Separation Science 2017 is a world-class international conference and exhibition, covering fundamentals, best practice and applications in chromatography, mass spectrometry and related techniques. Taking place 1-2 November, 2017 at the state-of-the-art Biopolis, Singapore, this educational event offers you the chance to learn from some of the world's top experts in these techniques in a genuine learning environment. The key technology focus of this year's event is hyphenated techniques and how their analytical power offers tremendous advantages for the modern laboratory scientist.
If you currenty use chromatography, mass spectrometry and related analytical techniques or plan to use it in the future, or if you need a better understanding of hyphenated techniques, how and when they should be applied, and how to interpret the results you obtain, then Separation Science 2017 is for you.
The programme will cover the fundamentals, practicalities, method development strategies and applications of hyphenated techniques, their use in both qualitative and quantitative terms, and applications in bioanalysis, pharmaceutical, food, energy and environmental analyses.
New Directions in Sample Preparation for Chromatographic Analysis
presented by Ronald E. Majors (ChromPrep, USA)
For many years, classical sample preparation techniques such as Soxhlet-, liquid-liquid- and solid-phase-extraction (SPE) have been widely used for chromatographic analysis. With the development of improved chromatographic separation columns and detection techniques such as LC-MS/MS, the principle of “Just Enough” sample preparation, where selectivity can be achieved anywhere in the sample preparation-chromatography-detection process, simpler and faster sample prep is now possible. Enhanced matrix removal systems are now being used to simplify multi-component samples by removing the interfering matrix rather than focusing on isolating the analytes of interest from the sample. The results are better sensitivity and analyte recovery. In this presentation, attendees will get an overview of some of the newer techniques for sample preparation that are faster, provide lower detection limits, better recovery, and less manual labor.
Membrane-Protected Liquid-Phase Extraction
presented by Hian Kee Lee (NUS, Singapore)
Very much like the way polymeric membranes have been used in the wastewater treatment and potable water production fields as filtration media for cleansing purposes, so can these materials be applied to the sample preparation of aqueous solutions in the analysis of water contaminants. Since about the late 1990s, we have been using membranes for the sample preparation (for single-step extraction, cleanup and enrichment) of aqueous matrices in the analytical chemistry of water. In this talk, we shall recount our experience of being pioneering proponents of the use of hollow fibre membranes in the liquid-phase microextraction (LPME) research field from its earliest days, to the present day, where modern autosampler technology can be exploited to allow the establishment of fully-automated membrane-protected LPME procedures, seamlessly integrated with gas or liquid chromatography-mass spectrometric analysis.
Novel Approaches to Coupling Orthogonal Separation Mechanisms in Pharmaceutical and Environmental UHPLC Analysis
presented by Deirdre Cabooter (KU Leuven, Belgium)
In this presentation, the design of a novel mixing unit is discussed that allows coupling orthogonal separation columns on a single ultra-high performance liquid chromatography (UHPLC) instrument. The set-up allows analyzing samples with a large variety in polarity in a single injection run by coupling hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) columns in series. Unretained peaks eluting from a HILIC column in first dimension are first isolated in a sample loop, before directing them to a RPLC column in second dimension for separation. Since the mobile phases employed in HILIC and RPLC are not directly compatible, a mixing unit is inserted in the set-up to alter the mobile phase composition before carrying out the second dimension separation. The entire set-up is implemented in a UHPLC instrument with a single pump, using commercially available equipment only.
Universal Generic (U)HPLC/UV/MS Gradient Method(s) for Multiple New Chemical Entities
presented by Michael W. Dong (MWD Consulting, USA)
This presentation chronicles the development of this universal generic method(s) by selecting the best column technologies for pharmaceutical analysis (superficially porous particles with bonding chemistries most compatible to highly basic compounds with simple mobile phases). The same methodology appears to work well for cleaning verification of multiple NCEs and serves well as a starting point for the development of critical stability-indicating assays for many new drug substances. Case studies are presented to illustrate the versatility of this method development approach by simply varying the gradient range and gradient time to customize the method for purity assays of many different NCEs including the separations of diastereomers for complex molecules with multiple chiral centers.
GC-MS and Fuels
presented by John Langley (Southampton University, UK)
Gas chromatography - mass spectrometry (GC-MS) is the go to tool for many aspects of fuel analysis.
The nature of the fuels, such as gasoline and diesel, require a high resolution chromatographic separation approach and the chemistry of these materials are perfectly aligned with analysis by electron ionization mass spectrometry. Fuel quality is forever changing due to pressures both legislative and advances in engine design. This presentation will show how different fuels can be differentiated through the use of GC-MS and how low level contaminants or additives may also be detected. Many will interpretation of these complex data sets can be time consuming and laborious. The simple be a reduction model will be shown as an example of how this process can be accelerated.
