Abstracts of papers presented at the 2002 Pittsburgh Conference

s of papers presented at the 2002 Pittsburgh Conference The following 125 abstracts form Part B of two issues of Journal of Automated Methods & Management in Chemistry devoted to abstracts of papers and posters presented at the Pittsburgh Conference held from 17 to 22 March 2002 in New Orleans, LA, USA. The papers and posters covered a range of topics and techniques, each of which provided valuable information to the conferees and exhibitors alike. The attendance ® gures remained disappointing, but Pittcon is still one of my favourite shows and has done a great job for Analytical Chemistry as a whole. People are tiring of New Orleans as a venue and it has become little more than a trade show. Hopefully, it will revive itself when the next show will be held in Orlando, FL, from 9 to 14 March 2003. Perhaps the attendees will spare some time from Disney and Sea World to visit us at the exhibition and conference. If you need further information about any of these abstracts, please contact the authors. It is my intention to publish full papers corresponding to some of these abstracts in future issues of the journal. Strategies for successful global laboratory information management systems implementations Stuart M. Miller, Taratec Development Corporation, LIMS & Lab Automation Solutions, 1170 US Highway 22, Ste 302, Bridgewater, NJ 08807-2933, USA Pharmaceutical and life sciences companies world-wide are under enormous pressure to transform their drug discovery and development processes in order to make decisions on whether to fail or promote compound development with more con® dence and in a much shorter period of time. Computing and software are playing a vital role in this transformation and as a ® rst step, many life sciences companies are establishing foundations of globally harmonized information systems that are intimately tied to the key business processes. LIMS are one of the key systems that provide much of the data required to make the go/no go decisions, and are often one of the ® rst areas of focus for these global harmonization initiatives. Successfully completing a truly global LIMS project involves the usual array of technical challenges but the regulated environments of the life sciences industry and the diverse make up of the global teams present additional challenges. Simultaneously satisfying FDA and international regulatory requirements with a uni® ed global system and establishing common goals and harmonization of the underlying business processes which meet the requirements of the local sites are typically two of the greatest impediments to the success of these projects. While life sciences companies are eager to reap the bene® ts of a truly global LIMS, special care must be taken to ensure the global teams are prepared to handle these and other challenges that often put the success of these critical projects at risk of producing merely another set of regionally diverse business processes and information systems. In this presentation, we will recommend proven strategies to adopt and pitfalls to avoid in the planning and execution of a global LIMS implementation in the evolving life sciences enterprise. Contrasting anonymous case studies will be used to illustrate the dos and don’ ts of this process. An LIMS to support recent developments in stabi-

Today' s fast moving corporate environment requires business decisions to be made at an ever increasing rate.To make such decisions, such as whether to move forward with a product in development or to release a product to a customer, professionals need access to information scattered across the corporate enterprise.Laboratory information is critical to these business decisions in companies such as pharmaceutical and chemical manufacturers.Commercially available Laboratory Information Management Systems (LIMS) have been available since the early 1980s.LIMS have been very eå ective in increasing the productivity and sample throughput of the modern laboratory.However, they are only one piece of the puzzle to building an eå ective bridge between the laboratory and the rest of the corporate enterprise.Additional information, such as that from statistical packages, MSDS, COA, presentations, supply chain management systems, spreadsheets or other sources of laboratory knowledge need to be integrated within the business enterprise.This knowledge bank protects the intellectual property of the corporation as well as enabling fact-based decision-making.To enable this corporate knowledge bank, an integrated laboratory Knowledge Management System (KMS) must be in place to aggregate and integrate information from all these diå erent sources.This electronic library must also be easily accessible to all those who need access to these knowledge assets for making fact-based decisions, insuring intellectual property, streamlining work ¯ows or integrating disparate geographic locations.This knowledge bank must also be able to store non-laborator y data and easily integrate with other enterprise-wide systems, such as ERP.For the regulated environment, the consolidation of this enterprise information must comply with regulatory requirements such as US 21 CFR Part 11.This paper will discuss and demonstrate the advantages of integrating all laboratory information into the corporate knowledge bank.Examples of integrating all laboratory information and data and the subsequent integration of these assets into the corporate knowledge bank will be presented.Searching, accessing and viewing of this integrated information as well as how organizations can improve compliance with 21 CFR Part 11 will also be discussed.

Using information technology to optimize product development in process industries
Andrew Cunningham, Formation Systems, Inc., 144 Turnpike Road, 310 Southborough, MA 01772-2121, USA The best way to optimize your Laboratory Information Management Systems (LIMS) productivity may be not to test!See how product lifecycle management can reduce redundant testing and allow testing groups to focus on the highest ROI opportunities.To increase product performance, move from unique formulations to product platforms or just increase testing throughput.Companies are looking to automate sample creation using robots and move toward commentarial testing techniques.Both investments will increase the volumes of tests processed.These investments are based on assumptions that all test requests are required and these tests are not duplicate eå orts.When formulation, material and test history are not readily accessible, formulators assume the shortest path is recreating the wheel.This leads to buying new raw materials when equivalent materials have been certi® ed or when creating duplicate formulas.The real impacts are felt in downstream intesting where redundant testing is performed that results in wasted costs, lost testing productivity and longer development lead-times.Too often R&D departments have con¯icting priorities and standalone systems that lead to gross ineae ciencies, higher costs and lost orders.To optimize your LIMS investment, eå ectively managing the product requirements process and reuse of existing formulas and test results can reduce the volume of test requests and often eliminate whole testing cycles.Testing can focus on highreturn opportunities rather than simply executing more tests.Formation Systems is the leading provider of Product Lifecycle Management (PLM) solutions for the CPG and process industries.Our Optiva 1 Software Suite automates product development and commercialization, reducing the time and resources required to take a product from idea through product launch.The results are accelerated revenue, lower costs, a simpli® ed supply chain and a product development organization that enhances your ability to compete.Optiva includes powerful formulation, regulatory, speci® cation and labelling functionality that complements a LIMS system.Formation' s CPG customers use Optiva in addition to commercial or in-house LIMS systems.Insure LIMS testing is focused on highest ROI PLM solutions include both commercial and technical scoring technique to prioritize highest ROI projects and kill low probability requests.By prioritizing highest ROI opportunities and insuring redundant testing is largely eliminated, testing capacity is eå ectively increased.

Rapid screening of environmenta l test wells by headspace mass spectrometr y
Brian G. Rohrback Gas chromatograph y is the preferred method for characterizing and quantitatin g low molecular weight compounds, but the separation process is often slow and often requires sample pretreatment.By eliminating the chromatograph and applying multivariate techniques to the bulk detector signal, composition can be determined quickly.Using a headspace sampling system with direct injection into a mass spectrometer, both qualitative and quantitative precision are demonstrated for a variety of environmental test applications.The short (3± 5 min) analysis time and the lack of sample preparation creates a fast screening technique for fugitive hydrocarbons .Although headspace technologies have gained some acceptance in the ¯avour and fragrance industry as a means of evaluating product and ingredient quality, little has been done to assess feedstocks, products or wastes in the petroleum industry.Data for this study are drawn from a set of more than 200 test wells positioned at various locations within the boundaries of a re® nery.For traditional quality assessment, classi® cation techniques (such as nearest-neighbou r and factor-based groupings) can be used to determine the contaminant origin even in cases where the candidate sources are chemically similar.Without resorting to GC separation, the concentration of classes of contaminants can be estimated over a four order-of-magnitud e range.

Automation of eld-deployed sensors for longterm monitoring
Scott R. Burge, Burge Environmental, Inc., 6100 S. Maple Avenue, 114, Tempe, AZ 85283-2872, USA The research was performed to ascertain if a variety of sensors including ion-speci® c electrodes, photo-ionization and a trichloroethene (TCE)-speci® c optrode could be deployed in the ® eld to perform long-term monitoring of contaminate d ground-water systems.A `universal' sensor platform was designed and fabricated which allowed the deployment of the sensors.The platform allows the sensors to be installed in the water sample or in the headspace above the water sample.The platform could be interfaced to accommodate a variety of ground-water pumping systems and was designed to derive its power from a solar cell.The platform could be installed adjacent to or inside the monitoring well.The operation of the platform includes sampling, purging and cleaning of the system, calibrating the analytical systems and quality control checks such as duplicates and spiked duplicates.The platform is an automated analytical instrument, with protocols similar to those designed by the USEPA (i.e.SW-846), capable of performing analyses in the ® eld with no operator.The system was ® eld-tested at Homestead Air Force Base, Florida.The system was con® gured to allow the TCE optrode to measure concentrations in the headspace.The system sampled ground water from a shallow aquifer and analysed for TCE at a concentration of 70± 100 ppb in the presence of several other volatile chlorinated compounds.No interference was observed from the other chlorinated compounds with TCE.The results of the system were comparable with the results of samples submitted for laboratory analysis (EPA Method 8260B.

Rapid screening for VOCs in soil using a portable membrane inlet mass spectromete r (MIMS)
Yoshihiko Arita 1 , Takashi Hirano 1 , Masahiko Fujiwara 1 , Daisuke Satake 1 , Koichiro Matsuda 1 and Steve J. Mullock 2 , 1 Horiba Ltd, 2 Miyanohigashi Kisshoin Min, Kyoto 601-8510, Japan, 2 Kore Technology Ltd, 291 Cambridge Science Park, Cambridge CB4 0WF, UK Soil pollution from industrial development has become a source of concern more recently, starting in the 1980s when toxins in soil began increasingly to be detected at industrial sites.In Japan, the signi® cance of the role soil plays in the natural environment has ® nally been recognized and the public is rapidly becoming more aware of the contaminated soil problem.In the soil analysis industry, both portability and sensitivity at high speed are essential for the success of preliminary site survey work.The traditional methods for site screening such as detector tubes and portable GC-PIDs are not suae ciently sensitive to detect ppb levels in soil.In addition, as many measurements are generally required to characterize a site, a more rapid analysis is highly advantageou s for the preliminary survey.The MS-200, portable time-of-¯ight mass spectrometer with membrane inlet designed by Horiba and Kore Technology allows much more rapid analysis in < 5 min, with higher sensitivity than GC, for VOCs in soil and semi-real-time analysis in ambient air.The MS-200 was evaluated for a number of diå erent soil samples.In particular, rapid analysis, using mixture analysis to perform a least-squares ® t of library data for complex mixtures instead of separation by gas chromatography, will be discussed.The most striking feature of the MS-200 is undoubtedly the speed, but its sensitivity at ppb levels and accuracy should also be noted and will be discussed.The results of these experiments will be pre-sented, backed up by data from a GC.In this paper, we will discuss the future potential, as well as the current performance, of MIMS.

Monitoring atmospheric particulate matter through cavity ringdown turbidimetry
Jonathan E. Thompson, James D. Winefordner and Benjamin W. Smith, University of Florida, Department of Chemistry, PO Box 117200, Gainesville, FL 32611-7200, USA The study of micrometer-and nanometer-sized suspended airborne particulate matter has become increasingly important in recent years.The interest in the aerosol has been driven by the revelation that airborne particulate matter adversely aå ects human health, as well as its possible role in the global energy balance.In this work, we have developed an instrument based on the cavity ringdown technique capable of monitoring atmospheric extinction in real-time.As the majority of atmospheric extinction is often caused by aerosol scattering/absorption the technique is useful for monitoring suspended particulate matter.We have applied this technique to monitoring changes in extinction following smoke from a nearby wild® re and have also correlated extinction measurements with levels of particulate mass at our location.

Rick Parry
1 , Evaldo de Armas 2 and Jack Cochran 3 , 1 LECO Corporation, Separation Science Applications, 3000 Lakeview Avenue, and 2 Marketing, 4000 Lakeview Avenue, Saint Joseph, MI 49085-2319, USA, 3 LECO Corporation, Separation Science, 815 Pilot Road, Ste C, Las Vegas, NV 89119-3739, USA Flavonoids found in fruits and vegetables have been shown to provide a variety of medicinal bene® ts in the areas of cancer treatment and prevention as well as the reduction of allergic response.The analysis of ¯avonoids is complicated by the complexity of the food matrices in which they are monitored.The rather low concentrations of these analytes are easily masked by other matrix components signi® cantly complicating the accurate iden-ti® cation of the ¯avonoids.A variety of chromatographi c techniques have been used to attempt to address this analytical problem.More recently, LC-MS and LC-MS-MS approaches have been used to minimize further matrix interferences.However, other LC-MS tools such as accurate mass measurement, nozzle-skimmer fragmentation, and a combination of peak location and mass spectral deconvolution algorithms may provide signi®cant advantage s in the detection and proper identi® cation of the ¯avonoids in complex matrices.In this paper, a group of seven ¯avonoids has been successfully identi-® ed in a citrus matrix using a commercially available LC-TOFMS system.Unique peak location and mass spectral deconvolution algorithms were used to simplify signi® cantly the process of identifying the chromatographic positions of all analytes in the matrix and obtaining a pure spectrum for each analyte.Accurate mass measurement was used to determine potential empirical formulas for each analyte.Simple spectral interpretation techniques were also applied to determine reasonable elemental ranges for the formula calculator.Potential formulas were generated and then searched against a variety of commercial databases to obtain possible structures for the analytes.Finally, nozzle-skimmer fragmentation was used to minimize the number of potential identi® cations.

Rapid, solvent-free fat analysis for food and dairy applications
Vince Collins and Bobbie J. Haire, CEM Corporation, Applications, 3100 Smith Farm Road, Matthews, NC 28104-5044, USA Industries producing food or dairy products typically test for fat content.Many of these companies rely on the traditional methods such as Babcock, Mojonnier or Soxhlet extractions.These methods are long and laborious and require the use of various solvents.These procedures are not bene® cial for process control needs.The SMART Trac System uses a new technology to provide rapid fat analysis for the food and dairy industries.This technology combines the use of microwave drying with nuclear magnetic resonance (NMR) to provide an accurate moisture and fat result in < 3 min for most samples.Prior work performed with NMR technology was subject to oversaturatio n of the signal with high moisture products.The use of microwave drying to remove moisture combined with NMR for measurement of fat protons allows accurate moisture and fat data.This test can provide valuable and timely information for the plants to use for process control.This paper discusses results comparing traditional methods and the new microwave/NMR technology for ice cream mixes, cream and dried dairy powders.

Automated enzymatic food analysis
Wilfried Liekmeier and Paul-Heinz Sollboehmer, Ascanis OHG, Theodor-Lachmannstr 17, Ueberlingen D-88662, Germany Enzymatic food analysis methods are highly speci® c and eae cient and are indispensable for food analysis according to national and international norms and recommendations (e.g.IFU, EN, IDF and ASBC).The technique allows one to quantify important food constituents like sugars, fruit acids, cholesterol and others with minimal sample preparation.The practical implementation however sometimes seems tedious and complex.The technique is illustrated and it is shown how a new application software package substantiall y facilitates enzymatic analysis with commercial test kits and how it improves its results.The EnzLab software guides the user through the analysis, controls the measuring wavelength and times, it automaticall y reads the spectrometer data and performs all required calculations.Prede® ned analysis methods, con® gurable sample documentation and operators directions as well as a comprehensive analytical report are further items of rationalization of the technique.The improved timing accuracy will improve the result quality.A complete automated system is for example based on a standard double-beam UV/Vis-spectro-photometerÐ optionally with a 13-cell automatic cell changer for parallel sample measurements.The system can further be expanded with a piston pump for precise enzyme injection.High photometric accuracy of the system will decrease detection limits and reduce sample preparation requirements.For existing photometers without PC-communication, the `manual' software version is applied.The software prompts the user according to the selected method/analyte and samples to enter the photometric readings manually via keyboard or clipboard.The analysis of chlorinated pesticides is routinely performed on an electron capture detector (ECD) with a con® rmational analysis by GC-MS.Since the ECD is a semi-selective detector for chlorinated pesticides, sometimes a false-positive will occur.Subsequent analysis by GC-MS is necessary to con® rm the presence of the pesticide.The two analyses are usually done on two diå erent GCs and by diå erent analysts.Considerable time is spent in reviewing the data to determine which samples will require the con® rmation by GC-MS.A software program has been developed to program the autosampler to inject the sample on the ECD for screening and then on the GC-MS for con® rmation, only if a target compound is found.Intelligent software makes smart screening possible by combining an ECD and Mass Spec analysis on one GC.The Trace GC may be con® gured with an ECD, PolarisQ Ion Trap Mass Spec, dual split/splitless injectors and a fully programmable AS2000 autosampler.The injection of the sample on the ECD is made ® rst to screen for the presence of pesticides.Then at the end of the run the data are processed.If a target compound is detected, the autosampler will re-inject the same sample on the GC-MS.A duplicate column is installed in the second injector to the PolarisQ Ion Trap.The mass spec is set up for analysis by MS/MS.In this mode of operation, the matrix interferences are excluded from the trap and a precursor ion is isolated.Then energy is applied to fragment the ion to form a complete spectrum of unique product ions for con® rmation.This fast screening double injection technique has previously been used for analysis with dual ECDs where a screening method and analytical method or combined in one GC [1].

Smart screening of pesticides using double pro injection on the electron-captur e detector and ion-trap GC-MS/MS
1. Clay, D., Automatic screening and analysis of samples using a single autosampler in double injection mode.ThermoFinnigan Corporation .Pittsburgh Conference, 2001Conference, (2002)).

