/v) and column operation conditions (40 g/l, conductivity <1.0 mS/cm) to resolve apo-Tf from IgG and albumin. The single step purifies apo-Tf to >90% and provides an efficient means to obtain commercial quantities of the protein.

Acid glycosidases from hen oviduct and egg albumen

Activities of seven acid glycosidases: β- N-acetylhexosaminidase (β-HEX), α- and β-galactosidase (α- and β-GAL), α- and β-mannosidase (α- and β-MAN), α-glucosidase and α-fucosidase in magnum region of hen ( Gallus gallus domesticus) oviduct, and four acid glycosidases: β-HEX, β-GAL, α- and β-MAN in egg albumen, were investigated. β-HEX from magnum and egg albumen hydrolysed 4-methylumbelliferyl-β- N-acetylhexosamine-6-sulphate (4-MeUmbGlcNAc-6-SO 4) like mammalian β-HEX form A. Multiple forms of magnum and egg albumen β-HEX, β-GAL, α- and β-MAN were separated by strong anion exchange chromatography and chromatofocusing method. Chromatofocusing of the magnum resulted in the appearance of multiple forms for β-HEX with pI of 6.18, 5.43, 5.55, 5.34, 5.27 and 5.16, for β-GAL with p I of 4.98, 4.84, 4.77, 4.64 and 4.68–4.63, for α-MAN with p I of ≥ 7.4, 6.75, 6.62 and 6.26, and for β-MAN two forms with p I of 6.37 and 5.77. Chromatofocusing of egg albumen yields multiple forms for β-HEX with p I of 6.24, 6.08, 5.55 and 5.35, for β-GAL two forms with p I of 5.10 and 4.86–4.80 for α-MAN multiple forms with p I of ≥ 7.4, 6.80, 6.60 and 6.30, and for β-MAN forms with p I of 6.30 and 5.77. In conclusion, this study was the first to show β-HEX activity against 4-MeUmbGlcNAc-6-SO 4 in the magnum and albumen of bird eggs, corresponding to β-HEX A activity in mammals. Main multiple forms of β-HEX, β-GAL, α- and β-MAN occurring in the magnum were revealed in the egg albumen. Comparison with a cock of the same breed showed that hen egg magnum and albumen has the same multiple forms of the enzymes that are found in the epididymides and seminal plasma of the cock.

High-performance anion-exchange chromatography combined with intrinsic fluorescence detection to determine erythropoietin in pharmaceutical products

A high-performance anion-exchange chromatography (HPAEC) method was developed for determination of recombinant human erythropoietin (EPO) in pharmaceutical products. A fluorescence detector was added to the HPLC system as intrinsic fluorescence detection compared favourably to UV detection regarding sensitivity and selectivity. The HPLC method has been successfully applied to analyse erythropoietin products even in the presence of albumin as excipient. The intrinsic fluorescence chromatograms of both proteins revealed various peaks attributed to either micro-heterogeneous erythropoietin or albumin variants. The intrinsic fluorescence signal was linear over the range 10–200 μg/ml erythropoietin corresponding to pharmaceutically relevant concentrations. The HPLC method appeared to be a suitable method for differentation between recombinant human erythropoietin epoetin-alpha and -beta as they revealed different intrinsic fluorescence elution profiles. In conclusion, this study contributes to the development of a straightforward physicochemical method for specific quantification of recombinant human erythropoietin in pharmaceutical preparations.

An experimental investigation of accumulation and transmutation behavior of americium in the MOX fuel irradiated in a fast reactor

Americium isotopes generated in the MOX fuel irradiated in the experimental fast reactor JOYO were analyzed by applying a sophisticated radiochemical technique. Americium was isolated from the irradiated MOX fuel by a combined method of anion-exchange chromatography and oxidation of Am. The isotopic ratios of americium and its content were determined by thermal ionization mass spectroscopy and α-spectrometry, respectively. The americium isotopic ratio was similar for all the specimens, but was significantly different from that of PWR-MOX. On the basis of present analytical results, the accumulation and transmutation behavior of americium nuclides in a fast reactor is discussed from the viewpoints of neutron spectrum dependence and the isomeric ratio of the 241Am capture reaction. The estimated isomeric ratio is about 87%, which is close to the latest evaluated value. A rapid estimation method of Am content by using the 240Pu to 239Pu ratio was adopted and proved to be valid for the spent fuel irradiated in the fast reactor.