Recent Advances in 2D-LC for Deep and Efficient Characterization of Biopharmaceutical Materials
presented by Dwight Stoll (Gustavus Adolphus College, USA)
Over the past ten years, significant strides have been made in the sophistication and robustness of commercially available systems dedicated to 2D-LC, and how this technology is being adopted rapidly in the biopharmaceutical application space. In this presentation I will give an overview of the most important technology developments in this space in the past five years. I will then share recent results from my group and others that show where we can take this technology in the near future. 2D-LC concepts are being used at all levels of characterization, including peptide, subunit and intact level analysis.
Hyphenation in Gas Chromatography: Improving Analyte Characterisation
presented by Philip Marriott (Monash University, Australia)
Today we have an ‘extravagant’ array of mass spectrometry (MS) tools for both liquid phase and gas phase separations. MS plays three main roles: to provide sensitive analysis; to reduce or remove response to matrix in order to improve selectivity; and/or to provide a mass spectrum of the analyte to serve identification purposes. Sometimes, MS fails to provide adequate characterisation. This is most commonly seen for isomers, where the mass spectrum is equivocal as to which compound we have. It is especially problematic for essential oils. Against this background, we have developed a range of approaches to address improved identification of compounds. Thus prep-GC allows collection of pure analyte for off-line NMR analysis. GC hyphenated with MS and FTIR is an on-line approach that provides dual MS and FTIR spectra to be obtained. Software allows comparison of these various spectrometric tools to provide improved identification.
What are the Benefits of LC-MS for Accurate and Robust Analysis of Protein Biopharmaceuticals?
presented by Davy Guillarme (University of Geneva, Switzerland)
The aim of this presentation will be to review the possibilities and trends of LC-MS using different modes of chromatography for the characterization of biopharmaceuticals at the protein level. The historical chromatographic modes, namely size exclusion chromatography (SEC), ion exchange chromatography (IEX) and hydrophobic interaction chromatography (HIC) having been successfully combined in our laboratory with MS using a 2D setup. On the other hand, reversed phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) were directly combined to MS, and HILIC was found particularly useful to assess glycosylation at the protein level of analysis.
Comprehensive 2-Dimensional HILIC × RPLC: Theory, Practice and Application to Phenolic Analysis
presented by André de Villiers (Stellenbosch University, South Africa)
Comprehensive 2-dimensional liquid chromatography (LC×LC) is finding increasing application in the analysis of complex samples, such as encountered in the fields of proteomics, metabolomics and natural product analysis. In this presentation, an overview of the fundamental and practical aspects of LC×LC will be presented, with particular emphasis on the on-line hyphenation of hydrophilic interaction chromatography × reversed phase liquid chromatography (HILIC×RP-LC). Theoretical aspects such as the sampling rate and second dimension analysis times, measures of orthogonality, dilution factors and estimation of the practical performance of LC×LC separations will be addressed. These concepts form the basis of method development in LC×LC, which involves the relationship between a large number of experimental parameters and is aimed at achieving three conflicting goals: maximising separation performance (peak capacity), minimising analysis time and minimising dilution.
Unlocking Complexity with API and UHPSFC-MS
by John Langley (Southampton University, UK)
Whilst gas chromatography-mass spectrometry is a powerful tool used for the analysis of different fuels types it can also be blind to some of the fuel additive packages used. These different additives are used for a number of different reasons and so identification of these individual materials can be important in understanding the performance of different fuels. Use of soft ionisation techniques, in this case a range of atmospheric pressure ionization techniques together a way as ionization enhancing additives and supercritical fluid chromatography mass spectrometry have been shown to access these different chemistries are not observed by a normal GC-MS analysis. Ultimately the complementarity of GC-MS and SFC-MS will be discussed and proposed as the ideal approaches for comprehensive two analysis.
Electrostatic Interaction Modified HILIC (emHILIC) and ERLIC for Analysis of Post-translation Modifications in Aging Research
presented by Newman Sze (Nanyang Technological University, Singapore)
In order to pave the way for future effective new therapies that can protect against aging and disrupt degeneration diseases, we developed We propose that these degenerative protein modifications (DPM)-containing peptides with different charge states and hydrophilicities can be separated from the unmodified peptides in an ion exchange HPLC column operating in electrostatic interaction modified hydrophilic interaction (emHILIC) mode, thus improving the sensitivity to detect these structures by LC-MS/MS. Furthermore, the unmodified and modified peptide variants will be eluted from the ion exchange column in a predictable order based on their charge densities in LC-MS/MS mobile phase. Many DPMs associated with degenerative diseases and aging are discovered using this method.