Simultaneous gas chromatography /mass spectrometry and gas chromatography / ame ionization detector
James D. Hudson Interest in having both Gas Chromatography /Mass Spectrometry (GC/MS) and Gas Chromatography / Flame Ionization Detection (GC/FID) data has created the demand to perform both types of analyses simultaneousl y using a single injection.This set-up gives the best of both worlds: GC/FID, which is deemed superior for quantitation, and GC/MS, which is advantageous for con® rmation of compound identi® cation based on the mass spectrum.A procedure to use dual columns from a single injector port of an HP 6890 GC coupled to two diå erent detectors is described.The detectors are a FID and an HP 5973 mass spectrometer.The GC was interfaced to both the mass spectrometer and the FID.Two 100 m £ 25 mm i.d.£ 5 m m ® lm thickness HP-1 (crosslinked methyl silicone gum) columns were connected to the rear injector port using a two-hole ferrule (Agilent Part # 5062-3580) .The detector ends of the columns were connected in the usual manner.No other modi® cation was necessary.The Chemstation software for the HP 5973 mass spectrometer is capable of handling the acquisition of both the FID and mass spectral data provided that the computer is of suae cient speed.For the analysis described below we used a 1 m l injection and 100:1 split ratio.Since the sample is split between two columns, we injected approximately twice the amount desired per column.GC/MS and GC/FID traces of a gasoline sample, for the peaks elute earlier in the GC/MS trace.The diå erence in retention times is attributable to diå erent column lengths that might have occurred upon manufacture or cutting during installation.Despite this diå erence, peaks are easily matched by comparing the pattern of peaks; therefore, we did not try to adjust column lengths to make these retention times coincide.It is possible that the vacuum of the mass spectrometer may draw more sample toward it than the FID, but this does not pose a problem because of the greater sensitivity of the FID.

Alex Niewland
1 , Steve L. Morgan Over the last years, the injection of larger-than-norma l volumes in capillary gas chromatograph y (GC) has attracted an increasing amount of research attention.The objective of large volume injection (LVI) is the achievement of an increased signal-to-nois e ratio by the introduction of more sample onto the column.This is particularly important in detecting analyte in trace amounts, such as in toxicological applications.However, LVI is still not common practice in most toxicology laboratories.Many large-volum e injectors are commercially available.These injectors are designed to inject over 100 ml of sample volume, as compared with the conventional 1± 3 ml.The injection port is generally set below the boiling point of the solvent, while the split vent is opened to divert the solvent ¯ow.Subsequently, the injector temperature is rapidly ramped to a high temperature, with the split vent closed, and the analytes are swept onto the GC column.Some work has been reported on improving GC sensitivity by injecting several 100 ml of sample with a conventional split/splitless injector.A packed injection liner, with either silanized glass wool or Tenax TA, was used for the analysis.Also, the septum purge ¯ow was set high throughout the injection and the initial oven temperature was set at least 20± 30 8 C above the standard solvent boiling point to facilitate removal of the solvent.This method worked well with the various hydrocarbons tested.However, large losses of volatile sample components could occur [1].Another viable method is to inject 1± 2 ml of sample multiple times using a conventional split/splitless injector.For example, a splitless injection of 1 ml of sample can be repeated every 20 s for a total of 10 ml of sample volume, thereby improving GC sensitivity by a factor of ® ve to 10 times.This improvement in sensitivity is often suae cient for many applied environmental and toxicological applications, especially with sample volumes of only 25± 50 ml.Examples of this injection method are demonstrated for pesticides and drugs.

On-line electrolyti c eluent generators and their applications in ion chromatograph y
Yan Liu Traditionally, these eluents are prepared oå -line by dilution from reagent-grade chemicals.The oå -line preparation of chromatographi c eluents can be tedious and prone to operator errors, and it often introduces contaminants that can cause undesirable chromatographic baseline artefacts and even irreproducible retention times of target analytes during the ion chromatographi c separation.Therefore, there is a need for improved methods of preparing high-purity chromatographi c eluents for ion chromatography .We have recently developed novel electrolytic devices that can be used to produce electrochemically high-purity electrolyte solutions for use as ion chromatographic eluents using de-ionized water as the carrier stream.In this presentation, we will describe the principles and operation of the automated on-line electrolytic eluent generators and demonstrate the advantages of using these eluent generators in isocratic and gradient ion chromatographi c separations.When coupled with electrochemical techniques, In Situ Raman spectroscopy is powerful tool for investigating electron-transfer processes in electrochemical reactions.This combination has proven very advantages in studying reactions occurring at the interface between electrode and electrolyte solutions.In this study, In Situ Raman investigations at potentiostatic condition were applied to a monolayer of 4-nitroazobenzen e (NAB) molecules on glassy carbon (GC) electrodes (NAB-GC) in the acetonitrile (ACN) solution of 1 m tetrabutylammoniu m tetra-¯uoroborate (TBA-BF4).A special cell was designed and constructed for the simultaneous use of In Situ Raman and electrochemical measurements.The NAB-modi® ed electrode preparation procedure in this work was similar to that described in the literature.The variation of NAB-GC Raman spectra in 1 m TBA-BF4 and ACN at the electrode potential (a) + 400 mV, (b) ¡1200 mV and (c) ± 1500 mV (versus Ag/Ag + ) shows no spectral changes in the potential range between + 400 and ¡800 mV.However, at a more negative potential range between ¡800 and ¡1800 mV, we could see spectral changes in the frequency range between 1100 and 1600 cm ¡1 .These spectral changes are attributed to reduction of the NAB monolayer to a chemisorbed NAB anion.We shall discuss such changes of in situ Raman spectra.

Advances in mass-directe d automated highthroughput sample processing
Christopher P. Loran High-throughpu t screening (HTS) methodologies greatly reduce the time and costs associated with drug and chemical development.The many synthesis products generated from combinatorial chemistry techniques require puri® cation and analysis before screening of their bioavailability, pharmacokinetics, toxicity and other biological activities.A fully automated highthroughput system for analytical and semi-preparative HPLC has been developed to incorporate mass-directed fraction collection for the rapid identi® cation and collection of important fractions.New developments include fraction collector triggering based on any combination of detection parameters (including time, threshold, peak slope, and sensitivity) to ensure complete collection of the analytes of interest; and sample-speci® c control variables (e.g.mass) used to make real-time changes to control parameters without changing instrument control programs.The chromatograph y management system provides a single platform with consolidated control panels to control all the components of the HTS chromatographi c system, track samples, collect data, and compare results from multiple screen-ings, with integrated system checks in a 21 CFR 11compliant environment.In this presentation, we will show how recent advancements in instrumentation and software were applied to HTS methodologies.Examples of high-throughpu t LC/MS, and fast method development will be presented using ultra-fast gradients with mass-directed fraction collection from 96-or 384-well microtitre or deep well plates.

A. Paul Zavitsanos and James D. McCurry, Agilent Technologies, 2850 Centerville Road, Wilmington, DE 19808-1610, USA
The formation of various product ions observed in a mass spectrometer by collision induced dissociation (CID) is a function of variety of factors: energy (voltage and target gas molecular weight), collision frequency (pressure, cross-section of gas), time and the structure of the compound under study.This presentation will focus on technologies that allow the creation and use of meaningful API spectral libraries with the Agilent LC/MSD.A novel tune procedure is described and search results for a variety of compounds of environmental interest are demonstrated.The unique characteristics of the Agilent LC/MSD that make it such an ideal platform for this approach are discussed.

A system for automated method development using liquid chromatographi c mass spectral detection
Eduard Kolovanov The principles behind computer-assiste d chromatographic method development are well established.Experiments are performed in which one or more parameters are modi® ed, and the changes in retention time for each peak are used to predict the best conditions under which to work.Predicted experiments are attempted, and the model is re® ned as necessary.The result is generally a better method developed in a shorter time frame.However, for particularly challenging samples, the experiment/prediction cycle may involve a large number of iterations, and a concurrently large amount of time and eå ort for the chromatographer .Attempts have been made to automate fully this process, hampered by inability to track (particularly impurity) peaks.Advanced Chemistry Development has designed a solution to this problem, by using secondary detection techniques.ACD/AutoChrom uses both UV-Vis and MS detection to track peaks unequivocally, developing methods resolving even trace impurities.The software is designed to interface with any of a number of compatible LCMS instruments, and is fully documented to allow integration with custom-built software.

A rapid high-performanc e liquid chromatograph y method for the simultaneous analysis of multiple sunscreens in cosmetic products
Gaspare A. Albanese It is well known that long-term repeated exposure to UV-A and UV-B radiation from the sun is extremely harmful.Long-term exposure is known to cause skin photo-ageing , sunburn, photosensitivity and an increased risk of skin cancer.As a result, many chemical and physical sunscreens have been incorporated into cosmetic formulations to combat the eå ects of UV-A (320± 400 nm) and UV-B (290± 320 nm) radiation.Therefore, the analysis of sunscreens in cosmetic formulations has become an important issue.Many recent publications have addressed the issue of sunscreen analysis.Most methods involve performing multiple chromatographi c runs and the use complicated buå er systems to acquire the desired separation of the analytes of interest.We have developed and optimized an HPLC method for the separation and quantitation of several of the most commonly used sunscreens: benzoresorcinol , 2-hydroxy-4-methox y benzophenone, oxybenzone, avobenzone, octyl methoxycinnamate , octocrylene and octyl salicylate.Our new approach reduces the actual number of chromatographi c analyses needed into a single 25-min run.The method uses a nonbuå ered gradient system with a reverse phase C8 column.Use of a scanning photo-diode array detector enables each component to be extracted and quanti® ed at its respective wavelength maximum.Accurate quantitation is accomplished using an external standard and identi® cation is con® rmed by UV spectral analysis.The use of this newly developed method has dramatically increased productivity and eae ciency in the laboratory .

Gabriel Levin
1 , Igor Nazarov The issue of drug quality and security has been the focal point for endless eå orts ever since drugs were manufactured.NIR has been implemented in several ways from incoming materials inspection to blend uniformity control.Implementation was hampered by low speed, sensitivity to ambient light variations, vibrations.Over the past several years, Brimrose cooperated with major companies in providing successful on line solutions.The following are some examples.
. Drying in a ¯uidized bed is a complex process, realtime control is essential for reproducible and quality product.The result is reproducible product and consistent tableting.
. Infrared spectroscopy has an important and unique role in the analysis and quality control of pharmaceutical products.Its ability to provide a unique ® ngerprint of a material makes it ideal for the de® nitive identi® cation and characterization of drug materials and associated raw materials.The technique can be, or has the potential to be, applied successfully to all facets of the industry, from raw material quality control to ® eld characterization of formulated ® nal products/dosage forms.The role of infrared spectroscopy has traditionally been limited because of the industry requirement for absolute reproducibility of measurements, a factor that has been an issue with traditional infrared measurement techniques.The use of diamond ATR simpli® es sample preparation makes it practical to sample materials equally in all forms: from liquid reactants to solid excipients, and from aqueous-base d injectables to tablet and capsule formulations in ® nal dosage forms.The diamond-based concept of sample preparation now provides a single method of sample handling that can be applied essentially to all materials without imposing changes in form or composition.Furthermore, the technique is very reproducible and requires no special skill in its operation.This paper will discuss the use of diamond ATR in an integrated, portable system for the following.
. The screening and testing of incoming raw materials, which can now take place at the point of delivery, the warehouse facility and/or the product blending area. .The role as an R&D and production tool in various areas of the plant, including reaction vessels, fermentation vats and product dryers will be covered.In this latter case, it is possible to move the system on a cart to the speci® c point of the process, and provide fresh data, without the delays associated with sending samples back to a remote analytical laboratory. .Finally, dosage forms can be tested for consistency, quality, and even identity from the warehouse, to the distribution centre, to the drug store or the hospital pharmacy.It will be demonstrated that this approach, with a combination sample handling and instrumentation , can provide a universal tool for the characterization of raw materials, intermediates and ® nal products in the pharmaceutical industry.The development of techniques and methods for eaeciently separating and detecting analytes in biological ¯uids becomes increasingly important in the post-genomic era.We have previously described a novel platform coupling self-encoded metal particles with mass spectrometry in the ® eld of immunoassays, relying on highaae nity capture of analytes from biological matrices before MS analysis.Herein, we report an expanded application of these particles as a solid-phase extraction (SPE) medium in biological analysis.Nanobarcodes identi® cation tags (NBCs) possessing varying surface chemistries are used to extract rapidly classes of metabolites/proteins from biological ¯uids in the presence of high levels of salts and abundant proteins.A large variety of stationary phases are tested, ranging from traditional chromatographi c materials to biomolecules covering a wide range of aae nities.Captured analytes, including small molecules, peptides and proteins, are detected using both MALDI and ESI mass spectrometry after elution and are identi® ed by MS/MS.Thus, multiplexed comprehensive pro® ling of biological samples is carried out using NBCs derivatized with a combination of diå erent stationary phases.In addition, an automated system is evaluated to improve the throughput of sample processing.The results presented will further illustrate the capability of this novel NBC-MS interface to achieve highly multiplexed, sensitive and selective measurements in small volumes of biological samples.

A novel molecular aptamer beacon for real-time protein recognition
Jinawei Je¡ery Li One of the most pressing problems facing those attempting to understand the regulation of gene expression and translation is the necessity to monitor protein production in a variety of metabolic states.Yet, there is no easy solution that will either identify or quantitate proteins in real-time.Recently molecular beacon-based assays have shown promise for real-time protein detection.Here we introduce a novel approach to construct an aptamer beacon for real-time protein recognition and quantitation.An aptamer beacon based on a thrombinbinding aptamer was prepared by labelling the two ends of the aptamer with a ¯uorophore and a quencher, respectively.The aptamer beacon combines the signal transduction mechanism of molecular beacons and the molecular recognition speci® city of aptamers.Signi® cant ¯uorescent signal change was observed when aptamer beacon was bound to thrombin, which is attributed to a signi® cant conformational change in aptamer beacon from a loose random coil to a compact unimolecular quadruplex.The aptamer beacon recognizes its target protein with high speci® city and high sensitivity in homogeneous solutions.Ratiometric imaging has been conducted with aptamer beacon labelled with two ¯uorophores , which makes it feasible for protein quantitation in living specimen.The unique properties of the aptamer beacon will enable the development of a class of protein probes for real-time protein tracing in living cells and for eae cient biomedical diagnosis in homogeneous solutions.

Katrin Batereau, Baldur Barczewski, Norbert Klaas and Martin Muë ller, University of Stuttgart, Pfa¡enwaldring 61, Stuttgart D-70550, Germany
The main objective of the research is the development of ® eld-screening instruments for on-site and in-situ measurement and assessment of contaminated sites to increase the statistical certainty of site characterization (by renunciation of detail precision of single-point measurement) and decrease costs for investigation.The used sensor arrays consist of diå erent sensor types of varying sensitivities.Data analyses by pattern detection give information about chemical composition and the concentration of contaminants.Metal oxide and quartz oscillators are suitable for measurement of VOC in the subsurface.The prototype that contains both sensor types is ® tted to a geotechnical driving rod and showÐ in laboratory and ® eld testsÐ suae cient sensitivity to detect of contamination typically found in the ® eld.The ® rst attempt of pattern detection showed that the distinction between chlorinated and mineral oil hydrocarbon s is possible.Laboratory and ® eld tests showed that ® breoptic sensors are suitable for the detection of free-phase organic contamination and of dissolved organic contaminants (e.g.PAH).Further tasks are investigation and application of diå erent sensor principles (surface wave resonators , heat conductivity sensors, etc.).

A real-time ratiometric method for the determination of glucose inside living cells using optical nanosensors
Hao Xu are made and demonstrated here to enable reliable, real-time measurements of intracellular glucose.The PAA nanosensors are prepared using a microemulsion polymerization process.The sizes of these spherical PEBBLE sensors range from about 20 to 200 nm.These sensors incorporate glucose oxidase, an oxygen sensitive ¯uorescent indicator (Ru[dpp(SO 3 Na) 2 ] 3 )Cl 2 , and an oxygen insensitive ¯uorescent dye, Oregon Green 488dextran or Texas Red-dextran, as a reference for the purpose of ratiometric intensity measurements.The enzymatic oxidation of glucose to gluconic acid results in the consumption of oxygen, which is measured by the oxygen-sensitiv e ruthenium dye.The small size and inert matrix of these sensors allow them to be inserted into living cells with minimal physical and chemical perturbations to their biological functions.Compared with using free dyes for intracellular measurements, the PEBBLE matrix protects the ¯uorescent dyes from interference by proteins in cells, enabling reliable in vivo chemical analysis.Conversely, the matrix also signi® cantly reduces the toxicity of the indicator and reference dyes to the cells, so that a larger variety of dyes can be used in optimal fashion.Furthermore, the PEBBLE matrix enables a synergistic approach in which there is a steady-stat e with local depletion of oxygen due to enzymatic oxidation of glucose.This cannot be achieved at all by pushing free enzyme and dyes into a cell.The work presented here describes the production and characterization of glucose sensitive PEBBLEs.The sensor response is determined in terms of linear range, response time, sensor stability and reversibility.The methods for loading PEBBLEs into cells will be discussed, including gene gun delivery and liposomal delivery.Applications of these PEBBLEs inserted in mammalian cells for real-time intracellular glucose analysis will also be demonstrated.