On the use of anion exchange chromatography for the characterization of water soluble organic carbon

An increasingly popular anion exchange chromatography method [Decesari et al., 2000] was used to separate organic matter in fog samples and water soluble organic carbon (WSOC) extracted from aerosol samples according to acidity. Analysis of both fog and aerosol WSOC samples showed results similar to previous studies, with peaks typically identified as corresponding to neutral/basic compounds, mono‐ and dicarboxylic acids and polyacids. In one example this approach was shown to classify a total of 82% of the WSOC into these three chromatographic fractions. Challenges to the classification scheme were made by injection of single compound solutions. Compounds were chosen to be representative of compounds observed in atmospheric samples. In many cases test compounds eluted in fractions other than expected based on classifying compound structure according to the three classes outlined above. The impact of classification errors is impossible to quantify without understanding the complete organic speciation of a sample, but is serious enough that researchers relying on this method to provide a suitable model for organic aerosol composition should interpret results with caution. The most severe problems are likely for fog samples, due to a prevalence of low molecular weight carboxylic acid and carbonyl compounds which exhibit greater tendency to be misclassified.

Membrane Purification of an Antisense Oligonucleotide

This contribution describes a membrane chromatography purification of a therapeutically useful antisense oligonucleotide (ASO). Sample self-displacement purification conditions were optimized for a 20-mer ASO on a 10-mL bed volume, high-resolution, strong anion-exchange membrane chromatography unit. The scale-up experiments were performed at 177-g scale on a 1-L bed volume unit that resulted in 84.4% yield and 95.0% (analyzed by LC−MS) pure full-length ASO following the membrane chromatography process. The purified ASO was subsequently significantly concentrated and diafiltered into water using a 650-Da MWCO ultrafiltration membrane. This robust, high throughput, membrane-based, anion-exchange chromatographic (AX) method simplifies the process-scale purification of phosphorothioate oligonucleotides by eliminating the need for column packing and packing validation.

Determination of phytic acid and inositol pentakisphosphates in foods by high-performance ion chromatography.

A high-performance anion exchange chromatographic method was adapted for the quantitative determination of phytic acid and inositol pentakisphosphate isomers (excluding enantiomers) in foods. Because of the cost and limited availability of inositol phosphate standards, a phytic acid sodium salt standard was used for the calculation of an average relative response factor for the quantification of inositol pentakisphosphate isomers, and the purity of phytic acid sodium salt standard was also accurately established. The detection limits (S/N = 3) for phytic acid and inositol pentakisphosphates were in the range of 1.5-3.4 microM (0.1-0.2 microg/100 microL). This method has been successfully applied to the determination of phytic acid and inositol pentakisphosphates in a variety of beans and nuts after extraction with 0.5 M HCl and cleanup with solid phase extraction cartridges. The results demonstrated that there was a strong correlation between either the phytic acid content or the total content of phytic acid together with inositol pentakisphosphates and the total dietary fiber content in the group of all raw dry beans and in the group of raw dry black beans but not in the group of raw dry red kidney beans, which was probably due to the insufficient number of the raw dry red kidney bean samples.

A comparison of pulsed amperometric detection and spectrophotometric detection of carbohydrates in cider brandy by liquid chromatography

A high-performance anion-exchange chromatographic method that uses an alkaline mobile phase (20 mM NaOH) on an anion exchange column (RCX-30 Hamilton) at 30 °C coupled with pulsed amperometric detection was applied to the analysis of the glucose, galactose, xylose, arabinose, ribose, fucose, rhamnose and fructose in cider brandies. Data were compared with those obtained from aldoses- and uronic acids- p-aminobenzoic ethyl ester derivatives analysis by HPLC on a C 8 column at 45 °C with spectrophotometric detection. Both methods were validated and their advantages and drawbacks taken into account.