HILIC-MS and RPLC-MS in Quantitative Metabolite Measurement
presented by Lu Yonghai (NERI, Singapore)
Hydrophilic interaction chromatography (HILIC) is an emerging separation mode of liquid chromatography (LC). Using highly hydrophilic stationary phases capable of retaining polar/ionic metabolites, together with high organic content mobile phase that offer readily compatibility with mass spectrometry (MS) has made HILIC an attractive complementary tool to the widely used reverse-phase (RP) chromatographic separations in quantitative metabolite measurement. The combination of HILIC and RPLC coupled with an MS detector provides more comprehensive metabolite coverage than use of only RP. Attendees to the presentation will learn characteristics of HILIC and RPLC in separation, and recent applications of HILIC-MS/MS and RPLC-MS/MS in quantitative metabolite measurement, ranging from amino acids, fatty acids, acylcanitines, and metabolites of specific nature.
Online Coupled MicroSPE-LC for Pseudo LC-LC Separations
presented by Yoshihiro Saito (Toyohashi University of Technology, Japan)
Fine fibrous materials have been introduced, especially as the extraction medium in miniaturized sample preparation and the separation medium in microcolumn chromatography. Downsizing of the column length was also demonstrated with a short polymer-coated fiber-packed capillary. In this presentation, miniaturized sample preparation capillary packed with a bundle of fine fibrous materials is presented along with several applications in liquid chromatography (LC). The extraction capillary could be regarded as both extraction cartridge and column for high-temperature LC separation. Introducing the fractions eluted from the fiber-packed sample preparation capillary to a conventional microcolumn LC system via a home-made modulator, pseudo-two-dimensional separations of aromatic compounds have been demonstrated. In addition, it has been confirmed an excellent stability of the extraction capillary, allowing repeatable use for more than 100 times in the typical temperat ure-programmed elution with pure water.
Capillary Electrophoresis: Versatile Tool for Chemical Analyses
presented by Bahruddin Saad (Universiti Teknologi PETRONAS, Malaysia)
Since it was first described by Jorgensen and Lucas in 1981, capillary electrophoresis (CE) has progressed from being a new technique of academic curiosity into a real world problem solver. CE users are quick to note how easy it is to perform the various modes of separations by just manipulating with the composition of the background electrolyte (BGE). In this presentation two research topics will be covered: analysis of chiral antimalarials (primaquine and quinocide) in pharmaceuticals and trans fatty acid in margarines. Fundamental studies (e.g., binding constant measurements, computer modeling) that provided complementary information that are key for deeper understanding of the CE migration behavior of some of these compounds will be provided. The direct analysis of trans fatty acid was made possible by the use of the capacitively coupled contactless conductivity detector (C4D), does not require derivatizations, thus offering considerable savings in manpower time. The compositions of the BGE for these separations will be discussed.
High pH Stability of Columns Packed with Hybrid Fully Porous Particles and Hybrid-Coated Solid-Core Particles
presented by Moon Chul Jung (Waters Corporation, USA)
Mobile phase pH is a powerful tool to alter the retentivity of ionizable analytes in reversed-phase separations, and thus to modify the selectivity. This strategy is especially useful for improving the retention and separation of basic analytes by using high pH mobile phases. The use of high pH mobile phases, however, can be limited by the instability of the stationary phase. Notably silica particles, commonly used for chromatographic stationary phases, are unstable in basic conditions. Organic/inorganic hybrid materials are a solution for such pH stability concerns. Waters C18-bonded BEH hybrid particles, introduced in 2004, show excellent stability over a wide pH range (pH 1 through 12). Advancements in solid-core particle technology have enabled improved chromatographic efficiency and faster analyses. Because the majority of currently available solid-core particles are made of silica, these benefits may not be realized for applications that require high pH mobile phases. We have recently investigated the application of organic/inorganic hybrid coatings onto silica-based solid-core particles, in order to impart improved high pH stability. This presentation summarizes our research effort while evaluating the high pH stability of a few commercially available columns containing hybrid-coated solid-core particles.
Topics and trends of separation science at JASIS 2017 and the latest developments of Japanese Separation Science Industry
presented Takeshi Kawamoto (Japan Analytical Instruments Manufacturers' Association (JAIMA))
This year at JASIS 2017, there are about 350 presentations in the new technology seminar, and 54 of them are in the category of separation science. In this presentation, topics and trends of separation science at JASIS 2017 will be presented to provide comprehensive and up-to date information on Japanese technologies and supportive analysis technologies in separation science. Topics such as advancement of column technologies in HPLC, new hardware technology for GC, and new development of SFC/SFE as a result of the recent deregulation of high pressure gases in Japan will be presented. In addition, recent developments and market situation of Japanese separation science industry based on the statistics of JAIMA and other market data will be explained. Some examples of the latest products/technologies from Japanese separation science industry will also be presented.
