Vendor Sponsored
Educational ProgramsNortheast Regional
Chromatography
Discussion Group
Fall 2001 Meeting
October 17, 2001 MEETING
Summary of Accepted Papers
(click on the title to view the Abstract)
ABSTRACTS
Submicron Organic-Inorganic Hybrid Particles for HPLC and
As the particle diameter is reduced, high separation efficiencies can be obtained in HPLC and capillary electrochromatography (CEC). Because of the electroosmotic flow (EOF) in CEC, efficiencies in CEC are expected to be higher than in HPLC (about twice). Our laboratory has developed a protocol to synthesize silica-based particles with diameters of = 1 µm. The material can be fabricated with unique characteristics suitable for HPLC and CEC. For CEC, EOF is generated on the silanol groups present on the particles or on specific moieties present on the particle, which are introduced during the particle fabrication process. The stationary phases incorporated on the particles can include moieties for reversed phase, ion exchange, and chiral recognition, for example. This presentation will include a chromatographic evaluation of the synthesized materials using HPLC and CEC. The advantages and disadvantages of using small particles will also be discussed.
Chip-Based Analytical Chemistry Coupled with Electrospray Mass Spectrometry
Jack Henion, Gary Schultz, Tom Corso, Geoffrey Rule and Simon Prosser
Advion BioSciences, Inc., 15 Catherwood Rd., Ithaca, NY 14850
and
Jack Henion, Timothy Wachs, Nicholas Barbarin, Doug Mawhinney, Tom Rozek, and Jun Kameoka
Analytical Toxicology, Cornell University, 927 Warren Dr., Ithaca, NY 14850We are exploring new strategies to provide chip-based electrospray mass spectrometry applications amenable to the pharmaceutical industry as well as other applications. At Advion BioSciences, Inc. we have developed a novel microfabricated nanoelectrospray nozzle as a monolithic device in a silicon substrate. This nozzle can be coupled with a variety of analytical techniques including CE, HPLC, and CEC as well as integrated sample handling via microfluidics and micro chemical or biological techniques. The nozzle may be fabricated in arrays on the flat surface of the chip and in a variety of arrangements due to its small size. It can also be effectively coupled with electrospray mass spectrometry without the need for fragile capillary tips or glued pieces of other devices. Since mass spectrometry is a valuable analytical tool for the pharmaceutical industry, coupling the ESI-Chip with other miniaturized analytical devices could provide exciting opportunities for this important industry.
This lecture will describe recent developments which demonstrate that our ESI-Chip may provide a powerful new analytical tool for studies in small molecule drug determinations as well as in other important areas including proteomics and genomics. Examples will be shown which demonstrate the potential of these devices in the pharmaceutical and clinical industries.
E. Peter Maziarz, X. Michael Liu, Dan Ammon, George L. Grobe, and
David Heiler
Bausch & Lomb Inc.
Global Scientific Affairs RD&E
1400 North Goodman Street
Rochester, New York 14603
Development of a product, such as an implantable device, is enhanced from a more complete understanding of the underlying principles that govern the molecular components within the product and their interaction with bodily fluid. The prerequisite to approach such an understanding involves analytical methods that rapidly and unambiguously identify all relevant components in complex mixtures.
By merit of its high sensitivity, specificity, selectivity, and information-rich nature, matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) time of flight (TOF) mass spectrometry (MS) can be an indispensable analytical tool for characterization of protein and polymeric materials. The crux of this study is two-fold. First, we demonstrate the unique potential ESI and MALDI-TOF MS offers toward synthetic polymer analysis. These studies include hyphenation of gel permeation chromatography (GPC) with ESI and MALDI-TOF MS to evaluate polydisperse polymers and complex mixtures thereof. We obtain detailed information including repeat unit sequence, existence of impurities, and end group chemistry of selected polymeric materials. The second part of this report demonstrates the unique ability MALDI-TOF MS offers toward analysis of proteins directly from a contact lens. These studies include in vitro and in vivo analysis. Efforts toward utilizing MALDI-TOF MS to quantitate proteins are discussed. We compare MALDI-TOF MS data to more conventional spectroscopic assays for lysozyme content.