Autonomous environmenta l monitoring devices: can potentiometric sensors play a role?
Dermot Diamond, School of Chemical Sciences, National Centre for Sensor R, Dublin 9, Ireland The merging of wireless communications with chemical sensors and biosensors opens the way to the realization of minaturized autonomous sensing devices.While there are many potential areas of application for such devices, environmental monitoring is particularly attractive.Key characteristics for these devices can be summarized as able to function independently in the ® eld for at least 1 year and capable of providing reliable analytical information throughout this period via wireless communications.Despite the emphasis in wireless communications on broadband systems for high-quality video and audio data exchange (e.g.Bluetooth), there have been signi® cant developments in low-power, lowbandwidth systems that are much more suited to sensor applications.It is now possible to obtain relatively small transceivers commercially that can run for many months or even years on button batteries.However, for the concept to be realized, robust analytical systems are needed.Most attention is currently focused on lab-ona-chip devices as they oå er routes to on-line calibration, low reagent consumption and assays based on wellknown chemistries.While potentiometric sensors have some attractive characteristics such as very low power consumption, signal independent of size (can be made very compact), ¯exibility in shape/size and good selectivity, there are formidable obstacles to be overcome if these sensors are to play a signi® cant role in the emerging area of autonomous environmental monitoring devices.Chief among these are device lifetime, calibration, limit-of-detection and reference electrode/liquid junction design.These issues and the overall concept of autonomous monitoring devices will be discussed in this presentation.

Raoul Kopelman
1 , Murphy Brasuel Embedding) optical sensors have been produced in three classes of biocompatible matrices: hydrophilic, hydrophobi c and amphiphilic.In addition to the original pH and calcium probes, these include now ratiometric potassium, sodium, magnesium, zinc and chloride nanosensors .While the hydrophilic (polyacrylamide) robes are based on selective ¯uoro-ionophores (pH, Ca, Mg, Zn), the hydrophobic (polydecylmethacrylat e liquid polymer) probes enable highly selective operation via ion correlation (ion exchange or co-extraction).The hydrophobic nanospheres are stabilized (in solution or cell) by appropriate dispersers, as are the amphiphilic (sol-gel) PEBBLE nanoparticles.In spite of their nano-dimensions, the ion-correlation-base d sensors exhibit calibration curves identical to those derived from thermodynamic equilibrium theory.While individual 200-nm sensors can be used, an ensemble of many such sensors is more practical for most bio-applications (and obligatory for 20-nm sensors).Reversibility and photostability are excellent.The overall intracellular lifetime of these disposable nanosensors is signi® cantly longer than the required application time (cell lifetime), while the (dry) storage time is 6± 12 months, or longer.Cell delivery is straightforward (by gene gun or liposomes) and can be directed toward subcellular compartments.Most importantly, these sensors are minimally cell invasive, both physically and chemically, while immune to chemical perturbations from the cellular environment.Real-time applications to brain cancer cells demonstrate the utility of chemical analysis and imaging by these nano-explorers .The transport, adsorption and binding of organic molecules within porous-silica particles is important technology for chemical separations, bioanalysis, combinatorial synthesis and development of ¯uorescently labelled materials.The performance of these nanomaterials depends on understanding their chemical structure and reactivity, which are dominated by interfacial processes due to their high speci® c surface area.In this talk, several optical spectroscopy tools are discussed that can probe the structure of sol-gel silica materials and chemical reactions that occur within them.To observe the infrared spectra of adsorbates and bound molecules on silica surfaces, thin sol-gel coatings to an internal-re¯ection element, which provides a high surface area substrate for in situ ATR-FTIR experiments.Adsorption and binding of molecules to sol-gel silica surfaces have been investigated using this approach.For observing molecular transport within sol-gel materials, time-dependent ¯uctuations in ¯uorescence from a small population of molecules can be observed and related to diå usional kinetics within the sol-gel ® lm.To gain a more detailed understanding of molecular transport in these materials, ¯uorescence of individual molecules can be imaged, and the trajectories of their random walks within the ® lm can be mapped and characterized.Finally, the development of high-throughpu t optical microscopies allows individual particles to be observed and characterized.Fluorescence microscopy is employed to report dye labelling of individual nanoparticles at the single-molecule level.Confocal Raman microscopy is used to investigate the chemical structure and reactivity of individual, optically trapped submicron particles.

A fast, accurate, on-line dilution accessory for ame atomic absorption spectrometr y
Steve Morton, Brian Clarkstone and Paul Neal, Thermo Elemental, Solaar House, Mercers Row, Cambridge CB5 8BZ, UK Flame atomic absorption spectrometry (FAAS) is a wellestablished, versatile analytical technique that is used widely in many industries.Although sensitive and selective, it suå ers from a limited dynamic range.To overcome this, many types of sample require pretreatment and dilution to bring them into the measurement range of the technique, adding to the cost and complexity of the analysis.There have been several attempts to design an automatic dilution system that will perform this step of the analysis automatically at the point at which the sample is measured.These fall into two classes: those that mimic manual dilution, by mixing accurately measured volumes of the sample and diluent solution before passing the resultant mixture into to the spectrometer for measurement, and those that operate on a controlled ¯ow principle, in which a sample stream with accurately known ¯ow rate is dynamically mixed with a similar diluent stream at the sample inlet of the spectrometer.This paper is concerned with the second, dynamic mixing, and technique.Methods of creating ¯owing sample streams with accurately known and reproducible ¯ow rates will be reviewed, and the advantages of a newly developed type of pump will be illustrated.The considerations involved in designing a practical dilution device based on these principles will be discussed, and the performance of such a device will be quanti® ed with reference to some typical FAAS analyses.
Determinatio n of arsenic in environmenta l and biological samples using an electrotherma l cell with tungsten coil in a hydride generation-atomi c absorption spectrometry-ow system Solange Cadore The determination of trace elements by using tungsten coil as electrothermal atomizer and vaporizer has been made and shows to be a feasible alternative for the graphite tubes.Tungsten ® laments show some advantageou s characteristics such as: use of high temperatures (about 3500 K), non-porous coil surface and no memory eå ects, electric suppliers are only 150 W (e.g. 15 V, 10 A), no formation of refractory carbide and low cost.The aim of the present work was the determination of arsenic in natural waters and biological samples by hydride generation-atomi c absorption spectrometry coupled with a ¯ow injection system.The developed system uses a new electrothermal cell that employs a tungsten coil for the arsenic atomization, after the arsine generation.The parameters involved in the HG-AAS and merging zones ¯ow injection systems were optimized: acid concen-tration (hydrochloric acid, 1 mol/l), reduction reagent concentration (sodium borohydride, 0.7% w/v), sample and reagent volumes (1 ml), atomization temperature (1900 K), reaction coil (60 cm long £ 0.8 mm i.d.), ¯ow rate for reduction reagent, HCl and sample (4.5 ml/min) and ¯ow rate for carrier gas (250 ml/min).To improve the sensitivity the water vapour must be removed with a continuous system using a Na® on membrane, that allows a 92.7% eae ciency.The calibration graph is linear up to 320 ng/ml arsenic (R = 0.9999).The limit of detection, calculates as three times the standard deviation of the blank signal, was 1.9 ng/l with relative standard deviation < 6%.The analytical frequency of the proposed system was 60 determinations per hour.It was possible to use four diå erent types of tungsten coil with similar performance related to sensitivity and limit of detection.The proposed system did not showed interference from other elements, considering the species usually present in environmental and biological samples.The accuracy was evaluated with reference biological and environmenta l samples and the achieved results were in good agreement with certi® ed values.The recovery of arsenic in mineral water and seawater, using the proposed system, showed values from 99 to 103% [FAPESP, CNPq].

Direct and rapid determination of heavy metals in human blood and plasma by Zeeman modulation polarization atomic absorption spectrometry with high-frequency modulation
Evgenia M. Iakovleva Information about concentrations of elements in biosamples can be useful for medicine if we know the background levels, the ranges of changing and trends of the elements concentrations for healthy people and patients.It is desirable to use direct and rapid technique for this data obtaining.New technique Zeeman Modulation Polarization Atomic Absorption Spectrometry with High-Frequency Modulation allow one to determine many elements directly (without additional compounds and reagents or with there minimum use) in blood, plasma.The highest selectivity of this technique allows one to determine the elements in the presence of substantial background absorptionÐ up to 98%.These parameters of the device allow carrying out the analysis of biological tests without preliminary pretreatment.For biosamples analysis (if we use atomic absorption technique with electrothermal atomization) two usual problems must be decided: removing the matrix eå ects and compensating on of background absorption.The matrix eå ects can be reduced in great deal with the aid of L' vov platform, sample pyrolysis and palladium modi® er using.A high level of background absorption can be corrected by high selective Zeeman technique only.NIST blood standards were used in our work.The contents of metals were determined in human blood and plasma.One of these elements was Ca, but Ca concentrations in blood and plasma are very high and the sensitivities for this element are also very high.It demands a high degree of dilution.For ions resonant lines the sensitivities are determined by atomization temperature since the sensitivities one can change in high degreeÐ up to 100 times.In this case, one can use rapid and high-precision technique almost without dilution.Because we have using ionic absorption spectrometry for analysis Ca.The data for trends (Pb, Mn, Cu, Al and Ca) are also presented.Copper and Al trends are almost absent.Mn concentration decreases in 1.5± 2.0 times from morning to night.For 60% of subjects, morning Pb concentration is 60± 90 ppb.Night concentration is low: from 30 to 70 ppb (because of liquid exchange).Subject no. 5 was working with leadÐ for this case, Pb concentration was high (168 ppb).Some metals (e.g.Ni, Cu, Al) were determined in the urine of children from two St Petersburg areas.The same metals we determined in the urine of children living in a resort area of the city.These results are discussed in the report.

The determinatio n of ultra-trace mercury in environmental samples by cold vapour atomic absorption and mercury concentrator
Christine M. Rivera This talk will describe the determination of mercury at the subparts-per-billio n level using the technique of concentrating mercury onto a gold trap to form a gold± mercury amalgam.A hydride generator will be used to form the atomic mercury vapour, which will then pass to a gold ribbon.The mercury is vaporized from the surface and passed to a quartz cell for atomic absorption measurement.By adding the amalgam stem into the system, the sensitivity can be increased by 50 times.The technique will be explained including hardware schematics, as will the analytical procedures required for ultra-pure determinations of mercury.Sediments, standing waters and soils will be analysed.Sample data, detection limit and sensitivity will be presented.

Determinatio n of Hg in environmenta l samples using a spectrometer with dual atomic uorescence detectors to meet the demands of US EPA Methods 1631 and 245.7
David L. Pfeil and Arthur Reed, Leeman Labs, Inc., 6 Wentworth Drive, Udson, NH 03051-4918, USA Eå orts to understand and control mercury pollution have expanded investigations into relatively pristine environments, pushing detection limit requirements into the subpart-per-trillion range.The Environmental Protection Agency has promulgated purge and trap methodology (1631) capable of achieving such limits.Method 1631, as published, employs manual sample and reductant introduction, demanding signi® cant analyst interaction.Furthermore, with the improved sensitivity of Method 1631 comes an increased risk of contamination by samples that have an unexpectedly high concentration of mercury.In this work, we evaluate the analytical capabilities of an automated cold vapour atomic ¯uorescence system with the ability selectively to use gold amalgamation to enhance detection limits.By using two independent ¯uorescence detectors, the analyser provides an unparalleled working range for mercury determinations (from 0.5 to 200 parts-per-trillio n without gold amalgamation and from 0.05 to 25 parts-per-trillion with gold amalgamation).In addition, each sample can be screened using the traditional sensitivity detector (normally used for Method 245.7 determinations) .Only if the ¯uorescent intensity is below a preset level, will the instrument then analyse the sample using the amalgamation units and the high sensitivity detector, thus preventing contamination of the critical amalgamation units and the high sensitivity ¯uorescence cell.The system is capable of routinely providing detection limits typically µ0.05 ppt for compliance with US EPA Method 1631 and < 0.5 ppt for compliance with US EPA Method 245.7.

Doug Shrader
1 , Cathleen Zimmerman 2 and Christine Sartoros For over 30 years, the Cold Vapor Atomic Absorption Spectrometry (CVAAS) technique has been used for the determination of low levels of mercury in environmental, biological/clinical, mining and industrial samples.It has been the method of choice for analysts around the world.Recently the cold vapour technique has been modi® ed to include ¯uorescence detection.Despite pressure from these Cold Vapor Atomic Fluorescence Spectrometry (CVAFS) methods, which can achieve ultra-low detection limits, CVAAS continues to be an extremely sensitive and reliable technique for mercury determinations.A new enhanced CVAAS instrument and software module will be discussed.This dedicated atomic absorptionbased mercury analyser demonstrate s improved mercury detection limits and linear dynamic range.These improvements have been made possible by the optimization of spectrometer hardware components and experimental parameters.A detection limit of 0.2 ng/l is achieved without the use of gold amalgamation, which can lead to memory eå ects when higher-level samples are measured.Analytical results from a variety of environmental and biological reference materials will be presented showing the enhanced capability provided.

Monitoring brain metabolism in critical care patients using an on-line electrochemica l assay
Mark C. Parkin We have successfully monitored levels of glucose and lactate in the brains of head injury patients over periods of up to 5 days in the intensive care unit.During this prolonged period following the initial injury, profound secondary damage to the brain can occur.Levels of extracellular glucose and lactate could provide an important marker of the energy state of the at risk tissue.Measurement of rapid changes in these levels could give vital information about the dynamics of energy supply within the brain.In patients, the levels are sampled using clinical microdialysis with the probe placed in `at risk' brain tissue.Joining the probe to the on-line assay is a length of low volume tubing giving a 9-min lag from patient to assay.To achieve the high time resolution required, we designed a rapid sampling ¯ow injection analysis system (FIA) coupled to low volume enzyme microreactors and electrochemical detection [1].At the heart of the system is a custom-built dispersion valve that alternately injects nanolitre segments of dialysate stream into the independent glucose and lactate assays.Each assay consists of immobilized substrate oxidase and horseradish peroxidase enzyme.The enzyme turnover is mediated by a ferrocene derivative and amperometric detection is accomplished by a downstream electrode.At a microdialysis ¯ow rate of 2 l/min this gives a sampling interval of 6 s for each pair of glucose and lactate measurements.Basal dialysate glucose levels varied between 100 and 1300 mm with a typical daily variation of 500 mm .
Basal dialysate lactate levels varied between 300 and 2000 mm with a typical daily variation of 200 mm .Superimposed on these global changes were rapid increases and decreases in levels with a sub minute time scale.We think these changes re¯ect the brain capacity to resist both physiological and pathological challenges.

Luminescent zinc nanosensor for real-time monitoring of cellular processes
James P. Sumner The development and characterization of a ¯uorescent PEBBLE nanosensor for the detection of cellular zinc is detailed.Zinc has been implicated in a number of events including the onset of Alzheimer' s and as a neurosecretory product.Presently, the most common method to measure zinc involves (toxic) histochemical staining.An analytical PEBBLE sensor has been fabricated that incorporates two dyes, one is a reference and the other is sensitive to zinc, which enables the sensor to be ratiometric.This scheme is extendable to enzyme-based biosensing.The sensing components are incorporated into a polymer or a sol-gel matrix by either a microemulsion process or a modi® ed sol-gel method.The nanosensors produced are in the size regime of 20± 200 nm.Cellular measurements are made possible by the small sensor size and its biocompatibility.The eå ects of reversibility, photobleaching and leaching have been examined as well as the selectivity of zinc to other cellular ions such as Na + , K + , Mg 2 + and Ca 2 + .Because of their fast response time, these sensors are capable of real-time inter and intra-cellular imaging.

Robert B. Je¡ers and Xiaohong N. Xu, Old Dominion University, Chemistry & Biochemistry, 4541 Hampton Blvd, Norfolk, VA 23508, USA
The function of cytokines as intercellular messenger molecules that initiate biological activity by binding with receptors on target cells is not well understood.We are particularly interested in the study of the dynamics of cytokine± receptor interactions on living cells in real-time.
Little is known about these processes at the molecular level.Single-molecule analysis of cytokine function presents the potential for tracking and manipulating of individual steps in a sequence of biochemical events involved in the immune response.This understanding is essential to the design of immune-based therapies for vaccinations or to treat diseases.Nanoparticle probes were developed and employed to follow the real-time binding interactions of various cytokines with their receptors on living cells.These probes demonstrate the possibility for monitoring individual cytokine± receptor interactions on the surface of living cells.By following these interactions in real-time it is possible to monitor cellular response.Real-time analysis also allows for the determination of reaction kinetics, i.e. binding aae nity, at the single-molecule level.The experimental con® guration, updated research results and prospective applications will be discussed in detail.

Michael Markelov
1 , John Kroczek Both static and dynamic methods can be used for preparation of mercury standards in air.Static methods call for the establishing of equilibrium between mercury metal and the con® ned vapour phase at constant temperature.These are the most accurate methods because they are based on the fundamental properties of matter and are independent of ¯ow rates or geometrical shapes of vessels and samples.However, they can produce only limited amounts of vapours.The dynamic methods can produce practically in® nite volumes of mercury vapours.However, their accuracy is always in question.Permeation tubes and similar devices do change their permeation rates with time and have all kinds of connecting tubing, valves, and joints where traces of mercury can be randomly lost.They are calibrated by observing their weight loss.This takes a very long time and there is no guarantee that weight loss is only due to loss of mercury and not the impurities in the polymeric membrane of the tube, especially, at the ng/M3 range.This paper will present the RA-915 + atomic absorption spectrophotometer with Zeeman correction, which can be calibrated using sealed mercury containing cells and ¯ow through cells and to relate one to the other.The sealed cells can be placed directly in the optical path of the instrument avoiding any losses of mercury associated with vapour transfer.Therefore, it allows one accurately and quickly to determine the concentration of mercury in a ¯ow through cell to calibrate dynamic vapour generators.The calibration approach using water-based standards will be discussed as well.