High throughput LC-MS
presented by Martin Gilar (Waters Corporation, USA)
Various definitions of high-throughput (HT) LC-MS analysis can be found in literature. Some authors define HT analysis as the throughput of 384 samples per 24 hour period (3.75 minutes/sample), while others would consider a HT analysis to take place in time scales faster than 1 minute per sample. The second dimension of comprehensive 2D LC usually falls in the category of HT analysis, where it is desirable to analyze the elution fractions from the first dimension at a pace faster than one minute each – ideally in the span of tens of seconds or less.
HT LC-MS analysis is often practiced in a simplified setup, where a liquid chromatography column is used purely for sample trapping and desalting. Such an instrumental setup enables fast, ten second cycle times, but does not permit for chromatographic separation of sample components.
We have evaluated the capability of UPLC systems to perform HT analyses, and found that Acquity UPLC pumps can run 0.25-1 min gradients. When working with sub-minute cycle times, the auto-sampler and data acquisition platform can become a throughput bottleneck. We have evaluated a setup permitting faster LC-UV and LC-MS analyses, using 96- or 384 sample data acquisition in a single file.
The performance and peak capacity of HT LC analysis for short, narrow-bore columns is discussed in terms of theoretical and practical performance. The impact of extra-column peak dispersion and peak capacity is evaluated.
Rapid Isocratic Speciation of Eight Arsenic Compounds
presented by Kenneth Ong (PerkinElmer, Singapore)
While separation of inorganic arsenic species can be easily achieved with a simple isocratic method, present of various arsenic compounds in real samples make it impossible for accurate determination of toxic inorganic arsenic. A common gradient flow technique used by most testing lab face two issues; long analysis time and potential overlap of high concentration level of AsB on inorganic Arsenic species.
In this work, the isocratic method would be a better choice for seafood sample which usually contain high AsB while toxic As(V) and As(III) were eluted much earlier. This allows toxic species to be detected accurately without worrying of high AsB tailing into next species.
Attendees to this presentation will learn a novel speciation technique for rapid analysis of Arsenic compounds.
Applications of Ion-Exchange/Reversed-Phase Mixed-Mode Stationary Phases in Combination with Charged Aerosol Detection for Separating Pharmaceuticals
presented by Joachim Weiss (Thermo Fisher Scientific, Germany)
Silica-based mixed-mode stationary phases combine both anion- and/or cation-exchange with reversed-phase properties. The packing material of the trimodal stationary phase is based on Nanopolymer Silica Hybrid (NSH) technology consisting of high-purity porous spherical silica particles coated with charged nanopolymer particles. The inner pores of the silica substrate are modified with an organic layer that provides both reversed-phase and anion-exchange properties. The outer surface is modified with cation-exchange functionality. This arrangement ensures distinctive spatial separation of the anion-exchange and cation-exchange regions, which allows both retention mechanisms to function simultaneously and be controlled independently. Thus, the selectivity of this stationary phase is orthogonal to reversed-phase columns. Mixed-mode stationary phases are ideal for the separation of anionic and basic compounds with different hydrophilicities as well as for simultaneous separation of basic, acidic, and neutral pharmaceuticals. However, the simultaneous analysis of pharmaceutical drugs and their counterions requires alternate means of detection, because nonchromophoric counterions cannot be detected with traditional absorbance techniques. Charged aerosol detection (CAD) has been introduced to overcome traditional nonspecific detection shortfalls. It can be utilized to characterize active pharmaceutical ingredients (APIs) together with their counterions.In this presentation, I will discuss column chemistries, chromatographic properties, the principles of charged aerosol detection, and some exemplary pharmaceutical applications.
Rethink GC-MS & LC-MS: Towards a Simple and Accurate Analysis to Reveal Unknown Compounds
presented by Chua Chun Kiang (ChemoPower Technology, Singapore)
Hyphenated chromatographic technique (GC-MS and LC-MS) is an increasingly important analytical technique for the separation and identification of chemical compounds in complex mixtures. The analysis of complex mixtures is often challenging as the co-elution of two or more components results in overlapping chromatographic peaks. Various instrumental and experimental modifications sought to address this concern but a one-fit-all solution is still not readily available. This has thus spurred the development of computational approaches to extract pure component signals from overlapping chromatographic peaks. Herein, we demonstrate the deconvolution and reconstruction of pure component mass spectra from overlapping GC-MS and LC-MS peaks based on an entropy minimization algorithm. Our approach computes extrema from raw mass spectra data to provide accurate qualitative and quantitative analyses in overlapping chromatography peaks without the need for any prior information. Moreover, pure mass spectra of minor components can be recovered with significant signal-to-noise enhancement. We demonstrate the application of our approach in several case studies. Our approach surpasses mass spectral processing software such as AMDIS and Agilent Masshunter. The integration of our approach into the analysis of GC-MS and LC-MS techniques is a significant step forward towards the development of a rapid and high-throughput chemical analysis.