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| HPLC 2010 SCIENTIFIC PROGRAM |
HPLC is the largest meeting in the world dedicated to liquid phase separation science. This International Symposium is multi-disciplinary in nature and brings together many of the world's leading authorities to address practical solutions to today's challenges related to all aspects of liquid phase separations and related techniques.
Click below to view HPLC 2010 Scientific Program and Plenary Speakers:
HPLC 2010 Scientific Program
HPLC Plenary Speakers
HPLC 2010 Plenary Speakers
The Importance of Second Dimension Speed in Two Dimensional Liquid Chromatography
Peter W. Carr
University of Minnesota, Minneapolis, MN USA
It is self-evident that the overall time of on-line LCxLC is set by the speed of the second dimension. This time is simply the product of the time needed to do a single second dimension run times the number of fractions taken from the first dimension chromatogram. However, a detailed analysis of the loss in first dimension peak capacity resulting from the deleterious under sampling that is a concomitant of excessive second dimension analysis time clearly indicates that the second dimension cycle time can and usually does cause considerable loss in effective 2D peak capacity. In fact, at sampling rates two fold slower than the well known Murphy-Schure-Foley limit the second dimension peak capacity has a significantly greater impact on the effective 2D peak capacity than does the first dimension peak capacity. This is so significant that unless the first dimension is run very slowly or the second dimension is extremely fast the first dimension peak capacity become almost an irrelevancy. Under realistic conditions assuming a first dimension time of 60 minutes and second dimension cycle times of 30 seconds. it makes no real difference if the first dimension delivers 150 or 300 units of peak capacity. Thus use of extremely small particles and very high pressures or very long monolithic columns as the first dimension in LCxLC is neither necessary nor advisable.
Thermodynamics and Kinetics of Solute Transfer in HPLC
Attila Felinger
University of Pecs, Pecs, Hungary
The study of the details of solute transfer in liquid chromatography is of central interest. We apply the microscopic (or molecular dynamic) model of chromatography to study the reversed phase separation of small and large molecules. The microscopic theory of chromatography describes the evolution of a chromatographic peak as the random migration of the molecules along the column combined with adsorption–desorption processes that occur at random, too.
The molecular dynamic model is rather straightforward to comprehend and it can furnish direct answers when one tries to understand the development of chromatographic peaks. We show that the microscopic model can be rather simply used to estimate the fundamental characteristics of the separation process. We can estimate the rate a molecule is adsorbed on the surface of the stationary phase while it migrates along the column.
When combining the general rate model with the molecular dynamic model, one can consider and compare the kinetics of the transfer of solute molecules between the flowing and stagnant zones of mobile phase, pore diffusion, etc.
We analyze the peak shapes recorded under linear conditions, and can characterize the heterogeneity of the surface of the stationary phase. With a peak shape analysis that is based on the molecular dynamic model of chromatography, we can identify the presence of heterogeneous mass transfer or adsorption kinetics. We can, furthermore calculate the amount of retention due to the individual adsorption sites.
The general rate model of chromatography, which is a macroscopic model, offers the most detailed description of the separation process. We compare the results provided by both microscopic and macroscopic analysis of the peak shapes and statistical moments.
We present results obtained on nonporous, fully porous, as well as on shell particles.
Droplet Technology for Microscale Separations: Improving Throughput, Fraction Collection, and Interface to MS
Robert Kennedy
University of Michigan, Ann Arbor, MI USA
Interfacing microscale separations to post-column reaction or detection systems has typically been complicated by the low volumes. Similarly, interface to automated sampling systems, such as autosamplers, is limited by the small volumes required. We have explored the use of multiphase flow wherein sample is formed into a series of plugs or droplets separated by immiscible liquid as a way to manipulate samples in microfluidic and microscale analytical systems. Methods for formation and manipulation of such plugs on the nanoliter scale have been developed and are increasing in sophistication so that it is now possible to perform many common laboratory functions such as sampling from and reagent addition to plugs in microfluidic systems. We have developed systems that allow droplets to be used for injection onto capillary electrophoresis and chromatography columns. The resulting systems can have extremely high throughput because of limited time required for rinsing between samples. We have also explored fraction collection by segmenting column effluent into droplets. In this way, complicated sample treatment and off-line interface to detectors such as mass spectrometry can be performed. Splitting droplets allows collected fractions to be stored and re-analyzed at a later time. This allows, for example, samples to be collected and then analyzed by multiple mass spectrometers. It also enables stopped flow (also known as “peak parking”) electrospray ionization-mass spectrometry analysis. The injection and fraction collection systems have applications for “separations-based sensing”, high-throughput screening and analysis, process analytical technology, hyphenating methods, and multi-dimensional analysis. In this talk we describe the formation and manipulation of segmented flows, interfaces to microscale separations, and their applications.