Justifying the purchase of a laboratory information management system (LIMS)
Howard J. Rosenberg, Thermo LabSystems, Sales, 100 Cummings Ctr, Beverly, MA 01915-6115, USA Justifying the purchase of a LIMS has been a topic that has received a great deal of attention since the institution of these automation systems.During the justi® cation process, it is imperative initially to establish the complete cost of ownership.With the adoption of client server technology into this space and most recently with the provision of these systems under an Application Service Provider (ASP) paradigm, fully documenting the cost of ownership has become particularly critical.The justi® cation process needs to take into account both quantitative and qualitative aspects.Within the quantitative portion of justi® cation, it is essential to document traditional time/motion study results for a number of key personnel.It is important to include all personnel who will be accessing the LIMS as well as those who are dependent on the information derived from these systems.Of course, it is also bene® cial to take into account any process cost savings that can be derived from the implementation of a LIMS, if applicable.Qualitative aspects, while not always easy to document are also a key element of preparing a complete justi® cation for the purchase of a LIMS.This paper will detail the complete cost of ownership of a LIMS while pointing out several areas of `hidden' costs that commonly overlooked.Both the quantitative and qualitative aspects of LIMS justi® cation will then be discussed and explored with an eye towards discovering non-obvious cost savings.The data derived will then be applied to the purchase of a LIMS operating within a client server environment and then contrasted to a LIMS operating within the ASP model.

Implementing LIMS and automation in an energy laboratory
Don Kolva, Lisa Goren£o and Christine Paszko, ATL, Inc., 496 Holly Grove School Road, West End, NC 27376-8412, USA Following an internal automation audit by a quality systems engineer at a fortune 500 ® rm of an analytical energy laboratory (gas and electric), several areas were targeted to provide automation and productivity enhancements.Following a systematic evaluation of the current laboratory processes, the following areas were identi® ed to enhance turnaround time; sample login (automatic ID), instrument integration, reporting, (approval and transmitting results), and rapid result dissemination.Improvements that were implemented included, instituting bar coding and label generation to assist in streamlining sample login.This insured proper identi® cation of samples throughout the laboratory and saved time over manual label generation.The decision was made to implement instrument integration to enhance data quality, reduce transcription errors and increase productivity.Reporting was enhanced in several ways.First, signatures of laboratory managers were digitized and placed on reports.Next, when managers viewed and approved results, the reports were automatically faxed from the LIMS to various clients, and hard copies were automatically printed.Finally, in order to rapidly disseminate information on any ® eld samples (such as those for gas leaks) that exceed warning limits, the LIMS automatically sends e-mail to responsible managers and the cc list of other group members to act upon the results, the e-mail is forward to the managers cell phone for immediate action.

Ensuring customer satisfaction with an LIMS customer relationship management module (CRM)
Kim Skrecz and Christine Paszko, ATL, Inc., 496 Holly Grove School Road, West End, NC 27376-8412, USA As the economy slows and the business climate continues to become more competitive, ® rms that gain a reputation of excellent customer service and support have a strategic advantage over their competitors.Word quickly spreads and these ® rms can gain increased market share.This paper will focus on the implementation of an CRM module integrated with LIMS (Laboratory Information Management System).Regardless of the type of laboratory or the types of tests performed all laboratories receive complaints or inquiries on the testing performed or the ® nal product.A water quality laboratory will be used as the model in this case study, but this module can be applied to any laboratory type.The laboratory receives calls on the aesthetics of the water quality, the taste and odour, the colour and other attributes.The ASTM (American Society of Testing and Materials) has a procedure for analysing water aesthetics and reporting on the ¯avour pro® le.The results of this testing can be entered into the database and users can follow the standard procedure for performing the testing.In addition to internal testing, users are also able to enter complainants received from customers, the client name and contact information, the nature of the complaint, the location, the data and time.With this information, a sample can be collected for analytical testing.The results of the testing are held in the LIMS database integrated with the CRM module so that results can be compared and investigated.For example, if a water utility customer reports a strong chlorine odour in their water, a sample may be collected, analysed and perhaps con® rm high levels, there may be a problem with the distribution system.With this information in the LIMS, it is possible to investigate rapidly and possibly resolve the problem.Users can graph the results and monitor trends; print regular reports on open issues and assist managers is achieving customer satisfaction goals.

Robert Pavlis and Steve Bolton, Labtronics, Inc., 546 Governors Road, Guelph, N., Canada N1K 1E3
The balance is one of the most prevalent instruments in the world of analytical chemistry.Its wide spread usage in a vast array of analytical procedures generates tremendous amounts of data.Automation of the capture and processing of balance data is sure to increase accuracy, consistency, eae ciency and productivity in any laboratory.Surprisingly, balances can be among the most diae cult instruments to interface.Although the data are simple, compared with virtually any other instrument, most users normally want to do more than just transfer the numbers.Finding the right balance automation solution has more to do with the application and the user interaction than with the actual data.This presentation looks to the world of Chromatograph y Data Management as a model for the ultimate solution, one that will use client/server architecture to improve both the ¯exibility and the security of balance automation applications.This presentation unveils the world' s ® rst Balance Data System.A central server performs all of the processing, running many diå erent procedures at the same time, servicing any number of clients and storing all of the collected data.Storing the data on the server increases security and assists in meeting regulatory requirements such as 21 CFR Part 11.Because the client simply acts as an interactive display, the client hardware can be any computing device, a desktop PC, a laptop or a PDA.The system lets users de® ne their SOP, and then the control program carries out the SOP, stepping the user through each stage of the process.Controls can be added to check values, control editing, and even check the balance to ensure that the calibration schedule is followed.An SOP can be de® ned with speci® c hardware requirements and for speci® c hardware devices, so that if a ® ve decimal balance is required, only suitable hardware can be used.The SOP can also be assigned on a user basis.When a user logs on to a system, they will only have access to the SOPs assigned to them.Capabilities for reporting to LIMS and getting work lists from LIMS are built in.This ultimate solution addresses application and user requirements in a cost-eå ective manner, dramatically increasing the potential for balance automatio n throughout the laboratory.

The analysis of natural products by reversedphase liquid chromatograph y using a novel bonded silica C18 column following accelerate d solvent extraction
Andrea W. Heckenberg Consumer interest in natural products has exploded in recent years.The perceived bene® ts of these nutraceuticals covers a wide range, from relieving headaches to boosting the immune system.The therapeutic compounds that are responsible for these eå ects can be found in all parts of a plant, including the ¯owers, the leaves, the stems and the roots and include pharmacologicall y active compounds such as vasodilators, anti-oxidants , antibiotics and sedatives.Although this industry is not yet regulated, manufacturers of natural products are investigating analytical methods to produce and monitor consistent and clean product.Therefore, the separation and identi® cation of natural products by HPLC is receiving much attention.To extract the compounds of interest, the plant is often pulverized manually in a volume of solvent and then ® ltered before injection.This adds considerable labour-intensive time to the total analysis time.An alternative and less labour-intensive method to extract the nutraceuticals is Accelerated Solvent Extraction (ASE), an extraction technique that speeds the extraction process and reduces the total amount of solvent required.The system uses conventional solvents at elevated temperature and pressures, which results in improved extraction kinetics.In this paper, novel bonded silica reversed-phase columns are used to characterize the composition of a series of nutraceuticals, following ASE extraction.These columns have been optimized for eae ciency; hydrophobicity and low column bleed for use with mass spectrometers, for positive component identi® cation.

Semi-automati c sample deposition and its application to attenuated total re ection/Fourier transform infrared spectrometri c analyses
Jessica L. Jarman Fourier transform infrared (FT-IR) spectrometry is a useful tool for structural analysis.When microanalysis is required or desired, attenuated total re¯ection (ATR) elements may be used in FT-IR analyses to accommodate smaller sample quantities and lower concentrations.With the development of these sensitive and compact analysis systems, the need for automated sample deposition has arisen.Unfortunately, smaller sample volumes increase the diae culty of sample delivery.Characteristics of a reliable deposition system include reproducible results such as the total volume deposited and the placement of the deposits.Due to limited sample quantities, analyses are made more diae cult because of sample manipulation that requires precise placement of minute volumes.These small volumes favour single bounce ATR techniques if spectral analysis is carried out.We have been successful in the development of a semi-automated liquid sample depositor for use with a single bounce ATR accessory, speci® cally a Harrick SplitPea.The depositor is fashioned from a series of valves and a glass nebulizer made to custom speci® cations.The system can be used to deposit small quantities reproducibly onto an area about 400 m m in diameter, which is ideal for the SplitPea.The internal re¯ection element has an active site of only about 250 m m in diameter.When comparisons between traditional manual deposits and semi-automated deposits are made, the semi-automated deposits are superior in deposit thickness, shape, and splatter characteristics.The design has been characterized for deposit size; precision of placement, and average deposit thickness.Additionally, variables such as sample volume and solvent elimination techniques can be easily altered.Due to this ¯exibility, the system is applicable to many analyses and sample types.

Andrew M. Wolters
1 , Dimuthu A. Jayawickrama Providing structural data unattainable by other techniques, nuclear magnetic resonance (NMR) spectroscopy represents an intriguing detector for capillary electrophoresis (CE).Wrapped around capillaries, microcoils, the best NMR coils in terms of mass sensitivity, aå ord easy on-line integration of NMR to CE.Recent improvements in coupling NMR to two diå erent types of CE, capillary zone electrophoresis (CZE) and capillary isotachophoresis (cITP), are presented.Through high levels of sample stacking, cITP greatly increases the eå ective concentration sensitivity of microcoil NMR for charged species.With the added bene® t of electrophoretic separation provided by cITP, this coupling oå ers a powerful means to analyse trace charged natural and synthetic organic impurities.If a charged trace impurity possesses a greater electrophoretic mobility than the main component, it can be eae ciently isolated from the main component and simultaneously concentrated for NMR detection.As a demonstration , a 9.4 m l injection of 200 m m (1.9 nmole) atenolol, a charged pharmacological agent, obscured by a 1000-fold excess of sucrose (200 m m ) is analysed by cITP/NMR.By completely separating the atenolol from the sucrose and stacking it 200-fold (up to 40 m m ), cITP enables rapid 1 H-NMR detection of the isolated trace component (10 s observation time).In comparison with the same microcoil probe operating without the bene® t of sample stacking, cITP reduces experimental time by a factor of 40 000.For this particular probe and sample, greater stacking eae ciencies than that achieved here would not improve NMR observation as most of the injected atenolol sample occupies the microcoil during the peak maximum.NMR probes consisting of multiple microcoils arranged in series facilitate loading and trapping of focused cITP bands for stopped-¯ow NMR analysis through the use of a scout coil.Using multiple coil technology in a slightly diå erent manner, NMR probes consisting of multiple microcoils arranged in parallel can successfully avoid spectral degradation experienced during continuous-¯ow CZE/microcoil NMR.By cycling the electrophoretic ¯ow from a separation channel between diå erent outlets, high-resolution stopped-¯ow NMR spectra can be obtained from a continuous CZE separation.

Gregory M. Banik, Eric Melanson, Victoria Rafalovsky and Re¢k Karabay, Bio-Rad Laboratories, Informatics Division, 3316 Spring Garden Street, Philadelphia, PA 19104-2552, USA
A number of systems for prediction of 13 C and 1 H-NMR spectra are currently available.This paper reviews commercially available systems used in the characterization of compounds in drug discovery.Parameters impacting the quality of the predictions are enumerated, and the scope and limitations of each system are compared.

Recent advances in the automated structure elucidation of a series of related natural products from the Cryptolepus family
Antony J. Williams Signi® cant literature exists about the elucidation of a number of related natural products from the Cryptolepus family.This literature has allowed us to test recent developments in our automated structure elucidation development.Our previous work has used an expert system based on a database of 13 C-NMR spectra and a library of molecular fragments and their 13 C-NMR subspectra.We have recently implemented the ability to use information obtained from 2D spectroscopy including direct and long-range heteronuclear correlations for both C13 and N15 nuclear centres.The PC-based software solution presently delivers the ability to process raw 1D and 2D data, elucidate possible structures using the fragment databases, predict the 1 H-and 13 C-NMR spectra of the potential structures for comparison and train the prediction database s in order to use fragments from related structures for the automated elucidation process.We will review recent developments and successes in the application of automated elucidation to natural products, which in some cases have reduced the manual task of elucidation from many days to just a few minutes.

Daniela Held, Guë nter Reinhold and Thorsten Hofe, PSS Polymer Standards Service, PO Box 3368, Mainz D-55023, Germany
SEC is the most used technique to characterize polymers.The most important items in SEC instruments are the columns for separation.PSS introduced HighSpeed SEC columns in 1999 which are able to separate all kinds of polymers in < 2 min, cutting down the time requirements of traditional SEC runs by a factor of ® ve to 10.The ability to generate a 12-point calibration curve in 10 min is unparalleled.With the availability of such high-speed columns SEC results can be given extremely fast, which is necessary on one hand for in-line production control systems (quality control) and high-throughpu t screening devices and on the other hand for high-end applications like 2D chromatography .Compared with their analytical counterpart (8 £ 300 mm) HighSpeed columns have larger diameters and a smaller length (20 £ 50 mm).Therefore, they permit higher ¯ow rates with equal linear ¯ow, equal resolution and polymer information.The ¯ow rate is more limited by instrumentation (e.g.detectors, pumps) than by the column itself.The HighSpeed columns are studied concerning performance (monodisperse and polydisperse samples), accuracy and reproducibility in aqueous and organic solutions.The results obtained in just 20% of the time are comparable to analytical columns.Low shear forces on the HighSpeed columns result in less stress for the characterized samples, which is important for high molecular weight products.Nevertheless, there is still room for further SEC column development.It requires the skill and dedication of the chemists who create the packing materials and the profound understanding of the underlying principles and applications.

Keeping archived data active
Michael Elliott, Scienti¢c Software, Inc., 6612 Owens Drive, Pleasanton, CA 94588-3334, USA Archiving data for increased security and/or long-term storage does not have to mean that the data are no longer an integral part of a scientist' s daily work.Finding new meaning in old data or relationships between new and old data can save a signi® cant amount of research and development time.The limited disk space of some permanent storage media and the high cost of read/write devices often mean that archived data must be stored oåline.This paper will describe a software package that makes archived data actively available for searching, viewing and retrieving to users, whether stored on-line or oå -line.The physical transfer of data from active to archive is an important step in the life cycle of any company' s records.This transfer of custody does not have to mean that the data ceases to be useful.Cyber-LAB Knowledge Engineering System, from Scienti® c Software, makes archived records available whether stored on-line, near-line, or oå -line.When electronic records are uploaded into the CyberLAB system, keywords, text and results values can be extracted and stored as part of the record' s metadata.These metadata can be search through any of CyberLAB' s search mechanisms.During the archive process, these metadata are exported as an XML ® le and is archived with the data, however the database retains a copy of this metadata, so the data ® le remains available for searching.When searching for data with any of CyberLAB' s search tools, both archived and non-archived data can be returned.Even when the data have been stored on removable archive media, such as CD or DVD, and been removed from the system, the metadata can be searched.The only indication that the end user has that the data has been archived is a small `lock' icon next to the ® le name.CyberLAB does not alter the original ® le during the archive process.This means that an archived ® le stored on-line can be viewed immediately simply by clicking on the ® le link.Should the ® le be stored oå -line, the user has the option to send an e-mail request to the archive administrator to load the required archive volume.With this `active archive' feature researchers can search for new meaning in previous work, or correlate preceding studies with current.

Automating data management
Soheil Saadat, Scienti¢c Software, Inc., 6612 Owens Drive, Pleasanton, CA 94588-3334, USA The increasing automation of laboratory instrumentation and processes has led to a substantial increase in the amount of data produced.This data must be transformed into meaningful information as quickly and eae ciently as possible.Extracting more knowledge from existing information without overburdening scientists depends on the power of the automation method.This paper describes an automation method for the knowledge engineering process.To be an eå ective automation tool, this system must monitor the entire enterprise for new or altered data to be uploaded into the systemÐ no user interaction should be required.
This software system can monitor speci® c computers, drives and/or folders for general or very speci® c ® les to transfer.Once maintained and protected inside the software, ® les can be monitored for automated archival based on time schedule or ® le age.A variety of archive devices and archive schedules can be supported simultaneously.On ® le upload, options must be available for automated metadata extraction whether results values or searchable text.This information can be extracted from ® les as they are transferred and is available for searching immediately.During the lifetime of any piece of data, the information needs will change.It is essential that users be able to update a ® le' s metadata with additional information or searchable keys quickly and easily.Once the data has been added to the system users must be able to retrieve it in such a way that new relationships between data can be discovered and explored.Simple keyword searches can produced lists of ® les related by content.Advanced search features allow deeper similarities between diå erent data types to emerge.Both simple and complex searching mechanisms are crucial components to eå ective data mining.

Tim Meehan, Todd Stewart and Joel Hunter, RTS Enabling Technology, 3200 George Washington Way, Ste D, Richland, WA 99352-1664, USA
The Standish Group Report determined that the success of software projects is incredibly low; whereby 31% of all software projects are cancelled before completion, 53% of projects will cost 189% of estimates, 9% on time and on budget (large companies), and 16% on time and on budget (small companies).Success was gauged on restarts of projects, cost overruns, time overruns and percentage of functionality.The high rate of failure was largely attributable to lack of user input, insuae cient requirements and changing requirements.With the demands of the Internet as well as federal regulations from the FDA and DOE, quality assurance in software is increasingly becoming the main focus of software development.To mitigate the failures of past software development, a number of methodologies have arisen to address these issues.This seminar will discuss two of these methodologies, which are gaining considerable favour in the software industry: the Rational Uni® ed Process and Xtreme programming.The similarities and diå erences of the two methodologies will be discussed, and how they ® t the needs of the software owners and the software developers.

