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SEC-Light Scattering for Biomolecular Characterisation
The area of biomolecular characterisation for molecular weight determination has increased rapidly with the requirements for a complete picture of the behaviour of biomolecules in solution being both critical not only for the academic understanding, but also from a regulatory viewpoint for safety and efficacy. For example with bioconjugates such as pegylated interferon, used in the treatment of chronic hepatitis, it is essential for the molar masses to be above that of the kidney cutoff to incre...
Gel Permeation Chromatography: an Evolving Technique for a Changing Polymer Industry
Stimulated by the sustainability agenda, the polymer industry is actively engaged in developing new materials that combine excellent performance with low carbon footprint. Molecular weight is a defining characteristic of all polymers and often is one of the parameters manipulated to achieve desired performance. Gel permeation chromatography (GPC) is the method of choice for molecular weight characterisation. This paper provides an introduction to GPC and examines how it is evolving to meet the c...
Review of CSI Activities for the Year 2011
Soon after a very successful symposium on the ‘Essential Oils, Flavors and Fragrances’ in November 2010, Chromatographic Society of India (CSI) entered yet another exciting year of activities. It was promised during the above symposium that there will be a workshop conducted on GC and GCMS for the benefit of those in the Essential Oils industry. Therefore, a two-day workshop on GC and GCMS was conducted in collaboration with the Shimadzu Analytical Centre in Mumbai. Only a limited number of 16 p...
Optimising Core-Shell UHPLC Columns for Improving Protein and Peptide Separations
The introduction by several chromatography column manufacturers of sub-2 µm fully-porous as well as core-shell sub-3 µm UHPLC columns over the last several years have profoundly impacted the chromatography industry in the analysis of small molecules. These advances in silica particle technologies have for the most part focused on using increased efficiency as a means of reducing run times while maintaining resolution of existing separations.1,2 The fundamental concept for all new silica media (s...
The Challenge of Using Small Particle Packing Materials in SEC/GPC Risks and Possibilities
About 5 years ago, [1] the particle size of packing media in HPLC columns was reduced to 2 µm or smaller. This new column technology named U-HPLC or UPLC offers much better resolution and column efficiency compared to standard 5 µm packing materials. For SEC/GPC, the 2 µm range can also be quite interesting but very challenging as well. In contrast to HPLC, SEC mostly deals with very large molecules. The diameter of these large molecules can easily be in the range of the interstitial volume of t...
Characterisation of Polymers Using Gel Permeation Chromatography and Associated Techniques
Gel permeation chromatography (GPC), also known as size exclusion chromatography (SEC) is a technique for obtaining information on the molecular weight distributions of polymers. After polymer type (chemical composition), the molecular weight distribution is the next most fundamental property and this will determine if a particular material is well suited to any given application. In crude terms, the higher the molecular weight, the stronger and more resilient a polymer material is likely to be,...
Using Online Sample Preparation with LC-MS/MS to Speed Up the Process of Analysing Pharmaceutical and Personal Care Products
There is growing environmental concern regarding the health impact of trace levels of pharmaceuticals and personal care products (PPCPs) in water resources. Although the term PPCP was only recently coined, these bioactive chemicals have been in existence for decades, and their effect on the environment is now recognised as an important area of research. PPCPs include prescription and overthe- counter therapeutic drugs, cosmetics, and nutraceuticals such as vitamins. These substances can find the...
“Advances in Separation Science” The Chromatographic Society Spring Symposium 2011
As delegates weaved their way through leafy West Sussex on the morning of 11th May it was a bright sunny day. These were gloomy times though for R&D in major UK pharma but to all intents and purposes it was just like any other ChromSoc Spring Symposium at a major pharmaceutical site. There was a buzz in the air with just over 100 delegates attending and lively networking taking place in the exhibition area (15 exhibitors and/or sponsors: Waters (Gold Sponsors), Agilent Technologies, ARC Sciences...
Evolution of Mixed-Mode Chromatography
Mixed-mode chromatography is emerging as a powerful tool in separation of various molecules. Mixed-mode is defined as liquid chromatography in which at least two modes of interactions exist simultaneously, both controllable by mobile phase selection. In the last few years, several companies have introduced new mixed-mode columns with different stationary phases (SIELC, Dionex, Imtakt, Sepax, Agela, etc). Mixed-mode approach emerged due to a need for better retention control for both polar an...