Extended-Nano Channel for Chromatography on Chip
Takehiko Kitamori
University of Tokyo, Tokyo Japan
Micro channels and micro spaces on microchips have been utilized as novel chemical processing tools, and many kinds of chemically and biologically functional devices were developed. We also developed various chemical devices based on our original technological platforms, such as micro unit operation (MUO), continuous flow chemical processing (CFCP), and thermal lens detection of non-fluorescent chemical species (TLM). And the analytical, synthesis, and cell biological equipments in which the chemical devices were installed as chemical CPUs were developed and their excellent performances were proved. Now, we are moving from micro to extended-nano fluidics as a next step.
The size region of 101-102 nm, that is extended-nano space, is still unexplored area. In our group, we fabricated extended-nano channels into glass microchips, and succeeded in fluidic control and single molecule detection by our TLM. And the same methodologies MUO, CFCP, and TLM are shown to be available even in extended-nano fluidics. We have already characterized water and other solvents in extended-nano space and unique characteristics of water and other solvents were found.
We applied the extended-nano channels and spaces to pico-litter immunoassay and atto-litter HPLC. Pico-litter is smaller than the volume of a living cell, and therefore, single cell single molecule analysis will become available. Some results of pico-litter ELISA will be shown. The atto-litter chromatography is really challenging. We fabricated an injector and separation channel in extended-nano size. Some preliminary results of chromatograms were successfully obtained. The space of the extended-nano channel is much smaller than the gap in the packed-column, and well controlled as uniform straight space. These well controlled geometric merits contribute to reduce diffusion and circular flow effects on separation. Actually, we obtained much superior separation results to the conventional packed-column. There are still technical difficulties to complete the ideal conditions, and we experienced mechanical broadening of the sample zone. However, in case when the ideal operation of the extended-nano fluidics is completed, the theoretical plate is expected to reach to 106 orders.
Linking Glycome and Genome: Robotic HPLC Based Platform Establishes the Variability, Heritability and Environmental Determinants of Human Plasma N-Glycome
Pauline Rudd1; Ana Kneževic5; Igor Rudan2; Harry Campbell3; Caroline Hayward4; Alan Wright4; Margaret Doherty1; Jonathan Bones1; Niaobh O'Donoghue1; Matthew Campbell1; Gordan Lauc5
1Dublin- Oxford Glycobiology Laboratory, NIBRT, Dublin, Ireland; 2University of Split Medical School, Split, Croatia; 3University of Edinburgh, Edinburgh, United Kingdom; 4MRC Human Genetics Unit, Western General Hospital, Edinburgh, United Kingdom; 5University of Zagreb, Zagreb, Croatia
Glycosylation plays an essential role in the functions of the proteins that contain this common post-translational modification and it is a parameter which determines the efficacy, pharmacokinetics and safety of most biological drugs. These include erythropoietin, the pituitary hormones and monoclonal antibodies, of which 31 are currently licensed by the FDA and 200 more are in the pipeline. Glycosylation processing pathways are complex and not under tight genetic control, therefore monitoring and controlling glycosylation in bioprocessing plants is a significant challenge. Rapid, detailed, quantitative glycan analysis is required at all stages of bioprocessing, from quality by design, through process analytical technology to drug licensing and in vivo tracking. This robotics platform has been designed to be rugged in terms of cost, reliability, data interpretation and operator training. It can provide either glycoprofiling data or more advanced glycan analysis for research purposes such as identifying critical features of therapeutics, determining the factors that control glycosylation and understanding the implications of their structures. Plasma glycans were analyzed in 1008 individuals to evaluate variability and heritability, as well as the main environmental determinants that affect glycan structures. By combining HPLC analysis of fluorescently labeled glycans with sialidase digestion, glycans were separated into 33 chromatographic peaks and quantified. A high level of variability was observed with the median ratio of minimal to maximal values of 6.17 and significant age- and gender-specific differences. Heritability estimates for individual glycans varied widely, ranging from very low to very high. Glycome-wide environmental determinants were also detected with statistically significant effects of different variables including diet, smoking and cholesterol levels. Glycomics analysis combined with a genome-wide association study identifies loci involved in regulation of the human plasma N-glycome.