Alan M. Czyzewicz and Daniel E. Meils, Scientech, Inc., O&M, 2650 McCormick Drive, Clearwater, FL 33759-1005, USA
This paper presents a viable approach to ensuring an appropriate degree of con® dence to the precision and accuracy of the data.Thereby satisfying customer' s requirements, minimizing rework, inappropriate calibrations, maintenance and repairs.When you know and understand your customer' s valid requirements for analyses, Statistical Process Control (SPC) becomes the tool to lower costs.SPC allows for management by exception, thus focusing on work where it is needed.Shewhart control charts are needed to be at the foundation of SPC.To bene® t most from the appropriate chart, a methodical approach to its development is needed.Twenty to 30 data points from an appropriate sampling of technicians and conditions are at the foundation.Statistical tests for outliers, normality of data and % RSD need to be calculated and examined.Bias should be determined with respect to the target value and centreline adjusted accordingly.Once the chart has been developed, key indicators must be examined with each QC analysis.Finally, long-term trends should be examined via SPC by reviewing bias with respect to target value, change in precision and normality of data.Bene® t One Satisfy customer requirements by quantifying accuracy and precision through statistical analyses of your methods.Customers can chose the methods that yield the most appropriate accuracy and precision to meet their needs and budget.Bene® t Two Minimize rework by using SPC to indicate when the analysis is Out of Control (OOC) before the actual performance of analysis.Minimizing reanalysis produces costs savings.Bene® t Three Minimize inappropriate calibrations, maintenance and repairs by using SPC to determine when an instrument is OOC and corrective actions are required.`Don' t ® x what ain' t broke.' Statistical programs, such as Lab Stats Pack TM , are available to perform these calculations and reviews.Clearly, starting with the knowledge of your customer' s valid requirements and managing your instrumentation by statistical exception will create obvious bene® ts to your laboratory QA/QC program.

Kim Ahmed and Malcolm McGregor, LabVantage Solutions, 245 Highway 22, Bridgewater, NJ 08807-2560, USA
Traditionally science has been viewed as a handcrafted occupation.Skilled scientists are employed to plan and undertake experiments and report on their ® ndings.In academia, the end result is scienti® c papers and patents and in industry Quality reports or patents during the research phase.Modern discovery oriented sciences have made these methodologies as obsolete as Ford made the hand-built automobile.There are two fundamental aspects of discovery activities that have forced the changes.First, the volume of data being produced is overwhelmingly beyond a human' s ability to reduce to meaningful information without computerized automation and, second, in many instances it is the information itself that is the `product' of the laboratory.It could therefore be said that discovery laboratories are in the business of `Scienti® c Information Production' and should view their processes, workforce and product accordingly.This paper examines the proposition that `Scienti® c Information Production' is a valid model for these laboratories.The paper goes on to identify the pieces of automation required to achieve the necessary eae ciencies and ® nally to look at a software model designed to unify these disparate entities into a true production system.Each phase of the production environment is examined from customer interaction and experimental design through operations and data collection to packaging the ® nal data for sale or use.Key obstacles from the past such as the integration of existing expert systems and the portability and compatibility of various data formats are given special attention.

Specialty gas session: a fast, innovative infrared analyser for monitoring ultratrace moisture in semiconducto r FAB gases
Wen-Bin Yan, Tiger Optics, LLC, 250 Titus Avenue, Warrington, PA 18976-2426, USA Evidence of direct correlation between gas purity and process defects has been documented in ® elds such as silicon epitaxy, oxidation and annealing treatments, and ® lm deposition.This has driven equipment suppliers to develop new analytical detection systems for monitoring ultratrace gas impurities in bulk and specialty gases for the semiconductor fab.Moisture is a major contaminant in the semiconductor fab gas system.A trace gas analyser, the MTO-1000 has the detection capability to measure to 500 PPT moisture and expand to monitor for PPB or sub-PPB for other trace gas impurities such as ammonia, an airborne molecular contaminant that must be constantly monitored at ultratrace levels.The MTO-1000 uses a small, inexpensive diode laser that can operate in various parts of the infrared spectrum.The principle of operation is Cavity Ring-Down Spectroscopy (CRDS), which was jointly developed by Tiger Optics and Princeton University.In the gas phase, the spectrum of a molecular species consists of many sharp rotational lines that provide high selectivity for detection.This is the `molecular ® ngerprint' of that species.The diode laser provides the narrow bandwidth to focus on the major absorption lines for high accuracy and maximum absorption of a trace gas impurity.Data from gas suppliers will be presented and discussed on fast, trace gas impurity detection of trace moisture in both inert and corrosive gases.

Specialty gas analysis: analysis of trace-level moisture in corrosive and reactive gases using an evacuated FTIR gas analyser
Robert Klebba and Peter Zemek, Midac Corporation, 17911 Fitch, Irvine, CA 92614-6016, USA The demand for increasing purity in semiconductor gases is pushing the allowable moisture levels in these gases < 1 ppm and down to the low ppb level.For inert gases, there are relatively simple technologies for directly detecting moisture down to single-digit ppb concentrations.However, these techniques cannot be used for corrosive or otherwise reactive gases such as hydrogen bromide (HBr) or ammonia (NH 3 ).Fourier-transfor m infrared spectroscopy has been used for analysing moist-ure and other contaminants in semiconductor gases for many years.Gas cells made of corrosion-resistan t materials isolate the gas from the spectrometer components, allowing easy purity and concentration analysis.High-resolution FTIR spectrometers overcome problems due to spectral interference, and FTIR' s inherent photometric accuracy provides long-term calibration stability.Early attempts at FTIR moisture analysis were plagued with problems due to the unstable concentration of moisture inside the analyser.Even with UHP nitrogen purge, the analyser would take days to dry down, or leaks might occur allowing the in® ltration of ambient moisture.These and other factors kept earlier analysers from achieving > 500 ppb limits of detection.A diå erent approach involves removing all of the moisture inside the spectrometer by evacuating down to 10 ¡6 torr.This level of vacuum eå ectively removes all moisture inside the spectrometer, so that only the gas analyser detects the moisture in the semiconductor gas.In this presentation, we will discuss the special considerations for an evacuated FTIR gas analyser as well as the selection of speci® c components to optimize performance for moisture analysis.

Specialty gas session: temperature dependent infrared absorptivity data to support stack gas emission monitoring
Pamela M. Chu The infrared absorptivity of industrially relevant compounds methanol and sulphur dioxide are evaluated for temperatures ranging from 25 to 200 8 C at 25 8 C increments using an FT spectrometer with NIST primary gravimetric standards.This project was initiated to provide quality assured reference data for communities measuring stack emissions by passive FT-IR and other infrared-based technologies.To correct for the sample and sample cell emission eå ects, sample and reference data are acquired with the infrared source on and with the infrared source oå .The average relative expanded uncertainty for the uncorrected absorption coef-® cients is 2.0%.The emission correction tracks the blackbody emission of the sample cell and is both wavelength and temperature dependent.For temperatures up to 200 8 C, there is no emission correction for bands at ¶2600 cm ¡1 .In the ® nger print region, the emission correction be-comes signi® cant especially at temperatures > 100 8 C. At 200 8 C, the correction is approximately 10% for bands in the spectral region near 1000 cm ¡1 .To validate further the absorption coeae cient data, the spectra are compared with available data and models.

Monitoring proteins in real-time and in homogeneous solution
Weihong Tan, University of Florida, Department of Chemistry & The McKnight, PO Box 117200, Leigh Hal, Gainesville, FL 32611-7200, USA As the Human Genome is completing, researchers are turning increasingly to the task of converting the DNA sequences into information that will be highly useful in human medicine and healthcare.One of the key challenges ahead is to understand proteomics, the science of the cellular protein universe.It is expected that in the post-genome era, proteomics and the study of protein functions will play an increasingly important role in the understanding, diagnosis and treatment of the most challenging human diseases such as cancer, AIDS and heart disease.The development of new molecular probes for protein analysis is of great interest.We have developed novel protein recognition mechanisms for real-time protein monitoring using molecular beacons (MB) and molecular beacon aptamers (MBA).MBs are a new class of oligonucleotides that can report the presence of speci® c nucleic acids.They have an excellent signal transduction mechanism.Molecular beacon aptamer is developed by combining MB' s excellent signal transduction and aptamer' s protein aae nity for real-time protein recognition with extremely high sensitivity and excellent speci® city.We will report our progress in protein detection using MBAs.These proteins are important biomarkers for cancer diagnosis.

Huba Kalasz, Semmelweis University, Department of Pharmacology, Nagyvarad Ter 4, Budapest H-1089, Hungary
Each type of separation methods has limited speed.Both gas chromatograph y and liquid chromatograph y can be optimized according to the ¯ow velocity of the mobile phase.A de® nite optimum can generally be found which is determined by certain factors such as the eddy diå usion, mass transfer and longitudinal diå usion.Planarchromatograph y takes a special position among the separation techniques.The classical mode of thin-layer chromatograph y (TLC) is realized on stationary phase, which is dry (non-wetted) at the beginning of the separation.There is a gradient of the mobile phase to the stationary phase ratio that is increasing during the development.FF-TLC (forced-¯ow thin-layer chromatography ) kept the majority of features of TLC, and an essential speed-up of the mobile phase ¯ow velocity was given by the forced-¯ow.Also, the optimization of ¯ow velocity of the mobile phase was possible by the constant and regulated mobile phase supply.The optimum ¯ow velocity of the mobile phase was checked, and the plate high of the system at optimum ¯ow velocity was near to the particle diameter.Planar electrochromatograph y (PEC) is the technique of great promise.As the eddy diå usion is practically eliminated in PEC, it means really breaking the speed limit in planar chromatography .Separations may be performed in 10 to 100s of seconds instead of several to 10 min.PEC can be performed using a wide variety of stationary phases.The stationary phase is wetted before the PEC, thereby closing the electric circuit.The choice of the mobile phase depends on the solutes to be separated.Aqueous mobile phases generate higher current; therefore, a cooling system keeps the temperature constant.PEC in organic solvents generates low current, and both the solvent resistance of the set-up and thermostating at a constant temperature can be oå ered by the adequate construction of the system, which is made of glass.This project as sponsored by the grants of OTKA T025142, T032185 and T034677.

High-speed separation of biopolymers in microfabricated devices
Andras Guttman, Torrey Mesa Research Institute, 3115 Merry-¢eld Row, Suite 191, San Diego, CA 92121-1125, USA Rapid and large-scale genotyping, mapping and expression pro® ling require aå ordable, fully automated high-throughpu t devices enabling rapid, high-performance analysis using minute quantities of reagents.Electric ® eld-mediated separation methods in capillary dimensions, such as CE, ultrathin-layer electrophoresis and microchip-based techniques have been emerged in recent years to address rapid analysis of biologically important molecules, such as nucleic acids and proteins.These novel, high-performance separation techniques feature rapid separation times and very high resolving power, while requiring only very little amounts of samples and reagents.The applicability of electric ® eld-mediated microseparation methods in capillary dimensions will be discussed for rapid and high-resolution analytical and micropreparative separation of biomolecules, using replaceable polymers as separation matrices.Various ways of automation for large-scale analysis of biological samples (96-well plate format) will also be demonstrated.While electric ® eld-mediated separations in capillary dimensions are primarily considered as analytical tools, they can also be used in micropreparative applications.In micropreparative operation mode, nanomolar quantities of DNA fragments were collected for subsequent downstream processing, such as sequencing for rapid expressed sequence tag (EST) generation to establish unigene sets and for high-throughput cloning.

Julian F. Tyson
1 and Ahmed Youssef We have developed a procedure for the speciation of aluminium in natural water systems based on the reaction of `free' aluminium with 8-hydroxyquinolin e and extraction of the resulting complex into isobutlymethyl ketone (IBMK).To restrict the extracted aluminium to that which was only able to react rapidly, the kinetics of the formation and extraction processes were controlled in a ¯ow-injection system.To determine the low concentrations likely in natural waters, quanti® cation was by graphite furnace atomic absorption spectrometry.The problem of pumping the IBMK was overcome by the injection of a relatively large volume (2 ml) into an air carrier stream.The system contained a novel-phase separator device, which was fabricated in-house based on a design of Zhaloun Fang' s.This device has a conical Te¯on cavity mounted on a stainless steel base; the IBMK wets the Te¯on and is drawn out via an exit at the apex of the cone, whereas the aqueous phase wets the stainless steel.The system was evaluated by comparing the measured fraction of free aluminium with that calculated from the relevant equilibrium constants for several model ligands (EDTA, oxalate and malonate).Good agreement was obtained.The procedure was applied to the study of the aluminium binding to a humic acid material, the aluminium speciation in surface water, and to a study of the kinetics of dissociation of aluminium complexes and polymeric aluminium species.The procedure was applicable to concentrations down to 2 ppb.The surface water contained 60 ppb of dissolved aluminium of which 2.4 ppb was reactive.On acidi® cation, 20 h was needed to release the aluminium bound to the humic acid material.

Determinatio n of antimony by ow injection hydride generation atomic absorption spectrometry after photo-oxidativ e sample pretreatmen t
David J. Scott and Julian F. Tyson, University of Massachusetts, Department of Chemistry, 710 N. Pleasant Street, Amherst, MA 01003-9305, USA Increased analyte transport and the removal of interfering matrix eå ects are two advantages of hydride generation (HG) sample introduction in atomic spectrometry compared with standard nebulization.HG is widely used in the trace determination of arsenic, selenium, mercury and antimony with various atomic spectrometry detectors.As toxicity is directly related to chemical species, the trend in research has been towards speciation based on chemical or chromatographi c separation with subsequent element-speci® c detection.Recent reports describe the speciation of inorganic antimony by manipulation of the oxidation states before hydride generation, although the measurement of total antimony content with little regard to speciation is often reported.There is currently interest in organoantimon y [1] compounds, such as trimethylantimony, found in urine [2].The hydride generation chemistry of trimethylantimony has been the subject of discussion due to the demethylation [3,4] of the hydride species and subsequent molecular rearrangement resulting in broad peaks and poor detection limits.One way to overcome these problems would be to convert the antimony species separated chromatographicall y to SbIII by a suitable post-column reaction before forming stibine.Here we present our investigations into the photo-oxidation of antimony compounds before reduction to SbIII and subsequent hydride generation.

Atomic absorption determinatio n of inorganic and methylmercur y by ow injection chemical vapour generation and amalgam trapping
Julian F. Tyson Studies of biogeochemical transformation s are underpinned by the provision of reliable information about the chemical composition of relevant materials with respect to inorganic and organomoetallic constituents.With regard to mercury, while a number of organomercury compounds are known, it is the pathways involving inorganic mercury (i-Hg) and methylmercury (m-Hg) that are of current interest.We have been developing a procedure based on the reaction of the two species, i-Hg and m-Hg, with borohydride [1].Our hypothesis is that the species form elemental mercury (e-Hg) and methylmercury hydride (m-HgH), respectively (though this is contrary to some published results, but in agreement with others).As we think that m-HgH does not absorb radiation from a Hg hollow cathode lamp, the direct transfer of the vapours generated to an atom cell allows determination of i-Hg only.As both species are trapped by gold± platinum gauze, both are determined via the amalgam trapping route.On this basis, we have accurately quanti® ed the mercury species in some marine tissue reference materials from the National Research Council of Canada.Recently we have found that the extent to which e-Hg is produced from m-Hg is related to both the borohydride and the sodium hydroxide (added to slow the decomposition of the reagent in aqueous solution) concentrations, so that conditions may be selected under which both species form e-Hg.This opens a new possibility for a speciation method based on diå erent borohydride/hydroxide concentrations .Selectivity is also obtained by the judicious use of hydrochloric acid to extract the mercury species from the samples.At low HCl concentrations , only m-Hg is extracted.Samples may also be handled as slurries in the FI system, and we have obtained selective preconcentration by sorbent extraction on C-18 following complexation with APDC.Detection limits were improved by up to a factor of 10. ) is provided by a built in 10-m multipass cell.The selectivity of the instrument to mercury is assured by using a single mercury isotope lamp along with Zeeman correction.To address high levels of mercury contamination, the instrument is also equipped with a 6-cm single-pass cell.The instrument also has a built in cell for instant QC check in the ® eld.It can be operated via its own hand held controller/reader or using a notebook PC.The paper will also discuss approaches to calibration of Mercury analysers for analysis of air, water, soils and petroleum fractions.

Real-time in vivo study of single-molecula r biological events of bid
Qian Wan 1 and X. Nancy Xu Bid is one of the widely studied apoptotic proteins.Upon the induction of apoptosis, bid will be cleaved into two parts: 6.5 kD tn-bid and 15 kD tc-bid.Previous studies have showed tn-bid bound tc-bid in vitro using Western blot and tc-bid moved into mitochondria to promote apoptosis.However, it is unclear whether tn-bid and tcbid will bind in living cell and where tn-bid is located inside the cells.We study these cascades using real-time single-molecule chemical microscopy.This research aims to demonstrate the ® rst single-molecule measurements of sequencing events in vivo and uncover the roles of bid in apoptosis in vivo.The tn-bid and tc-bid genes are cloned using plasmids and fused with red ¯uorescence protein and green ¯uorescence protein, respectively.Then, the plasmids are transfected into human cells and monitored in real-time using single-molecule dynamics microscopy.