Ionic Liquids: Sensitivity Enhancement in Headspace Gas Chromatography
A relatively new application for ionic liquids, as solvents in static Headspace Gas Chromatography, is further investigated. Two ionic liquids were studied and the response towards a range of low boiling point residual solvents increased compared to when dimethyl acetamide was used as the headspace diluent. An attempt to correlate results with a suitable solvent model was made. The results highlight the potential of ionic liquids to enhance headspace sensitivity for residual solvent analysis....
Separations-on-a-chip? Scientists at Liverpool Put a Different Spin on Things!
We have all read about lab-on-a-chip (LOC) devices (or if we hadn’t already, we will now after reading this issue of Chromatography Today!) and how they may offer the possibility of miniaturising analytical instruments. However one thing continues to let them down and that is how do you move fluids around the chips? The pumping mechanisms within these devices can be very complex and difficult to implement reproducibly as it commonly uses external pumps or high voltages. Scientist at the Univers...
Droplet-Based Sample Injection for Chip-Based Analytical Separations
The advent of capillary and chip-based electrophoresis has opened up new possibilities for high-throughput analysis of a diversity of biological systems. The high-resolution separations typical of chip-based electrophoresis are in large part defined by the ability to introduce small sample volumes into the separation channel. This is not a trivial process and standard electrokinetic and hydrodynamic methods are far from ideal. Herein, we review recent studies that integrate droplet-based and con...
The mAb-Glyco Chip Kit - a Workflow Solution for Rapid and Fully Automated Characterisation of N-linked Glycans from Monoclonal Antibodies
Monoclonal antibodies (mAbs) are an important class in the group of new biological entities with about 30 antibody drugs licensed for treatment of various diseases [1]. These glycoproteins bear complex oligosaccharide moieties within their structure, whose presence, absence, and profile can have significant impact on therapeutic efficacy, pharmacokinetics, and immunogenicity. Glycosylation is influenced by many factors such as the cell line in which the mAb is produced as well as specific produc...
Microfluidics-Based Separations Technology for the Analytical Laboratory
A seminal paper was written by Manz et al. (1) identifying the use of possibilities of using ‘chip based technology’ within an analytical instrument. When did you as a scientist first think that ‘chip technology’ could be a useful addition to analytical instrument design and separation science in particular? In the early 1990’s we saw a lot of enthusiasm for the “Lab-on-a-Chip” concept. And at the time, the concept of integrating multiple laboratory processes like sample prep, cell lysing,...
Hydrophilic Interaction Liquid Chromatography - A Potential Alternative for the Analysis of Dextran-1
A method for the analysis of dextran-1 has been developed using hydrophilic interaction liquid chromatography (HILIC) with charged corona aerosol detection (CCAD) as a potential alternative to the size exclusion chromatography method described in the European Pharmacopoeia (EP). The EP method is in excess of 500 minutes and is considered long for routine analysis of multiple samples. The developed method retains resolution between monomers, whilst performing separation within 13 minutes. Introd...
Chromatography……..but not as we know it
The use of countercurrent chromatography (CCC) as a preparative technique is seen to occupy a niche area of separation science and is largely used to isolate natural products. The technique has however considerable untapped potential both at the laboratory preparative scale and also at larger scale…………. Introduction This article gives a chromatographer’s perspective of the potential of countercurrent chromatography (CCC) in the pharmaceutical industry, specifically relating to highperformance...
Chromatography Today talks to Howard Hill
Up until now, interviewees featuring in Chromatography Today have been drawn from individuals who have been recipients of Martin or Jubilee Awards of The Chromatographic Society. Illustrious though this group very much is, they do not have exclusive ownership of all comment that is worth making in the field of analysis and separation science. Accordingly, we are more than happy to turn to Howard Hill (of contract researchers HLS) who not only has his finger on the pulse regarding biopharmaceutic...