Though only preliminary steps toward a more complete understanding of contact lens materials, this report provides an overview of MALDI and ESI-TOF MS and demonstrates the ability of these techniques to evaluate polymer materials and probe for polymer-protein interactions.
"Chromatographic and
Mass Spectral Characterization of
Functionalized Organosiloxanes"
Martin Ruszaj
Occidental Chemical
2801 Long Road
Grand Island N.Y. 14072
Difunctional organosiloxane oligomers are used in the synthesis of organosiloxane block copolymers. These copolymers have proven useful in electronic applications that demand high thermal stability and good adhesion properties. These properties are dependent upon the chain length or molecular weight distribution of the organosiloxane portion of the copolymer.
This presentation focuses on the development of analytical techniques to characterize the molecular weight distribution of amino terminated polydimethylsiloxane oligomers. Discussion will include the use of high temperature GC, Gel Permeation Chromatography (GPC), HPLC and mass spectrometry using atmospheric pressure ionization (API) techniques. Improvements in the chromatographic and detection properties were achieved by the formation of 2,4-dinitrophenyl (DNP) derivatives of the amines.
Studies in Preparative HPLC and LC-MS
(Through the 80’s and 90’s into the 2nd Millenium - in Rochester)Hans F. Schmitthenner, Eastman Kodak Company
Preparative HPLC is a powerful tool for the purification of synthetic chemical entities and for the isolation of unknowns such as metabolites, process impurities, and various components in commercial materials. With roots silica gel chromatography it has grown from using simple devices with limited utility to using sophisticated instrumentation with remarkable capabilities. While the fundamental theories and methods are derived from analytical HPLC, prep HPLC introduces new challenges for the separation of components, and isolating them on a larger scale.
This seminar will illustrate some of those challenges and the manner in which they were solved. Examples will be drawn from previously presented studies in a pharmaceutical setting including the isolation of metabolites, enantiomeric separations, and purification of peptides. They will also be drawn from patented classes of photographic materials at Kodak and published mixtures of compounds. Techniques will include ion exchange, solid phase extraction, normal and reverse phase separations, and fractionation techniques including UV-Vis and MS triggered collection of components.
A short review of instrumentation for prep HPLC and prep LC-MS will be presented including a summary of current instrument manufacturers. This will be followed by a description of the HPLC labs developed at Kodak over the past two years and some of the commercial instrumentation we have available for open access use by synthetic chemists and custom separations for internal customers.
What is a TAG?
Debra Butterfield
Eastman Kodak Company
What is a Technician Affiliate Group (TAG) and how does it fit within the American Chemical Society (ACS)? This presentation will examine the connections between TAGs, the ACS, Local Sections (LS), the Division of Chemical Technicians (TECH), and the Committee on Technician Affairs (CTA). The benefits of belonging to a professional scientific society will be detailed. TAG objectives, opportunities, membership levels, and "What's in it for me and my employer?" will also be discussed.
Reverse Phase High Performance Liquid Chromatography Method for the Analysis of Amprenavir, Efavirenz, Indinavir, Lopinavir, Ritonavir, Saquinavir, and Nelfinavir and Its Active Metabolite in Heparinized Human Plasma
Keil, K.; Brewer, J.; Morse, G
Laboratory for Antiviral Research, Department of Pharmacy PracticeState University of New
York at Buffalo, Buffalo, NY 14260
There has been an interest in developing a method for the analysis of a variety of antiviral drugs used in the treatment of AIDS. Since many patients are treated with multiple drugs at one time, analysis of multiple species is desirable. A method for the determination of nine species, including one internal standard has been developed. This method is used to quantitate Efavirenz and the following protease inhibitors: Amprenavir, Indinavir, Nelfinavir and its metabolite, M8, Ritonavir, Saquinavir, and Lopinavir. The method uses reversed phase high performance liquid chromatography (RP-HPLC) for sample analysis and was validated using a liquid-liquid extraction process. Drug species are chromatographed using a Waters SymmetryTM shield RP8, 3.0 X 150-mm column and detected using a photodiode-array detector (PDA), scanning at four various wavelengths. The method is currently being used in the laboratory in support of clinical studies.