S. Alison Arnold, John Crowley, Linda M. Harvey and Brian McNeil, Strathclyde Fermentation Centre, Department of Bioscience, Glasgow G1 1XW, UK
The use of Near Infrared Spectroscopy (NIRS) to monitor both microbial and mammalian cultivations was investigated.NIRS oå ers a number of advantage s over other monitoring techniques in that it is fast, requires no pretreatment of sample, can monitor several analytes simultaneously and has the capability to be used on-line or in-situ; therefore, enabling analysis of both chemical and biological parameters in terms of real-time.Potentially, a real-time multi-analyte monitoring system could be very useful within the bioprocessing industry.In the present study, application of the technology was ® rst investigated at-line (where the analyser is near the bioreactor although not in direct contact with it).The cultures investigated included Escherichia coli, Streptomyces fradiae and Pichia pastoris.Successful models were built and validated for a number of critical analytes and products within these three very diå erent bioprocess ¯uids [1,2].Following the successful application of NIRS at-line the use of in-situ NIRS was examined.This was carried out using steam sterilizable ® breoptic probes in the bioreactor.Here the probe is in direct contact with the process stream inside the bioreactor allowing real-time analysis of analytes.Successful on-line models for biomass in a fedbatch E. coli system and glucose, glutamine, ammonia and lactate for a CHO-K1 cultivation were built and validated.The key issues in conversion from at-line to in-situ operation were discussed.The success of NIRS both at-line and on-line in monitoring such an array of diå erent analytes from very varying bioprocesses bodes well for its routine use in an industrial setting.As we use the term, a discriminator is a algorithm that can be used to classify a spectrum within de® ned sets of spectra.Each set is for a given chemical composition: a pure material or a mixture.It can be used to con® rm the identity of a material or automatically to identify a material.This concept is substantially diå erent from that of a search algorithm.In a search, the goal is to identify the most likely spectral match, but con® rmation of identity is done visually.Searches are not set up as discriminators, if they were then matches would seldom be obtained because of sample impurities and variations in spectrometers and spectral accessories.The greatest hindrance to setting up a discrimination package for a plant has been the lack of automation, with days or weeks of work to set up discriminators for the individual materials.This algorithm uses two variables for developing discrimination criteria from spectral data: a similarity parameter and a PCA model.The similarity parameter is calculated between a measured spectrum and a representative spectrum of each target material.The PCA models are used to con® rm the results.To develop the discriminator model the user is only required to enter the name of the material and the spectra of a series of measurements.The program develops the models with no user input, appropriate masks are calculated for each spectral region and inappropriate spectra are identi® ed.In use the program automatically classi® es the spectrum based on the appropriate criteria, which are set automatically when building the models.Examples of the use of this package with several data sets will be presented.

Applying equality constraints during alternating least-square s factor analysis
Mark H. Van  The use of constraint s during multivariate curve resolution is critical to ensure one obtains a reasonable and physically meaningful solution.Typical constraints are non-negativity , unimodality, closure and selectivity.Frequently, equality constraints are employed to ensure that a variable, whose value is known or well de® ned, is contained in the solution.A common practice to employ equality constraints, presumably for computationa l convenience, is to solve the least-square s problem without constraining the solution, followed by replacement of the constrained variables with their known values.While this method is easy to program and fast to implement, it most often destroys the least-squares result.We will present established methods of solving the constrained leastsquares problem.We will also discuss the bene® ts of using these methods and demonstrate how they can be employed in alternating least-square s factor analysis.The suitability of coupling an HPLC to an ICP-MS for the fully automated, routine analysis of bromate in drinking water as per the proposed EPA Method 321.8 was investigated.The necessity to monitor the carcinogen bromate in ozonated drinking waters at single ppb levels has led the USEPA to investigate HPLC-ICP-MS as an alternative technique to the ion chromatograph y with conductivity detection method currently speci® ed.During this investigation, a series of rigorous performance checks were used to assess the implementation of the proposed method including determination of the abundance sensitivity of the ICP mass spectrometer and the method detection limit, assessment of potential chromatographic interferences, and analysis of laboratory for-ti® ed blank and matrix samples.This was followed by the determination of bromate in a series of EPA disinfection by-produc t (DBP) standards.A description of the protocol used and the corresponding analytical ® gures of merit will be presented.

A consensus method for available cyanide using ligand displacemen t and ow injection analysis amperometry
Scott W. Stieg A new ASTM standard test method for available cyanide is described.ASTM Committee D19, Water Quality, developed this method.`Available Cyanide' has been de® ned by the USEPA to replace or more precisely de® ne the older term `cyanides amenable to chlorination' .It includes the species HCN, CN ± , and a set of metal± cyanide complexes, such as mercury and nickel cyanides, that are easily dissociated into free cyanide ions.The method does not detect the less toxic strong metal± cyanide complexes such as ferrocyanide and ferricyanide.A proprietary version of this test method (OIA-1677) was approved by the USEPA in December 1999 for NPDES and NPDWR compliance monitoring.This ASTM method will be a consensus version of this method, and the collaborative study will lead to approval for these Clean Water Act and Safe Drinking Water Act programs.The apparatus is commercially available from both Lachat Instruments and OI Corp.A water sample is digested by batch addition of ligand-exchange reagents tetraethylenepentamine and dithizone solutions at room temperature.After a few minutes, the digested sample is injected into a carrier or donor stream which is acidi® ed.The resulting HCN(g) diå uses across a hydrophobic membrane into an alkaline acceptor stream.This eliminates non-volatile interferents such as thiocyanate.The cyanide anion in the acceptor stream is detected at a silver electrode by oxidation of silver to silver cyanide and measurement of the resulting amperometric current at about 0 V versus Ag/AgCl.The amperometric method eliminates the pyridine used in the colorimetric version of this method.Weak acid dissociable (WAD) cyanide can also be determined by omitting the digestion with ligandexchange reagents.As required by ASTM and the USEPA a collaborative study organized by the Bayer Corporation Environmental Testing Services was carried out by ASTM D19 during 2001± 2002.The results of this interlaboratory study will be presented.

Discrete analyser technology applied to environmental analysis
Stephen C. Coverly In clinical chemistry, continuous-¯ow (CFA) analysers have been almost completely replaced by `discrete' analysers where each reaction takes place in a separate vessel.Discrete analysers provide more reliable analysis at lower cost and with lower manpower than CFA systems.However, the clinical analysers have found very limited application for water, food and industrial samples.One reason is that the requirements for accuracy and detection limit in clinical analysis are less rigorous than for industrial samples: another is the diå erence in techniques, colorimetric end-point analysis being the only technique widely applied to both ® elds.Furthermore, up to now no methods were available based on EPA approved chemistries.We have built a discrete analyser specially designed for environmental samples.Compared with a clinical analyser it has changes to the detector, sample and reagent holders and to the software.We have developed special hardware and software to automate the Cd reduction stage required by the EPA for nitrate analysis.All the 11 EPA water analysis methods commonly run with CFA have been automated.Reproducibility is > 1% RSD for each method, with detection limits (MDL) in the low g m l ¡1 region.The analysers can automatically rerun samples in diå erent ranges, with manual or automatic range selection.Sampling rate is method-dependent , and is 100± 200 tests per h for most methods.We present comparison data obtained on laboratory quality control and actual environmental samples of fresh, brackish, and wastewater.

Reno Cerra
1 and Mike Retzik A Total Organic Carbon (TOC) analyser using a unique design will be discussed.The bene® ts of this newly development method include the ability to achieve fast TOC analysis with extremely low running costs.The development and application of this method will be discussed in conjunction with the derived bene® ts.In a process application where continuous monitoring is required, the operating cost of any technique is important.The relatively high costs associated with some TOC measurements can make on-line (process) TOC measurements very expensive.A method of measuring TOC inexpensively and rapidly would be a signi® cant advantage in these applications.In this newly developed method, the oxidation is achieved through the use of titanium dioxide-based photocatalyti c oxidation.Titanium dioxide is extremely inexpensive and also very safe.The oxidation takes place in a low-volume photocatalyti c reactor that is speci® cally designed to provide highly eae cient and rapid oxidation of organic carbon-to-carbo n dioxide.Since the system does not require a carrier gas or other oxidizing reagents the operating costs are further reduced.The sensitivity of the system is vastly enhanced by the introduction of several miniaturized components, and an improved closed-loop ¯ow design.The NDIR detector used in this method provides enhanced sensitivity while maintaining a small system volume.The ® nal result is a unique TOC system that has a very low operating cost, hence making it particularly suited to continuous on-line measurements.

Ralf Dunsbach
1 , Joachim Kupka 1 and Scott Hughes 2 , 1 Elementar Analysensysteme GmbH, Donaustrasse 7, Hanau D-63452, Germany, 2 Elementar Americas, Inc., 520 Fellowship Road, Ste B-204, Mount Laurel, NJ 08054-3408, USA Total Organic Carbon (TOC) analysers originated about 40 years ago: two techniques currently dominate, one using UV and the other high temperature for oxidation of the carbon in the sample.The current development uses the high temperature oxidation method for measuring TOC but is unusual because a multipurpose reactor compactly and eae ciently houses several of the traditional chemical processes required in this analysis.The sample up to 2 ml ¯ows into the vertical reactor.Acid is combined in the reactor converting the inorganic carbon (IC) to CO 2 , which is then measured by the NDIR detector.Next a dynamic oven that surrounds the reactor heats the reactor from room temperature to 850 8 C. in just < 2 min.Upon heating the non-purgeable organic carbon (NPOC) volatilizes and rises into the cerium dioxide catalyst housed in the upper chamber of the reactor heated by a second, static oven.The catalyst completes the oxidation so the CO 2 can be detected for the measurement of NPOC at the NDIR detector.Water samples with salt or other particles are by nature inhomogeneous thus demand high injection volumes.The reactor chamber can eae ciently accommodate these high injection volumes.This approach oå ers a highly eae cient system because the salt or other particles never come into contact with the catalyst avoiding typical problems of plugging and poisoning.

Aaron S. Beatty, Mandel Scienti¢c, Product Development, 2 Admiral Place, Uelph, ON, Canada N1E 5Z1
The task of ensuring that drinking water is safe has become increasingly important as fresh water supplies diminish and public awareness of the potential health hazards grow.Turbidity, the cloudiness of water, is an important general indicator of the quality of water.This cloudiness can come from suspended solids such as silt, clay, microorganisms, and organic and inorganic matter.Large amounts of these solids in drinking water reduce the ability of chemicals and radiation to neutralize disease-causing bacteria and viruses.The US Environmental Protection Agency (US EPA) highlights the importance of turbidity with regulations requiring that drinking water must have turbidity that is < 1 NTU, reported to the nearest 0.055 NTU, before being released for public use.The US EPA has also recently increased the number and frequency of turbidity samples required for municipal water treatment plants.Therefore, there is now a need for a precise and quick method for measuring turbidity.This need can be met with an automated turbidity analysis system.This paper will illustrate the performance of the automated Turbidity Assay-Plus TM meter, as an integrated module of the PC-Titration Plus TM Conductivity, pH and Alkalinity Analysis system.The parameters of time per sample, precision and detection limits will be discussed in detail.

Gerald J. DeMenna
1 and James Kababick to treat everything from topical infections to mental disorders to menopause.Almost a dozen companies a month are opening their doors and producing products from the plethora of materials currently available WITHOUT restriction (soon to change with the upcoming QC requirements being proposed by the United States Pharmacopoeia ); ranging from Echinaceia for treating colds to soy iso¯avonoids as a hormone replacement therapy for menopause.Many of the active components for these complex formulations derive some of their activity from sterol-based compounds.A routine analytical methodology for both identi® cation and quantitation of these materials is high-resolutio n Capillary Gas Chromatograph y using medium-to high-polarity column phases.Such runs typically take 30± 60 min, and are prone to sample degradation due to the prolonged exposure to high temperatures during the separation.A technique using a column interface for direct heating of the capillary allows the same level of separation to be done 10 to 25 faster, and has the added advantage of maintaining the morphological integrity of the sample since the temperature exposure is so short.The EZ-Flash GC accessory is available for upgrading most existing GC systems, and was used for this investigation.Data from standard GC and EZ-Flash GC runs will be shown for comparison.

Patricia A. Fowler, Michael E. Swartz and Frasier H. Charles, Waters Corporation, 34 Maple Street, Milford, MA 01757-3696, USA
The fast pace of the pharmaceutical industry requires laboratories to reduce the analytical burden of their test procedures, and increase productivity while still satisfying regulatory compliance.There are several ways to meet these challenges in the dissolution laboratory.By automating the dissolution process pharmaceutical laboratories eliminate the slight variations that may occur in manual methods, insuring reproducible data, higher throughput, and cost reduction.Validated single source software control of the entire system, as well as dissolution data acquisition, calculations, and reporting can further streamline the work ¯ow while maintaining FDA compliance with 21 CFR Part 11.Automated on-line HPLC dissolution systems can pool dissolution samples signi®cantly to save time.Similarly, automated sampling at shorter intervals and analysis of a large number of samples by on-line HPLC may provide a more complete solution for the decision making process in the early stages of drug development.In addition, these systems must be capable of handling increasingly complex formulations, such as multiple actives, and widely varying dosage levels, as well as diå erent media types, such as buå ers and surfactants.High-throughpu t applications including ultrafast chromatograph y and ¯ow injection analyses can also signi® cantly increase productivity.We will present new developments in an automated on-line HPLC issolution system that satis® es all of these challenges.Applications used to help reduce the analytical burden and increase the sample throughput and productivity in the laboratory will be highlighted.

Sergey Galushko
1 , Oleg Pylypchenko The aim of this communication is to demonstrate that an arti® cial intelligence program ChromSword Auto connected to an HPLC system can work as an autonomous virtual assistant developing HPLC methods.It plans experiments, runs an HPLC system, solves real method development problems and prepares reports.The program performs method development steps fully automatically, optimizing types and concentration of organic modi® ers in a mobile phase, linear and multistep gradient pro® les, temperature and even the choice of HPLC column.The program can solve automatically several separation problems.Typical examples are: development or optimization of ® nal HPLC methods for chemical and pharmaceutical industry when all components of a mixture to be separated are available as standards.Development of HPLC isocratic and gradient methods when only some components of a mixture are known and available as standards.During measuring performance the system worked successfully in parallel with quali® ed chromatographer s in pharmaceutical and chemical companies of Europe and USA carrying out real separation projects: (1) development of HPLC methods for quality control; (2) impurity and degradation products search and separation; and (3) related compounds separation.In many cases the unattended automated optimization led to better conditions than a trial-and-error procedure and requires much less human resources and user quali® cation then computer-assiste d method development.The system is build in ChromSword-Auto software for automated method development (Merck KGaA, Darmstadt, Germany) and can control Merck-Hitachi and Waters HPLC systems.

Masami Shibukawa and Kazunori Saitoh, Nihon University, College of Industrial Technology, Department of Applied Molecular, 1-2-1 Izumi-Cho, Narashino 275-8575, Japan
HPLC is one of the most powerful methods for separation of chemical compounds.However, the diå erences in retention factors of sample components on an HPLC column are often insuae cient for complete separation.One of the ways to enhance the separation selectivity is the use of a chemical reaction speci® c for analyte compounds as a derivatization reaction.The derivatization for enhancement of separation selectivity is usually carried out batchwise before HPLC separation.On the other hand, we have recently developed a new HPLC system consisting of two separation columns and one redox derivatization unit placed between them.The redox reaction proceeds in the derivatization unit chemically or electrochemically so that an analyte compound is separated as its original form on the ® rst column, while as its oxidized or reduced form on the second column.This system should enable one selectively to separate the analyte compound provided that only the analyte undergoes the redox reaction.We found out that a porous graphitic carbon was very useful as a catalyst for chemical redox derivatization.Its redox ability can easily be modi® ed by treatment with a solution containing a suitable redox agent.We have also employed an electrolytic cell of which the working electrode is made of graphitic carbon as an electrochemical derivatization unit and have obtained successful results.This paper deals with development of the on-line redox derivatization HPLC method and its application to selective separation of some metal ions.The eå ects of applied potential ¯ow rate and amount of analyte injected on the on-line derivatization eae ciency of the system presented will be discussed.

Richard Spragg
1 , Robert Hoult Two approaches to generating IR images are currently being used.The combination of an MCT array detector with a step-scan spectrometer has allowed the production of high quality images in relatively short times, but at high cost.At the other extreme, mapping with a single detector, although much less expensive, has been prohibitively slow except for small images.New approache s are now beginning to emerge with the development of detector arrays designed speci® cally for this application.The availability of lower cost systems is expected greatly to expand the use of FTIR imaging.This paper describes the development of a simpler and less expensive FTIR imaging system using a rapid-scan spectrometer with a linear MCT detector array.The custom-designed detector combines in a single package the linear array, with a range to 720 cm ¡1 , and a single element detector covering the range to 600 cm ¡1 .Switching between imaging and conventional single point measurement does not involve moving any optical elements.The image is built up by moving the sample on a stepping microscope stage.In imaging mode, the pixel size can be either 6.25 or 25 m m 2 .The system can image an area 2 mm 2 with 80 £ 80 pixels in < 2 min.This speed is comparable with that achieved with larger arrays, typically 500 times faster than point mapping.The image format can be matched to the application and the image size is limited only by the memory available.There are signi® cant advantages to using a linear MCT array because it can operate in photovoltaic mode and have external electronics.This allows a wider spectral range and gives better signal-to-nois e performance than larger arrays.The interferometer scan is linked to the movement of the stage so that data collection is uninterrupted.With simultaneous data collection from all the detector elements the duty cycle is considerably better than with larger arrays.One consequence of the excellent signal-to-noise performance is that images of specula re¯ectance can be recorded.Some issues that arise in mid-IR re¯ectance measurements will be discussed.

Richard D. Godec
1 and Rock Wickham A new on-line trace boron analyser was used in at a semiconductor-manufacturin g site to study the control of boron contamination in their ultrapure water.This presentation will review the new analyser technology, examine the data from the on-line boron analyser, analyse the eå ect of improved boron control, and compare laboratory ICP-MS boron measurements to the on-line analyser measurements of randomly collected samples.By using the new analyser, the manufacturer was able to show they could improve the control of boron contamination in the ultrapure water and save money at the same time.