Mass Spectrometry - Based Therapeutic Metabolomics from Biofluids and Tissues
Gary Siuzdak
The Scripps Research Institute, La Jolla, California USA
Quantitative global analysis of endogenous metabolites from cells, tissues, fluids or whole organisms - metabolomics, is becoming an integral part of functional genomics efforts as well as a tool for understanding fundamental biochemistry. Where the genome and proteome is largely enabled by the predictable fragmentation pattern of peptides, metabolomics is complicated by the tremendous chemical diversity of metabolites. The experimental aim in our global metabolomics studies is to obtain a comprehensive quantitative and unbiased view of the metabolome. We have explored multiple novel mass spectrometry platforms for metabolomics including both solution-based approaches and surface-based mass spectrometry, such as nanostructure-initiator mass spectrometry (NIMS) for tissue imaging. These platforms will also be presented in the context of its application to diseases such as chronic pain and multiple sclerosis.
Porous Polymer Monoliths with Tailored Porous Properties and Chemistry
Frantisek Svec
Lawrence Berkeley National Laboratory, Berkeley, California USA
The porous polymer monoliths emerged in the late 1980s and their first application was the separations of proteins. Since then, porous polymer monoliths found numerous other applications such as HPLC, solid phase extraction, enzyme immobilization, capillary electrochromatography, gas chromatography, and ion chromatography. Their advantages include ease of the preparation, robustness, high permeability to flow, mass transfer via convection, and a vast variety of chemistries. Recently, most of our efforts are focused on capillary format designed for micro- and nanoscale applications. Typical porous polymer monoliths exhibit a small surface area since they lack the mesopores. Although they are excellent stationary phase for the separation of large molecules, they do not work adequately for the separations of small molecules. To eliminate this weakness, we have now developed and demonstrated monoliths of second generation using a new two step approach to the control of porous properties. It includes the preparation of monolith with a typical porous structure and providing it with nano and mesopores via the in situ hypercrosslinking reaction. This technique enables the preparation of porous polymer monoliths in capillary columns possessing a large surface area thus making them useful for fast and efficient separations of small molecules as well as rapid size exclusion chromatography. Surface chemistry of the monoliths can be manipulated using copolymerization with monomers bearing the desired functionality, chemical modification of preformed monolith, and photografting of pore surface with polymer chains bearing the desired functionalities. Recently, we have introduced another approach consisting in modification of monoliths with nanoparticles. This method enables the preparation of monoliths in which both porous structure and chemistry are optimized separately. Applications of the new generation of monoliths will also be presented.
Simple, Low-Cost Diagnostic Systems: from Open-Channel to Paper
George Whitesides
Harvard University, Cambridge, Massachusetts USA
This seminar will describe the development and use of low-cost diagnostic systems, intended for immediate use in the developing world. It includes, inter alia, a new family of microanalytical devices based on paper, simple systems for magnetic levitation, and low-cost microelectronic systems. To fabricate micro-paper-based analytical systems (μ-PADs), paper is first patterned with lines of a hydrophobic polymer or wax; the hydrophobically bounded regions of paper then act as fluidic microchannels, through which water moves by capillarity. Stacking patterned paper with interleaved hydrophobic sheets (double-sided adhesive tape, with holes patterned in it) generates three-dimensional devices. In the developing world, low cost and mechanical ruggedness are important. These attributes are also valuable in a wide range of other capabilities and uses, some of which this talk will summarize.
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