Water Condensation Nucleation … a Universal detection technique opening up new HPLC applications
Introduction Water Condensation Nucleation (or Water Condensation Particle Counting) is a technology originally developed to detect aerosol particles in the application field of environmental monitoring. Using this technique air was drawn through an instrument and any particles counted to produce a global particle count for the environment. By introducing a liquid stream rather than a gas flow the technology has been now been refined for use in Liquid Chromatography. Since Water Condensation...
Correlation of Analyte Retention in Organic and Inorganic Mobile Phases to aid Liquid Chromatography Method Development
Many liquid chromatography methods are developed using organic mobile phase additives which allow compatibility with mass-spectrometric (MS) detection. However, these types of additives often give high UV absorbance which can lead to low level impurity quantitation issues. Additionally, these additives often have little or no buffering capacity at the pH they are typically used, which in turn can lead to variability in analyte retention time. A rational approach for the selection of phosphate...
Analysis of Aminoglycosides with a Zwitterionic HILIC Stationary Phase and Mass Spectrometry Detection
A method was developed to analyse three model aminoglycosides (neomycin, apramycin, and kanamycin) using a zwitterionic HILIC (hydrophilic interaction liquid chromatography) stationary phase and mass spectrometry (MS) detection. High separation efficiency and good peak shape were obtained with an optimal combination of mobile phase composition and gradient profile. Gradient elution proved to be reproducible and a good way to combine both HILIC and electrostatic interactions to obtain unique sele...
Use of Fortis HILIC stationary phases
Hydrophilic interaction liquid chromatography or HILIC is a technique that has been around for quite some time, based somewhere between reversed-phase (RP) and normalphase (NP) chromatography. Understanding HILIC A highly polar stationary phase, often bare silica, is used with largely a non-polar mobile phase system (water acting as a very strong solvent in HILIC) to provide retention of polar or hydrophilic molecules. The mechanisms of HILIC are still being fully explored but it is quite wel...
HILIC After the Hype: A Separation Technology here to Stay
Today Hydrophilic Interaction Liquid Chromatography (HILIC) is by far the fastest growing HPLC separation mode, currently being used by almost 20% of the HPLC instrument owners [1], although the relative number of applications still is considerably fewer. Does this mean HILIC is yet another technology-hype on the separation science scene, and that the interest will soon vanish? We believe not. This paper reviews the reasons behind the remarkable rise of HILIC and based on that, tries to predict...
Advances in Separation Science - The Chromatographic Spring Symposium & Annual General Meeting
The Chromatographic Society’s Spring Symposium and Annual General Meeting will be held at the Novartis site in Horsham (West Sussex) on Wednesday 11th and Thursday 12th May 2011. The theme for the meeting is “Advances in Separation Science” with a particular focus on pharmaceutical drug discovery. The Spring Symposium will be held over one-and-a-half days allowing for an extensive range of presentations from academics, industrial speakers and vendors, as well as significant exhibition and networ...
Using a Triple Quadrupole GCMS/ MS System for Rapid Analysis of 303 Pesticide Residues in Green Beans
Pesticide analysis is extremely important due to the need to ensure that foodstuff is not contaminated with pesticide residues, which can be harmful to human health. Pesticide analysis poses a number of challenges for laboratories due to the wide ranging chemistries within the contaminants. As the variety of pesticides continually expands, techniques traditionally used for pesticide residue analysis in laboratories require that one sample be analyzed using several injections with different metho...
Separation of Fucosylated and non-Fucosylated Carbohydrates Associated with Monoclonal Antibodies using Capillary Electrophoresis
In order to gain a comprehensive understanding of therapeutic Monoclonal Antibody (MAb) function, it is necessary to critically characterize glycosylation associated with them. Carbohydrates, and therefore glycosylation, are known to play an important role in the structure, function, and clearance of MAbs and have been shown to be responsible for invoking immune responses in humans. Changes in carbohydrate composition or concentration can significantly impact the overall efficacy of therapeutic...
The Utilisation of Stereolithography Rapid Prototyping, to drive the design and testing of a new Chromatographic technique, Dynamic Field Gradient Focusing (DFGF).
In developing new analytical instruments and in particular chromatography instrumentation there is a need to modify or manufacture components to perform experiments and initiate the innovation. Usually this means anything from ordering bespoke parts to fabricating components and devices in house. At the University of Liverpool the development of a new technique in chromatography, Dynamic Gradient Field Focusing (DFGF), is moving forward with the aid of some of the latest advances in Stereolithog...