Robert A. Lodder, University of Kentucky, Department of Chemistry, A123 Astecc Bldg, Lexington, KY 40506-0284, USA
One branch of astrobiology is devoted to duplicating conditions of remote planets and moons on Earth, and identifying the life that survives there.Earth analogues for Mars are found in the deserts of Antarctica, and perhaps beneath glaciers.Cyanobacteria are one of the oldest forms of life on Earth, and similar forms of life may have developed on other planets like Mars as well.NASA has sent several missions to Mars to search for life, including the Mars Path® nder mission.While the Path-® nder mission transmitted some of the best data obtained from the Martian surface to date, the long Martian winters and rough terrain limit the amount of data that can be obtained from diå erent locations using a rover that must drive up to a target to sample it.The search for life near locations where water may be intermittently present demands ability to sense life farther from the rover.Hyperspectral imaging can be used to identify astrobiologically interesting sites from a distance.The rover can then drive up to the site and conduct further sampling.The cyanobacterium (blue-green algae) Gloeocapsa sp.produces a mycosporine-like compound that serves as a UV sunscreen in harsh environments.This compound can be used as a chemical marker for presence of colonies in the UV, visible, and near-IR spectral regions.Re¯ectance measurements made by a UV/ visible-near-IR/IR imager mounted on an electrically powered robot were used to detect the growth of cyanobacterial colonies at distances up to 20 m.The image and spectrum below was obtained from a Gloeocapsa colony growing on a calcium carbonate substrate.
(The spectrum of the limestone surface was subtracted.) The baseline distortion at the high wave number end of the spectrum arises from coloration of the colony that can be observed in the visible region.Gloeocapsa tolerate hightemperature and multiply in summer.Endolithic communities of Gloeocapsa can also be found in colder regions, including McMurdo Dry Valleys in Antarctica.While the absorptivity in the visible region is greater than in much of the near infrared, the near-infrared region yields a better `® ngerprint' for identi® cation of spores.

Prediction of chromatographi c selectivity , retention times and peak widths for new capillary stationary phases and columns
Frank L. Dorman For many years gas chromatographi c (GC) stationary phases have been synthesized with no speci® c application in mind.Consequently, many analysts struggle to force a speci® c analysis to work on general-purpose chromatographic columns.The science of separation has been understood for some time, however, and capillary GC column scan be designed which achieve optimum separation for a known list of analytes.This paper will address the state-of-the-ar t design of capillary GCc stationary phases and describe how it can be used to improve radically the common separations that many chromatographers are faced with on a daily basis.Discussion will be given to the method of prediction of stationary phase selectivity, and how combining this with the calculation of peak width can drastically improve the phases that are used in modern separations.These techniques have the potential to allow the design of an optimum capillary column for any GC analysis.

Chip-based DC glow discharge
Andreas Manz, J. C. T. Eijkel, O. P. Naji, F. Bessoth, G. Jenkins and D. Reyes, Imperial College, Department of Chemistry, South Kensington, London SW7 2AY, UK Glass microstructures have been used for micro¯uidics, electrophoresis, ¯ow-PCR, FIA and other applications during the last decade.More recently, plasma emission spectroscopy has been made available on chip, too [1± 3].The power requirements are in the 0.01± 1.0 W range and atomic/molecular spectra can be obtained.Here we present the successful use of these chips for the analysis of Cl and Br containing VOCs, coupling them to GC, using a double plasma arrangement to create injected sample plugs [4], attempts to use them for water samples [5] and for analogue computing [6].

Miniaturizatio n of analytical instruments: micro scale atomic spectroscop y
Vahid Majidi, Los Alamos National Laboratory, CST-9, MS K484, Los Alamos, NM 87545-0001, USA The growing interest in process analytical and real-time chemical analysis for ® eld applications has resulted in a new trend toward development of small analytical instrumentation.Improvements in materials possessing and electronics have resulted in a new breed of analytical instrumentation for oå -the-shelf deployment.However, the strength of these instruments is mostly in the areas of spectrophotometry , electrochemistry and membranebased sensors.These devices, in large, are used for one of two main applications: air monitoring or solution measurements.Over the last decade, the rapid development in the computer and electronic industries has lead to an overabundanc e of faster, cheaper and smaller microprocessors and ultimately fully functional computers.Considering the impact that computer control has had on laboratory-base d analytical instrumentation , it is now possible to enjoy the same level of automation in ® eldbased instrumentation .In addition to the vast improvements in the computationa l powers, the electronic circuits are now available in prepackaged forms to allow for modular instrument development.While plasma spectroscopy has a strong foothold in laboratory-base d elemental analysis, it is nearly non-existent in ® eld-based instrumentation.The paucity of ® led deployable instruments for elemental analysis has been mostly due limitations in electronic, computational and optical technologies.Popular science magazines often depict future analytical instrumentation as microprocessor-size d devices, it is important to note that chemical analysis cannot be compacted to the same extent as their electronic counter parts.The size of an electron is in® nitely small with respect to the submicron-diamete r conductors in electronics.However, when considering miniaturization of chemical analysis systems submicron diameters of a capillary or sensor walls is not much larger than the size of the analyte of interest.For this presentation, two diå erent micro plasma sources will be discussed.One system is based on atmospheric pressure plasmas while the second system is based on low-pressure plasmas.The goal of this work is not merely to miniaturize the plasma source but rather to design an integrated system, which takes advantage of scaling laws to reduce the size of every component.Owing to the increasing concern over the threat of biological warfare, there is a need to develop sensitive devices capable of early warning detection of biological warfare agents before human exposure.In particular, Bacillus anthracis (anthrax) spores are of great concern since they can be produced in large numbers, can be easily released into the air, and are highly resistant to inactivation.The work presented here will include the detection of Bacillus globigii spores (a surrogate for B. anthracis) using a prototype biochip system with photosensors, ampli® ers, discriminators, and logic circuitry on board.To provide both a sensitive and selective analysis, an enzyme-linked immunosorbent assay (ELISA) for antibody-base d capture of the target was used in conjunction with the biochip detection system.In this work, we use a novel alkaline phosphatas e substrate, which produces a ¯uorescent product that is optimally excited at 635 nm upon enzymatic cleavage.Using an antibodybased assay with this substrate, we have demonstrated limits of detection of B. globigii of approximatel y 500 organisms.Similar results are achievable through enzyme ampli® cation with DNA-based detection illustrating the ability to perform a sensitive analysis without conventional DNA ampli® cation methods such as PCR.Since the product is conveniently excited at 635 nm, a small diode laser can be used.This along with the self-contained ORNL biochip design allows for a small, compact system for biological warfare detection.

A biochip system with bubble-jet spotting technology for the detection of the fragile histidine triad (FHIT) gene in mice
Leonardo R. Allain, Minoo Askari, David L. Stokes and Tuan Vo-Dinh, Oak Ridge National Laboratory, Bethel Valley Road, Oak Ridge, TN 37830-8050, USA The human fragile histidine triad (FHIT) gene has attracted considerable interest after its identi® cation, since there is growing evidence that it acts as a tumour suppressor gene.In this work, the fabrication of micro-arrays containing PCR-ampli® ed genomic DNA extracts from mice tumours on a Zetaprobe 1 membrane using a modi® ed thermal inkjet printer will be described.
The inkjet spotting constitutes a simple and cost-eå ective procedure for the fabrication of micro-arrays containing biological samples.The usefulness of the biochip micro-array platform is illustrated by the detection of FHIT gene.Subcutaneous carcinomas were induced with MKN/FHIT and MKN/E4 cell lines in immunode® cient mice.Several weeks into their development, the tumours from both groups of mice were removed, and subjected to DNA extraction by lysis of tissue samples.The extracted DNA samples were ampli-® ed by PCR (30 cycles) using appropriate primers for the FHIT sequence.The resulting solution was transferred to individual reservoirs of a three-colour cartridge from a conventional thermal inkjet printer (HP 694C), and arrays were printed onto a Zetaprobe membrane.After spotting, these membranes were used in a hybridization assay using ¯uorescent probes and detected with a CMOS biochip.

Micro uidics: a modular approach to developing an analytical instrument
Steve Hobbs, Gene Dantsker and Hugh McManus, Nanostream, Inc., 358 Sierra Madre Villa Boule, Pasadena, CA 91107, USA Micro¯uidic systems have the potential to revolutionize biochemical analysis and high-throughpu t screening.One major bottleneck in drug discovery is high-throughput separations.This talk will focus on the development of a micro¯uidic chip that can do multiple parallel separations with resolution approaching that of a standard HPLC.Data presented will demonstrate separations of proteins and small organic molecules.Detection with mass spectrometry and optical techniques will be discussed.

Advanced automation interfaces: new toolkits for adding instrument control to a chromatograph y data system
Dario Fiore and Kristi McKiney, Scienti¢c Software, Inc., 6612 Owens Drive, Pleasanton, CA 94588-3334, USA One of the greatest limitations of chromatograph y data systems is in the area of true (interactive) instrument control for diå erent types of instruments from disparate manufacturers.Because instrument manufacturers are in business to sell instruments, many feel it is not in their best interest to provide control for competitive instruments.Yet this means the user must learn to operate multiple data systems in a single lab, many of which might not be the data system of choice.Hundreds of man-hours are wasted in learning, maintaining, and validating diå erent data systems that essentially do the same thingÐ data acquisition, analysis and instrument control.Many companies (both data system users and instrument manufacturers) are realizing the time and cost bene® ts of using open-architecture data systems where instruments of many brands are supported.
Although adding control for an analytical instrument is not a task for the everyday user, new tools are now available that facilitate the interfacing of instruments to the EZChrom Elite Chromatograph y Data System, making it much easier and faster for instrument manufacturers and large companies to create an instrument control interface.The Toolkit automation interface provides the tools required for a quali® ed company to add instrument control, and the new Rapid Control product allows Active X control to be added to the Elite data system.This paper will describe these tools and give examples of their use.
A collaborativ e environment for developers of scienti c software Deborah Anne Kernan, Victoria Rafalovsky and Ty Abshear, Bio-Rad Laboratories, Informatics Division, 3316 Spring Garden Street, Philadelphia, PA 19104-2552 , USA Bio-Rad' s KnowItAll(tm) Analytical System oå ers an integrated environment for analytical techniques, such as IR, H-1 and C-13 NMR, UV/Vis, GC, MS, Raman, and NIR.Within this system, chemists can perform multiple tasks within the same interface, using software `plug-ins' that reside within the main KnowItAll architecture.This design allows the user to easily transfer information from plug-in to plug-in without having to open another program.Because of this unique design, new third-part y software plug-ins can be quickly and easily incorporated into the KnowItAll architecture.The philosophy of this unique architecture is one that built upon the ideal of getting solutions to scientists faster and giving them more options.Thus, through collaborations with database content providers and third-party software developers throughout the world, the system grows and adapts to meet the changing needs of the scienti® c community.For those parties with existing software that may ® t within the content of the system, Bio-Rad has developed a simple Software Developer' s Kit to convert software into a format that will `plug into' and work within the KnowItAll system.

Development and validation of analytical software in a regulated environment
Kevin Bynum, Gillian Raymond, Lane Gehrlein and Philip Palermo, Purdue Pharma LP, Pharmaceutical Analysis, 444 Saw Mill River Road, Ardsley, NY 10502-2605, USA The development and validation of an analytical software package for use in the collection of cGMP compliant data will be discussed.The system has been designed to collect data to support drug development, stability testing, and release testing in the Pharmaceutical Industry.The software is designed to collect dissolution data from a UV probe ® breoptic dissolution system.The software, written in JAVA and C ‡ ‡, uses an Oracle database to ensure data integrity and security.The design features, which make this software `validatable' will be discussed.The validation testing and implementation of the system on our corporate network will be discussed in detail.

Validation Manager 2.0: new developments in computer-assiste d assay methods validation
Jean-Marc Roussel For laboratories willing to meet the requirements of ISO 17025 regulations, analytical assay methods validation is an step important to consider.Recommendations for these method validations have been announced during the fourth International Conference on Harmonization (ICH 4) and the corresponding statistical calculation procedures are described in the ISO guidelines (i.e.ISO 5725, ISO 8466).Based on these recommendations, we have developed an assay methods validation software which allows validation planning, calculations and reporting according to the authorities requirements (USP, EP, FDA).Validation step consists in the study of the method characteristics as described in the guidelines and uses well-known and eae cient statistical tests such as Dixon, Fisher, Student or Cochran.Although these statistical tools are frequently used in the analytical laboratories, most questions come when preparing the validation planning or determining the correct calculation con® guration to be used.To help the analyst, we have implemented in the software ready to use validation templates which include, for diå erent analytical techniques, prede® ned statistical tests con® dence levels and default number of analytical results per validation characteristics, all these in conformity with the international recommendations and regulations.A validation document preparation wizard may be used in combination with these templates, in order to prepare the detailed validation planning in the easiest way.Electronic data security has become of the utmost importance, in this respect Validation Manager software proposes to the user a FDA compliant con® guration in which every single addition or modi® cation in data or result is fully documented and saved in a method logbook.Each versions of the validation document is saved in a speci® c folder and cannot be overwritten.Each data modi® cation and calculation creates a new results version, ensuring full data integrity.Moreover, in order to avoid transcription mistakes, an automatic data recovery function has been developed allowing direct copy of information from the Hitachi D-7000 HSM and SSI EZ-Chrom Elite chromatography data systems.Using analytical data from our laboratory, the presentation will describe the diå erent steps of method validation using Validation Manager.The next future of this development project, including the use of experimental designs for robustness study will also be presented.

An automated software approach to analytical method validation
Michael E. Swartz and Patricia A. Fowler, Waters Corporation, Pharmaceutical Marketing Lab, 34 Maple Street, Milford, MA 01757-3696, USA Method validation is a tedious process performed to determine if an analytical method meets the requirements for its intended purpose.In he regulated laboratory, method validation may take many days to perform the necessary analytical tests.Data reduction and the statistical analysis of results performed can be a very time consuming process.There is also a greater possibility of introducing error when calculations are performed manually.With the use of automated software to perform these calculations, method validation is much faster and easier, with less chance for error.In this presentation, we will show how an analytical method is validated using automated software.Chromatographic results are directly accessed from a relational database bypassing manual intervention.Statistical calculations are performed automatically and a report generated showing the results of the analyses from the Student, Cochran, Dixon, and Fisher Tests.Graphs are generated representing the results of the statistical analysis.In addition, we will show that the data reduction and statistical calculations necessary to validate the method, complete with the necessary documentation and report generation, are completed in signi® cantly less time.

An automated approach to developin g HPLC methods for stability testing and related compound analysis
Michael E. Swartz HPLC method development of complex pharmaceutical assays such as those used for stability testing or related compound analysis is a complex and time-consuming process.Trial and error is still a common approach, but many researchers prefer more eae cient approache s that use chromatographi c modelling software that relies upon theory to decrease the time and resources required.However, while automated HPLC systems exist to run the methods, there is a disconnection between the chromatographi c and modelling software, resulting in a manual process requiring operator intervention for interpretation and implementation.We will present a new approach to HPLC method development automating the entire process from method requirements/de® nition to method implementation.The automated method development approach consists of both software and hardware operated by an iterative decision engine driven by a graphical user interface.Following input of basic separation requirements, starting conditions are identi® ed and run, evaluated, optimized, and automatically implemented by the chromatographi c system; and repeated until the separation goals are achieved.The result is an automated method development system capable of unattended operation increasing throughput and eae ciency.We will demonstrate the use of this system for the development of complex pharmaceutical assays that take advantage of the selectivity aå orded by high pH mobile phases and columns speci® cally designed for that purpose.

Automatic HPLC method development with intelligent peak tracking
Wolf-Dieter Beinert 2 Institute of Bioorganic Chemistry National Academy of Sciences, Fine Organic Synthese, Im Wiesengrund 49-b, Muehltal D-64367, Germany, 3 Institute of Bioorganic Chemistry of National Academy of Sciences, Murmanskaya 1, Kiev-94, Ukraine An automated expert system has been developed to search for optimum conditions in HPLC.Controlled by an arti® cial intelligence module, the system provides fully automated unattended method development in reversedphase HPLC for mixtures of compounds and can search for the optimum concentration of an organic modi® er for isocratic separation, for the best linear and multisegment gradient pro® les and for the temperature optimum.For proper peak tracking, the ® rst version of ChromSword Auto required single standards of the compounds to be separated.However, often one faces the situation that not for all compounds to be separated suitable standards are available.An example is the analysis of impurities in a product, where usually only a few (if any) of the impurities are available as pure substances.This is usually the case with pharmaceutical quality control samples for purity and stability tests.Therefore, Chrom-Sword Auto has been equipped with intelligent peak tracking capabilities.Applying advanced mathematical procedures for peak assignment, it is now possible to perform the optimization procedure with the sample mixture.The number of standards necessary for the optimization procedure can be reduced to just two, regardless of how many compounds are present in the mixture.The objects of the automated optimization procedure are: (1) separation of the mixture into a maximum number of peaks (e.g.search for impurities) or separation of target compounds; (2) optimum resolution; and (3) minimum analysis time In this way it is possible to automatically optimize HPLC separations of chemical and pharmaceutical samples where only a few substances (e.g. the active compounds) are available as standards.ChromSword Auto thus makes possible dramatic timesavings in the method development process as well in routine analysis.Moreover, the resulting methods possess of this chromatograph y expert system will be explained by means of practical examples.