A Miniaturized Column Chromatography Method for Measuring Receptor-Mediated Inositol Phosphate Accumulation

Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP(3)), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen assays, offer higher throughput. However, these techniques rely on measurement of IP(3) itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP(3). The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP(3) and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.

La transferrine hyposialylée et le % CDT. Mesure par électrophorèse capillaire avec le coffret CEofix CDT (ANALIS) chez des sujets consommateurs modérés

Twenty-four years after its discovery, the marker of alcohol, carbohydrate deficient transferrin (CDT) is still controversial (explained by inappropriate patient recruitment and different methodologies). Today, reflecting the performance of anion exchange chromatography methods, the definition of CDT comprises asialo-, mono- and disialotransferrin and the results are expressed in percent of total transferrin: %CDT. However, recent studies sensitize us on isoform visualisation (interference by non-CDT isoforms) and their individual quantification (asialotransferrin as marker of alcohol?). Recently, with the Ceofix CDT kit (Analis, Belgium), capillary electrophoresis is easily applied. We measured the %CDT in 68 moderate drinkers on a P/ACE MDQ (Beckman Coulter) using the CEofix CDT kit. Asialo- and mono- were not detected (except in one man for asialo-) and the %CDT was disialo- on all isoforms detected with EC. The %CDT mean of our population was 0.6% (±0.3). Today, some technical modifications are realized and additional studies are in progress.

Separation of phytic acid and other related inositol phosphates by high-performance ion chromatography and its applications

A high-performance anion-exchange chromatographic method was developed for the separation of phytic acid and other inositol phosphates ( myo-inositol bis-, tris-, tetrakis-, and pentakisphosphates) with gradient elution and ultraviolet absorbance detection after post-column derivatization. With the acidic eluents, the combination of anion-exchange and ion suppression retention mechanisms led to the separation of 35 inositol phosphates (excluding enantiomers) into 27 peaks for the first time, and the retention behaviors of all myo-inositol bis- to hexakisphosphate isomers were studied. The whole separation procedure was completed within 65 min. Based on the investigations of nonenzymatic hydrolysis of phytic acid under different conditions by using this method, an in-house reference standard solution was produced, which can be used for method development. In addition, by applying this method to in vitro kinetic studies, at least one new enzymatic hydrolysis pathway of phytic acid was found, and one rule of enzymatic dephosphorylation of inositol phosphates (position effect) was proposed and another one (neighboring effect) was confirmed. The principle of the proposed identification approach for several inositol phosphate isomers based on hydrolysis products study will be applicable to other natural products analysis, for which standards are very expensive or not available.

Cloning and expression of the vegetative insecticidal protein ( vip3V) gene of Bacillus thuringiensis in Escherichia coli

A genomic library of Bacillus thuringiensis var. kurstaki ( B.t.k.) was constructed and a positive clone harboring the full-length gene encoding a novel vegetative insecticidal protein (Vip3V) was characterized. The vip3V gene was subcloned into pET-22b(+) vector and overexpressed in Escherichia coli to an extent of about 30% of the total protein. While transcription was influenced by the T7 promoter of the vector, synthesis of Vip3V in E. coli host occurred from the B.t.k. ribosomal binding site (rbs) found 917 bp downstream of the insert and not from the E. coli rbs of the vector. The expressed Vip3V protein was found in the soluble and periplasmic fractions as well as in the inclusion bodies. A simplified anion-exchange chromatographic method for the purification of Vip3V using step gradient or one-step elution was developed. The purified protein showed broad-spectrum activity against some of the lepidopteran larvae tested and did not show any activity against the larvae of silkworm ( Bombyx mori) and mosquito ( Culex quinquefasciatus).