Pellicular anion-exchange chromatography applied to RNAi assays for monitoring strand stoichiometry and RNA stability.
Abstract RNAi products require both single strand and duplex impurity assays. Duplex assays should allow titration of guide or passenger strands that may be in excess. These assays are chromatographic, and typically employ two steps: First, as is common to other single-stranded therapeutics, denaturing conditions must resolve both guide and passenger strand, and thus may reveal impurities formed during the annealing process. Next, native conditions, where the duplex is stable, are used to reso...
Ion Pair Reverse-Phase Chromatography: A Versatile Platform for the Analysis of RNA
Introduction The requirement for high throughput analytical tools that can readily separate, purify and analyse ribonucleic acids (RNA) are assuming increasing significance with the recent discoveries of the diverse and important roles RNA plays in biological systems. RNA consists of a long chain of nucleotide units made up of a nitrogenous base, a ribose sugar, and a phosphate. Cells contain three abundant classes of RNA including messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (r...
Preparation and LC/MS Analysis of Oligonucleotide Therapeutics from Biological Matrices
Due to their association with other biomolecules as well as their unique chemical properties, oligonucleotide therapeutics present unique challenges in their isolation and LC/MS analysis from biological matrices. A new isolation protocol using a mixed-mode SPE cartridge is presented that demonstrates high recovery and sensitivity into the low ng/mL range. Optimization of LC/MS mobile phase conditions as well as MS analysis parameters results in improved sensitivity and identification of oligonuc...
Oligonucleotides: The Next Big Challenge for Analytical Science
With the rapid growth of interest in oligonucleotides as therapeutic medicines, the Chromatographic Society recently held at a two day symposium, from 27 – 28th October 2010, on the challenge of analysing these molecules. The venue was the very comfortable GlaxoSmithKline Amenities Centre, Ware, UK. The presentations, stretching over two information packed days, included talks from Pharma companies, CROs, academic institutions and instrument and column vendors. Whilst the majority of presentat...
Oligonucleotide Biopolymers - Future Challenges for Chromatography
Synthetic Oligonucleotides as Therapeutic Medicines Synthetic oligonucleotides are an exciting new class of biomolecules capable of treating many disorders, which are currently not amenable to existing drugs, including viral infections [1], respiratory disorders [2], cancers [3] and rare diseases [4]. Current interest has been largely fuelled by two key events: firstly, Fire and Mello’s Nobel-prize winning discovery of gene silencing by RNA interference (which helped to improve our understand...
Chromatographic Society Meetings Round-Up - E A Adlard
The topic of this meeting was “Current Method Development Strategies in Separation Science” although a more accurate title would have been “Current Method Development Strategies in HPLC in the Pharmaceutical Industry” since this was the theme of all the papers except for one on SFC. To one who has been in the field for a long time and can now afford the luxury of an overview the current situation in HPLC seems to bear a resemblance to GC in the days of packed columns when there were hundreds of...
Recent Developments in Type C Stationary Phases: Exploiting the Versatility of Silica Hydride Materials - Joseph J. Pesek
TYPE C™ silica is a relatively new chromatographic material that has been finding ever-increasing use in the last few years. The properties exhibited by these stationary phases are often significantly different than the ordinary silica used for most commercial products. While all TYPE C phases can be utilized in the reversed-phase, organic normal phase and aqueous normal phase modes, there are some unique capabilities within each retention mode that have resulted in innovative method development...
Are Separation Scientists suited to Metabolomics? - Cristina Legido-Quigley and Norman W Smith
Metabolomics focuses on metabolite profile changes in diverse living systems caused by a perturbation. These metabolite signatures can be measured using numerous analytical instruments. Separation techniques used include gas chromatography, high-performance liquid chromatography, ultra-high-performance/pressure liquid chromatography (UPLC), capillary HPLC (cap-HPLC), capillary electrophoresis, and capillary electrochromatography, often linked to a mass spectrometer. These techniques are ideal fo...