Pharmaceutical Analysis Through Implementation of Contemporary Methodology
Frerichs, V.A.; DiFrancesco, R.D.; Morse, G.D.1*; Colón, L.A.2
The continuous progression of analytical technology often provides important benefits in the pharmaceutical laboratory setting. For routine analysis and method development, implementing new instrumentation and techniques has positive implications in terms of cost, versatility, and sample throughput. Throughout the past year, we have been introducing new methodology into our laboratory for the analysis of pharmaceutical compounds. Specifically, we have implemented liquid chromatography- mass spectrometry (LCMS) for the development and validation of a multi-drug assay for antiviral species used in the treatment of human immunodeficiency virus (HIV). Using this particular LCMS has provided greater detectability, faster analyses, and better selectivity. Also, we are currently investigating the utility of capillary electrophoresis (CE) for the enantioseparation of methadone and related drug species, to support antiviral studies in heroin addicts. Presented here will be details of the LCMS validated method, and current results from our work with CE.
*corresponding author
Improved Performance Of Overload Flash Chromatography To Isolate
J. Liu, P. C. Rahn and J. Bickler
Biotage, Inc.
While pharmaceutical synthetic processes generally incorporate crystallization techniques for purification, preparative chromatography can yield particular advantages, depending upon the stage of development. Few techniques are as easy, inexpensive, and quick as flash chromatography. Since its development by W. C. Still[1], flash chromatography has become a popular separation method for purifying pharmaceutical, biotechnology, petrochemical, and agrochemical samples.
To maximize the throughput, preparative columns are often overloaded. Under these conditions, purification efficiency depends on sample mass and loading volume. Definition of column loadability has been proposed in the literature[2-4]. These theoretical approaches, however, are seldom used in practice for flash chromatography.
This paper focuses on optimization of flash column purification techniques. The impact of factors including sample mass overloading, volume overloading and sample dissolution solvents upon sample resolution are discussed.
Continuous Gradient Purification Of Closely Related Drug Intermediates
J. Liu, O. Mneimne, P. C. Rahn and J. Bickler
Biotage, Inc.
Flash chromatography is a commonly used purification technique for synthetic chemists. Thin-layer chromatography (TLC) is usually used to develop a flash purification solvent system. This solvent system, typically implemented in isocratic elution with normal-phase flash silica columns, is often used to purify organic compounds. As first described by W. C. Still1, the Rf value (from TLC) of the desired component should be optimized for efficient isocratic flash chromatography. However, separating a multiple-component sample requires other approaches besides isocratic elution procedure to be efficient.
Gradient elution is a very common technique for reversed-phase purification. In this study, a new normal-phase flash chromatography gradient elution technique is presented. This new method is described for separating a small group of oxazoline intermediates. TLC was used for both reaction monitoring and flash gradient method development. The goal of this work was to develop a new normal-phase gradient elution technique, requiring minimal method development.
1
W.C. Still, M. Kahn, and A. Mitra J. Org. Chem. 1978, 43, 2923-2925.Purification of Organic Compounds Using a New FLASH System
J. Robert Bickler
Biotage, Inc.
Synthetic and natural product purification has historically been a manual operation using self-packed glass columns. Although an effective purification technique, this type of flash chromatography is time consuming. With normal-phase separations, the column needs to be emptied and cleaned between purifications. This laborious process needs to be repeated for every purification, reducing throughput and productivity.
In this poster, we show how the use of new, pre-packed FLASH+ cartridges coupled to the new Biotage Horizon™ FLASH system simplifies and speeds purification, allowing the user to program and run gradients, collect compounds by UV and analyze and store the purification data directly onto their PC or laptop computer. Examples of various organic mixtures are shown using this Instrument and FLASH+ cartridges to yield mg to gram scale products.
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