PDF Fundamentals and Applications of Anion Separations

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Polymer-Supported Reagents for Anionic Recognition. Grant C. Lukey, Jannie S. Influence of the Speciation of metal ions on their sorption on Chitosan.

Jan D. Miller, Gustavo A. Back Matter Pages Nearly 40 papers devoted to discussions on anion separation related to fundamental research and applications were presented. Box , Building.

Frontmatter - Ion Chromatography - Wiley Online Library

Separations constitute an integral part of chemical industry. Chemical products typically originate in resources that must be concentrated and purified, chemically transformed, and subjected to fmal purification. Effluent streams from the processes must be treated to recycle reusable components and to remove environmentally harmful species. Some industrial processes are devoted to environmental cleanup after pollution has occurred.

In addition, many analytical methods require a separation for preconcentration, or a separation may be an inherent part of the analysis itself. He will present recent work from the lab dealing with unfolding and aggregation of proteins in chromatography columns. Arch instituted numerous safety features into the new lab space in ChE using 6S principles he acquired during his internship.

Some of the changes include: tubing from the AKTAs, HPLC, and UPLC going directly to waste bins, new magnetic labels for all drawers and cabinet space, ,and standardized techniques for disposing of filled waste containers. Congratulations Arch.

Fundamentals and Applications of Anion Separations

Arch Creasy center along with Prof. Lucas Kimerer and Yiran Wang took a break from their research and conducted a live protein chromatography demonstration which illustrated the separation of a mixture of three proteins by gradient elution. Thanks to Lucas and Yiran for organizing and conducting an informative demo. Watch the full interview at the following link:. Shaojie was selected for his research in our group dealing with protein adsorption in Protein A and AEX resins in relationship to resin fouling and weak partitioning chromatography.

We congratulate Shaojie for this great honor. HPAE chromatography separates anions under high pH conditions. Carbohydrates typically have pKas in the range of 12— Once the pH rises above the pKa of the carbohydrate, these carbohydrates ionise more specifically, become oxyanions and are then separated.

Introduction

These separations require hydroxide-based eluents. For hybrid or complex carbohydrates, separations are accelerated and improved by using sodium acetate gradients in sodium hydroxide. The analytes are separated using one of an extensive Dionex CarboPac column portfolio developed specifically for carbohydrates ranging from mono-, di-, oligo-, and polysaccharides.

Principles of Ion Exchange Chromatography

These highly cross-linked, ethylvinyl benzene-divinyl benzene pellicular resins have broad pH stability ranging from 0 to 14, allowing separations at high pH conditions. Silica based stationary phases are not stable in high pH mobile phases, limiting their use in carbohydrate separations using an ion exchange mechanism [7]. Once separated, these non-derivatised carbohydrates are detected on a gold working electrode WE surface by PAD, which is a direct detection technique. Alternative direct detection techniques available for LC of carbohydrates are short wavelength ultra-violet light UV and refractive index RI , both of which lack the sensitivity required for analysing mono- and oligosaccharides from glycoproteins [7] [6].

Additionally, the detection of these non-derivatised carbohydrates using UV is limited to the choice of the solvent being used. As most of the solvents and carbohydrates absorb quite strongly below nm. PAD easily addresses the sensitivity issue, as it is able to routinely detect low picomolar amounts of carbohydrates. This method applies a series of potentials a waveform applied two times per second 2 Hz to a gold working electrode, at high pH, resulting in the oxidation of analytes bound to the working electrode surface.

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PAD only detects compounds that contain functional groups that can get oxidised at the detection voltage. Detection is sensitive and highly selective for electroactive species, as many potentially interfering species cannot be oxidised or reduced, and so are not detected. A comprehensive monosaccharide profiling is required for biosimilars to meet FDA regulations.

This comprehensive monosaccharide analysis helps in the identification and quantification of the different types of glycosylation occurring in these proteins.

Principles of chromatography

In the biosimilar development process, it is critical to analyse monosaccharides in earlier stages to address any issues related to their manufacturing process. This information will prevent the development of an inappropriate products that would not meet the criteria stipulated by the FDA for biosimilars. Sample Preparation: To prepare glycoprotein samples ahead of injection on the HPAE-PAD system for monosaccharide analysis - a sample with PNGase F were first hydrolysed with slightly varied conditions for neutral sugar or amino sugar analyses Figure 1. The acid-hydrolysed samples were dried in a SpeedVac concentrator Thermo Scientific equipped with an acid trap and reconstituted in a small volume of deionised water [7].

Samples were acid hydrolysates of specified proteins. Peak results are shown in pmol.


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The Dionex CarboPac PA20 delivers excellent monosaccharide resolution while minimising mobile phase consumption and waste generation. The eluent generator eliminates error associated with hydroxide mobile phase preparation e. Inclusion of a Dionex AminoTrap column Thermo Scientific, Sunnyvale, USA delays the elution of amino acids and small peptides from the glycoprotein acid hydrolysis so that they do not interfere with monosaccharide quantification.

After separation, the monosaccharides are detected on a gold working electrode. The amounts of galactosamine GalN and glucosamine GlcN have been determined as an indicator of O- and N-glycosylation, respectively. The identification of the monosaccharides present in both the treated and untreated PSA were compared with the monosaccharide standards, highlighting the sensitivity of the HPAE-PAD system. In both cases, glucosamine and galactosamine were present in roughly the same molar ratio of 0. This suggests that for this PSA sample, the galactosamine was not a result of O-glycosylation but is associated with the PSA N-glycans, or that there was a contaminating protein with O-glycosylation that is not removed during the sample preparation to separate protein from released N-glycans.

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Sialic acids are critical to therapeutic glycoprotein efficacy, bioavailability, function, stability, and metabolism. When present, they occupy terminal positions in glycosylated proteins, providing charged points of interaction essential in many biological pathways.


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Any unexpected sialylation in biosimilars can cause immunogenicity, so it is critical to monitor total glycoprotein sialylation, and the identification of the sialic acids when changing process and or conditions. Although over 50 natural sialic acids have been identified, two forms are commonly determined in therapeutic glycoprotein products: N-acetylneuraminic acid Neu5Ac and N-glycolylneuraminic acid Neu5Gc. Due to the potential immunogenicity of Neu5Gc, it is considered undesirable in therapeutic proteins and must be tested for [7].

Sample Preparation: Neu5Ac and Neu5Gc, are negatively charged at pH 7 and acid-labile so require acetate in the eluent and weak acid conditions for hydrolysis.