Professor Peter J. Schoenmakers: Winner of the 2011 Chromatographic Society Martin Medal - Dr Paul Ferguson
Professor Peter J. Schoenmakers of the Faculty of Science, van‘t Hoff Institute for Molecular Sciences at the University of Amsterdam has been awarded the 2011 Chromatographic Society Martin Medal. The medal is named after Professor A.J.P. Martin who in 1978 gave permission for his name to be associated with this award. The ’Martin Medal’ is the highest honour the Society confers and is awarded to scientists who have made outstanding contributions to the advancement of separation science. Pete...
Chromatography Today talks to Kevin Altria (GSK (Harlow) CE Expert and 2006 Chromatographic Society Jubilee Medallist - John Lough
In this latest in a series of occasional interviews with Chromatographic Society medallists it is only appropriate that for this issue the subject of the interview should be an individual who has made a substantial contribution to the field of electrophoresis. In the UK, and indeed much further afield, the name Kevin Altria and the technique capillary electrophoresis (CE) are inextricably linked. Perhaps it is an indication that CE is still in rude health that this is the second interview that K...
Capillary Electrophoresis coupling to Mass Spectrometry (CE-MS), an advanced technique orthogonal to LC-MS for high resolution separation and accurate molecule identification - Martin Greiner
Capillary Electrophoresis (CE) coupled with Mass Spectrometry (MS) has been under investigation for the past couple of decades1. Combining the high separation power of CE with MS promises to yield more sensitive detection and higher information content on the analytes than current methods such as standard UV detection. Since those early days, there have been many improvements and technology advancements on CE and MS instruments, as well as the interfacing devices. Modern Time of Flight (TOF) MS...
3-Dimensional Retention Modelling of Gradient Time, Ternary Solvent-Strength and Temperature of the Reversed-phase Gradient Liquid Chromatography of a Complex Mixture of 22 Basic and Neutral Analytes using DryLab® 2010 - Melvin R Euerbya, Gesa Schada,
The present paper describes a multi-factorial optimization of three critical HPLC method parameters, i.e. gradient time (tG), temperature (T), and ternary composition (B1:B2) based on twelve experiments for the separation of twenty-two pharmaceutically relevant analytes. Examining the effect of these experimental variables on critical resolution and selectivity was carried out in such a way as to systematically vary all three factors simultaneously. The basic element is a gradient time–temperatu...
200 years of Electrophoresis - David Perrett
This brief overview outlines the development of electrophoresis from its first observation some 200 years ago via conventional gels for macromolecule separation and capillary electrophoresis (CE) to current developments centred around lab-on-chip. By definition electrophoresis separates ionic molecules so it is ideal for the separation of simple ions to macromolecules, which are mostly ionic in nature. Most important classes of small biomolecules e.g. amino acids, nucleotides and sugars are high...
Separation, size and charge determination of small molecules using CE in combination with UV area imaging - David M. Goodall, Ed Bergström and Mebs Surve
This article shows how a capillary electrophoresis instrument may be used in conjunction with UV area imaging to determine mobility, diffusion coefficient and charge of components in a mixture of small molecules. The analytes lidocaine, phenylmethanol and benzoate are chosen as representative of small molecules of different charge type: cationic, neutral and anionic respectively in the background electrolyte used, phosphate buffer at pH 7.5. A capillary with three windows is used, with detection...
Chromatographic Society Meetings Round-Up - Advances in High Resolution and High Speed Separations - John Lough
Belying the general gloomy atmosphere prevailing over the developed world pharmaceutical industry, an encouraging number of approximately 116 delegates descended upon AstraZeneca’s new Alderley Park conference centre to engage with the latest advances in high resolution and high speed separations. Appropriately there was also a good turn out of exhibitors (12) led by Gold Sponsors, Waters. In terms of quality of scientific meeting the bar has recently been raised by Spring Symposia drawing speak...
Turning on the Heat in Liquid Chromatography - Tony Edge & Luisa Pereira
The use of temperature within liquid chromatography (LC) has traditionally been limited to isothermal studies up to 50° or 60°C. However, this does not realise the full potential that temperature can have within a liquid chromatographic system. In particular the ability to run with thermal gradients [1,2] or to run green LC [3,4], where there is no organic solvent used, is something that is not considered as routine within the liquid chromatography community. The advent of ultra-high pressure...
Using GC-MS/MS for Superior Sensitivity, Specificity and Precision in Free Testosterone Analysis - Rohan A. Thakur, Clark Williard and Agnita Rajasekaran
This article discusses biologically important steroid measurements which have, in the past, seen a lack of analytical sensitivity. Examples include determination of testosterone levels in women and children and estrogen levels in post-menopausal women. The article gives insight into the analysis of free testosterone at physiologically relevant levels in plasma ultra-filtrate, concentrating on the sensitivity, specificity, precision and accuracy advantages offered by GC-MS/MS.
From crude samples to highly pure isolated solids in 4 hours - Peter Barton, Scott Boyd, Steve Chapman, Clive Green, Sam Groombridge, Adele Loynes & Paul Whittamore
Delivery of pure, dry, free flowing solids by a customer focused, medicinal chemistry purification service, using a Lean Sigma approach. Over the past few years the concept and benefit of Lean Sigma has become increasingly valued within the pharmaceutical industry. The necessity to drive efficiency and streamline processes is critical to the delivery of compounds through the R&D process in the most effective ways. Aiding the reduction of turnaround times and costs for a purification service,...
Application of dynamic temperature gradients and profiles for capillary LC through the application of a new thermoelectric array based column heater - David Collins, Damian Connolly, Dermot Brabazon, Mirek Macka and Brett Paull
Having a defined and well regulated control of temperature during chromatographic separations is of critical importance for every separation method. Capillary columns exhibit rapid thermal equilibration and precise application and control of longitudinal temperature gradients along the capillary column could provide interesting insights into band broadening processes within the column itself. By using thermoelectric modules in a platform column heater, simultaneous heating and cooling is possibl...
Fundamental issues in the implementation of a two-dimensional LCxLC separation - Candice Grivel, Amélie Dechenaux, Sabine Heinisch
This article aims at presenting the fundamental steps when implementing a two-dimensional on-line separation (2D-LC) of complex samples. Firstly, an approach is proposed to make the search for orthogonal conditions easy. It is based on the treatment of retention data acquired from generic gradient separations of test mixtures in various systems (stationary phase / mobile phase / temperature). The regression coefficient of 2D retention plots and the practical peak capacity are both determined....
Analysing crystalline polymers by gel permeation chromatography at high temperature - Greg Saunders
Polymers used in engineering applications are particularly difficult to analyse - they are generally crystalline, tough, and often require elevated temperatures or exotic solvents for dissolution and to maintain solubility. For these applications a high performance, integrated GPC system is required that maintains the temperature of the sample throughout the analysis. Two classes of engineering polymers were investigated by GPC to illustrate the conditions and equipment required for their analys...
Oligonucleotides: The Next Big Challenge For Analytical Chemistry - George Okafo & Chris Bevan
Research and development of oligonucleotides as therapeutic medicines is experiencing exponential growth and interest within academia, the biotechnology and pharmaceutical industries [1]. This has largely been driven by several key historical events including: 1. The regulatory approval of the first antisense oligonucleotide (ASO) for cytomegalovirus infection Vitravene® (1998)[2], 2. Approval of Macugen®, an aptamer for treatment of wet macular degeneration (2005) [3] 3. The discovery by...
The Use of Temperature for Method Development in LC - Gerd Vanhoenacker , Frank David, Pat Sandra
The use of temperature as a tool for method development is gaining interest. In most of the reversed-phase LC methods the selectivity can be altered significantly by changing the temperature. Since temperature is an instrumental parameter it is easier to change than e. g. buffer pH and is more robust. This note demonstrates the potential of temperature variations for controlling the selectivity in LC method development. Additionally, the features of a silica based and a polymeric stationary pha...
High temperature liquid chromatography - a brief review about an emerging technique - Thorsten Teutenberg
This review is focused to present a general overview about high-temperature liquid chromatography. It starts with a brief definition and then explains the necessary requirements to make use of this emerging technique. Also, the advantages of high-temperature liquid chromatography such as the reduction in the mobile phase’s viscosity and the possibility to replace toxic organic solvents with water are outlined. Furthermore, the influence of temperature on selectivity is demonstrated. This mea...