The virtual assistant-a new HPLC system with expert knowledge and best performance
Reinhold E. Spatz HPLC is by far the most used instrumental technique in the analytical laboratory.The instrument speci® cations continuously need to be adapted to new analytical requirements such as diå erent column dimensions, high sample capacity, fast sample throughput, latest governmental regulations or state-of-the-ar t information management.The paper describes the concept of the newly developed modular LaChrom Elite HPLC instrument system.It is suitable as well for standard HPLC applications as well for special application segments in HPLC, like semimicro HPLC or high-throughpu t separations with monolithic stationary phases.The same system can be modi® ed by the user without compromising speci® cations for the application ® eld in target.This ¯exibility is due to a new hardware concept and due to integrated unique software options that act like a `virtual assistant' in the background to deliver highest expertise whenever necessary for a certain application.Typical examples that require optimized hardware are semimicro HPLC with 1 mm i.d.columns or high throughput HPLC with monolithic columns and ¯ow gradients in the 10 ml min ¡1 ¯ow rate range.Typical examples for Virtual Assistance are integrated software modules such as `ChromSword Auto' for fully unattended HPLC method development or `AutoValidation' for automated Operation and Performance Quali® cation (OQ, PQ) of the HPLC system for regulated Quality Control Laboratories.The paper will describe the new hardware and software concept.Many evaluation results and typical applications will prove the high technical level of this new development.

Brandon T. Ruotolo, Kent J. Gillig, Earle G. Stone and David H. Russell, Texas A&M University, Department of Chemistry, PO Box 30012, College Station, TX 77842, USA
There have been several theories proposed as to the mechanism of folding processes in proteins.Among them, the idea that certain peptides segments of a protein exhibit intrinsic stability and contain the site(s) of helix nucleation within the protein, i.e. autonomous folding subunits, has been proposed and observed in a few isolated cases.However, with the introduction of relatively new methods for probing the conformation of biological molecules in the gas phase, such as ion mobility spectrometry, additional information can be obtained on the validity of this hypothesis.Proof-of-concept experiments have been performed on tryptically digested proteins, which are then screened by MALDI-IM-TOF MS.For example, a peptide signal from a tryptic digest of horse heart myoglobin was observed to deviate by more than 10% in total drift time from the other peptides in the IM-MS map.The peptide was sequenced using tandem mass spectrometry and identi® ed as the majority of the E helix of solution phase myoglobin.This peptide was found to exhibit helical structure in simulated annealing molecular dynamics simulations.This presentation will focus on our recent eå orts to probe the ability of MALDI-IM-TOF MS to screen for the presence of these extraordinar y peptides.The updated screening protocol involves digestion with a number of diå erent proteolytic agents and in diå erent solvent systems in order to produce a more complete map of the protein under investigation.These results will be discussed in light of insight gained on protein folding mechanisms.

Quantitative in-line measurement s of paper coatings by near infrared
Jon G. Goode Leading manufacturers in the paper industry are looking for in-line methods to determine the physical properties, perform component analysis and determine applied coating weights in paper.The MATRIX-E, an in-line process control FT-NIR spectrometer, makes possible a noncontact diå use re¯ectance measurement with a large sampling area.We will discuss an on-line paper application to determine the silicone coat weights on labelstocks.This is currently performed in the laboratory by a time-consuming X-ray ¯uorescence method.The aim was to achieve an absolute value for the deviation from the target value of 1 g m ¡2 during continuous paper production at velocities of approximately 400 m min ¡1 .Concentrations of silicone between 0 and 2 g m ± 2 on various paper substrates were included in a quantitative model and in¯uences from the uncoated paper type due to supplier, colour, opacity, area densities, precoatings as well as diå erent compounds of the silicone agent were investigated.It was found that all these factors could be included in a single PLS-model.The fact that elemental silicone is present in clay-coated papers was found to be of no consequence to the measurements with MATRIX-E.Moreover, during in-line installations it was found that the variation of the moisture content in the moving paper due to variable machine velocities as well as the re¯ecting material of the cylinder had to be considered.It will be shown that the result of the in-line calibration has the same prediction capability as lab scale results (root mean square error of cross-validation RMSECV = 0.034 g m ¡2 ).

James E. Rodgers
1 , Rafael L. Barraza The moisture content and the quantity of ® nish applied to the surface of textile ® bres (Finish-on-Fibre, or FOF) are often critical process and quality control variables, for they can signi® cantly impact physical properties, manufacturing processes, quality, and productivity.A recurring problem for textile bobbin products is the rapid detection and identi® cation of outlier moisture and FOF bobbins during production.Non-contact, at-line moisture and FOF measurements directly on the bobbin in manufacturing would result in improved yields, process monitoring, and Quality Assurance.In this work we establish the feasibility of using a portable Near InfraRed (NIR) moisture analyser (Kett KJT100) to measure moisture directly on nylon tire and carpet yarn bobbins in manufacturing and demonstrate the feasibility of a subjugate process monitoring measurement of bobbin FOF during spinning.NIR moisture calibrations were developed for numerous tire and carpet products (different yarn type, size, ® nish, etc.) at three locations (laboratory, lag area, spinning).The NIR method successfully monitored moisture diå erences between bobbins in the laboratory and manufacturing areas.In spinning, a strong correlation was observed between NIR bobbin moisture and FOF.Subjugate FOF calibrations for tire and carpet products were developed in spinning, and excellent agreement was observed between the NIR and reference FOF results. of critical importance to manufacturing was the rapid at-line identi-® cation of several `outlier' bobbins, preventing their contamination of downstream processes.The impacts of yarn parameters, measurement location, and environmental and operational conditions on the NIR results were slight.

Novel applications of Raman spectroscop y to process monitoring and materials ' characterization
Brian J. Marquardt, CPAC, Center for Process Analytical Ch, FJ-20, Seattle, WA 98195, USA This presentation will focus on the use of Raman spectroscopy for the analysis of solid samples (powders, slurries, etc.) with emphasis on on-line process analysis applications.A novel high precision Raman probe for on-line process analysis will be described.The unique design of the Raman probe provides enhancements in measurement precision by increasing the reproducibility and accuracy of optical sampling of high solid content samples.The probe has been proven an eå ective sampling interface for the analysis of powders, suspensions, slurries, particles and solids.The ease of use of the Raman probe and the increased sampling precision has lead to its use in various proofs of concept and on-line process analytical applications.These applications include dry powder± powder mixing eae ciency, coating thickness measurements, and solvent drying analysis, reaction monitoring and various other analytical processes.In this presentation I will discuss the physical and optical design of the Raman probe and demonstrate its applicability as an on-line sampling tool.

Determinatio n of sulfhydryl residues in cysteinerich metalloprotein s using ow-injection quartz crystal microbalanc e
Alejandro Lopez Briseno, Alfred Joe Baca, Fayi Song and Feimeng Zhou, California State University, Los Angeles, Chemistry and Biochemistry, 5151 State University Drive, Los Angeles, CA 90032-4226, USA Metallothioneins (MTs) are low-molecular weight proteins having amino acid compositions rich in sulphur which strongly bind to metal ions such as Zn, Cd, Cu and Hg.Extensive research has been conducted concerning the two types of redox groups (metals and mercaptide groups) in these molecules.Particular emphasis has been aimed at determining the number of sulfhydryl groups involved in the electron transfer reactions of metallothioneins.However, less attention has been directed in the determination of sulfhydryl groups that are not directly involved in the redox properties of these proteins.We report here our recent eå orts in determining the number of sulfhydryl groups that directly participate in the redox reactions of Metallothioneins.It was found that approximately four cysteines per MT molecule are involved in the cysteine± mercury thiolate formation.This was accomplished by combining two well-known techniques: the electrochemical quartz crystal microbalance (EQCM) and the inductively coupled plasma atomic emission spectrometry (ICP-AES).Another objective of this study will be to determine the availability of nonparticipating cysteine groups by signal ampli® cation through chemical reaction to convert sulfhydryl groups into easily detectable moieties.A conceivable method of accomplishing this is by selective modi® cation onto cysteine moieties acting as probes to methoxy-polyethyleneglycol maleimide (MPEG-MAL).The quartz crystal microbalance (QCM) will be used for gravimetric analysis of the sulfhydryl groups.

Miniaturized ow-through surface plasmon resonance detector for the study of protein-protein interaction dynamics
Rebecca J. Whelan Surface plasmon resonance (SPR), which senses changes in the refractive index of a dielectric medium adjacent to a thin gold ® lm, is a powerful tool for label-free studies of biological molecules and their interactions in real-time.We employ SPR to study the interaction of the G-protein-couple d beta-2 adrenergic receptor with other molecules, including receptor agonists and antagonists, kinases, and G. protein.The most relevant information comes from receptors that are immobilized on the surface in a uniform way.To this end, we have developed a novel mutation of the receptor, with an additional cysteine at the extracellular terminus.Biotinylation of this mutant receptor and use of conventional biotin/avidin binding ® xes the receptor to the gold ® lm of the SPR sensor with excellent uniformity.An alternate immobilization protocol uses an antibody (M1) directed against the receptor' s amino-terminal FLAG tag.The receptors retain their function after immobilization, as con® rmed by ¯uorescence microscopy studies.After introduction of a reaction partner through a ¯ow cell (operating with or without continual ¯ow), information can be obtained about the extent of interaction, binding kinetics, and pharmacology .Other biological interactions we have investigated include antigen/antibody, glycoprotein/lectin, and ssDNA/ssDNA.Regardless of the analyte system, maximum sensitivity and eae ciency are achieved by minimizing the volume of sample above the sensing surface.To minimize the required sample, we have developed a miniaturized ¯ow cell.This small ¯ow cell also opens the possibility of coupling SPR with a miniaturized separations platform such as CE or micro-HPLC.

Screening for environmenta l contamination using liquid chromatograph y with mass spectrometry detection
Jim Krol and Kate Yu, Waters Corporation, 34 Maple Street, Milford, MA 01757-3696, USA When assessing unknown contamination of an environmental sample, where does the chemist begin?Which validated method is appropriate, a speci® c analyte method or several diå erent analyte methods?What if the validated method reports no contamination ; productivity decreases, analysis cost increase, and the contamination analyte remains unknown.Liquid chromatograph y (LC) has the capability to retain and separate numerous analytes based upon their chemical properties, but conventional PDA-UV detection has limitations in seeing all the analytes.For analyte identi-® cation, retention time alone is not suae cient.Chromatographic analyte coelutions will exist in complex samples and compromise UV spectra making identi® cation and quantitation marginal.If the analyte is UV inactive, analyte identi® cation and quantitation are impossible.Mass spectrometry (MS) is a more universal, yet speci® c detection technique that detects a signi® cantly greater number of environmental analytes, such as carbamate and urea-based pesticides and herbicides using positive electrospray.Combined with gradient reverse phase chromatography , an environmenta l sample can be screened for numerous analytes.The simultaneous use of retention time, m/z mass spectra, and PDA-UV are used as qualitative variables for analyte identi® cation.However, the low ppb semiquantitative results need to be con® rmed by speci® c validated methods.This presentation will present a novel carbamate and urea analyte screening method approach.The critical link is the reliability of the MS processing method, chromatographic reproducibility, and the percentage of falsepositives/false-negatives.Wastewater and ground water will be the evaluated matrices.

Fast gas chromatograph y with conventional instruments using direct resistivel y heated capillary columns
Paolo Magni, Giacinto Zilioli and Riccardo Facchetti, Thermo-Quest Italia SpA, Research and Development, Strada Rivoltana, Rodano, Milan I-20090, Italy In gas chromatography , there is an increasing demand for signi® cant reductions in analysis time.However performing high-speed separations while keeping enough separation eae ciency is a diae cult task.The combined use of fast temperature programming and short narrow bore columns may provide an optimal solution, particularly for samples containing simple mixtures with a wide boiling point range.For some applications a suae cient separation can be achieved even in < 1 min provided that, in the same time, the column is raised to an appropriate ® nal temperature.The system presented in this paper, consisting in a directly heated capillary column module mounted in a conventional GC instrument, permits to achieve very fast temperature programming rates (as high as 20 8 C s ¡1 ), which cannot be obtained with the use of conventional air circulating GC ovens.The tremendous heating rate power of the device permits to reduce the tight bandwidth sample injection requirements otherwise dictated by the small internal diameter of the column.The module, containing the capillary column, the heating element, and the temperature sensor, is housed in the GC oven and connected to standard GC split-splitless injector and detection systems, just as any conventional capillary column.Results obtained with both split and splitless injection techniques are examined using Flame Ionization Detector and Time of Flight Mass Spectrometer.The attached chromatogra m shows an example of fames analysis, ranging from n-C7 to n-C22 fames, performed in < 1 min.The separation was obtained using a 5 m, 0.1 mm i.d., 0.1 m m ® lm thickness RTX-5 column with a temperature-programming rate of 5 8 C s ¡1 (from 70 to 300 8 C).Others applications of the fast GC accessory in the chemical, petrochemical, environmental, and food and ¯avours ® elds are presented.

Modi cation of OSHA GC methods for continuous area monitoring
John N. Driscoll, Process Analyzers, LLC, 25 Walpole Park South Drive, Walpole, MA 02081, USA OSHA has developed hundreds of GC methods for analysing organic compounds in the workplace as part of the standards process.These methods were developed to provide a analytical methods for samples collected on adsorbent tubes in the workplace for a wide variety of industries.Typical sample volumes collected range from 250 ml (at the ceiling value) to 12 litres (below the TLV) of sample collected on the adsorbent tubes.Once the sample (250 ml) is diluted, it is equivalent to injecting a 1 cm 3 sample at approximately 5 the TLV.For area monitoring, we would like to have a detection limit that is 1/20th of the TLV or PEL.If these detectors were used in an Automatic GC for Area monitoring, many would have diae culty detecting half the TLV for those compounds with low TLVs.In other words, many of these methods would be `sensitivity challenged' because of the use of the ¯ame ionization detector (FID).These OSHA GC methods can still be used but it clear that a more sensitive method (or a concentrator for the FID) is needed in order to obtain the required sensitivity.The photo-ionization detector (PID) is greater than 50 times more sensitive than an FID for aromatic compounds.This will allow a smaller sample (0.1± 3 cm 3 ) to be used and still have adequate sensitivity for the method.In addition, the selectivity of PID can be varied by choosing a 9.5, 10.2 or 11.7 eV lamp.The sensitivity of the FID can be increased via an on-board automatic concentrator that can improve the detector sensitivity by 10± 100-fold.The compounds to be concentrated can be optimized by switching the trap materials.Another diae culty that occurs at low ppm or ppb levels with polar species is adsorption or reactivity on/with surfaces of the sampling system and lines.This results in serious problems with reproducibility and accuracy.Examples include amines, phenols, organic and inorganic acids, pesticides.A permeation tube or other diå usion device can be used to condition the entire system and prevent adsorption of polar or reactive compounds.We will evaluate a number of methods for amines, sulphur compounds, chlorinated hydrocarbon s and diphenyl oxide.We will compare these results of the FID/NPD detectors with the photo-ionization detector (PID) mounted on an automatic GC for area monitoring.This should provide an alternate method that has more sensitivity and/or selectivity and does not require support gases.

Modi cation of OSHA GC methods for continuous area monitoring
John N. Driscoll, Process Analyzers, LLC, 25 Walpole Park South Drive, Walpole, MA 02081, USA OSHA has developed hundreds of GC methods for analysing organic compounds in the workplace as part of the standards process.These methods were developed to provide analytical methods for samples collected on adsorbent tubes in the workplace for a wide variety of industries.Typical sample volumes collected range from 250 ml (at the ceiling value) to 12 litres (below the TLV) of sample collected on the adsorbent tubes.Once the sample (250 ml) is diluted, it is equivalent to injecting a 1 cm 3 sample at approximatel y 5 the TLV.For area monitoring, we would like to have a detection limit that is 1/20th of the TLV or PEL.If these detectors were used in an automatic GC for area monitoring, many would have diae culty detecting half the TLV for those compounds with low TLVs.In other words, many of these methods would be `sensitivity challenged' because of the use of the ¯ame ionization detector (FID).These OSHA GC methods can still be used but it is clear that a more sensitive method (or a concentrator for the FID) is needed to obtain the required sensitivity.The photoionization detector (PID) is > 50 times more sensitive than an FID for aromatic compounds.This will allow a smaller sample (0.1± 3 cm 3 ) to be used and still have adequate sensitivity for the method.In addition, the selectivity of PID can be varied by choosing a 9.5, 10.2 or 11.7 eV lamp.The sensitivity of the FID can be increased via an on-board automatic concentrator that can improve the detector sensitivity by 10± 100-fold.The compounds to be concentrated can be optimized by switching the trap materials.Another diae culty that occurs at low ppm or ppb levels with polar species is adsorption for reactivity on/with surfaces of the sampling system and lines.This results in serious problems with reproducibility and accuracy.Examples include amines, phenols, and organic and inorganic acids, pesticides a permeation tube or other diå usion device can be used to condition the entire system and prevent adsorption of polar or reactive compounds.We will evaluate a number of methods for amines, sulphur compounds, chlorinated hydrocarbon s and diphenyl oxide.We will compare these results of the FID/NPD detectors with the photo-ionizatio n detector (PID) mounted on an automatic GC for area monitoring.This should provide an alternate method that has more sensitivity and/or selectivity and does not require support gases.

Kerstin Thurow, Agnes Schubert and Christian Wendler, University Rostock, Institute of Automation, R-Wagner-Str 31, Rostock, MV, D-18119, Germany
The use of combinatorial methods in chemistry and life science has been developing rapidly within the last years.One of the `bottlenecks' in synthesis control in pharmacy and life sciences is still the characterization and speciation of biotechnological reaction products using chromatographi c methods in combination with speci® c detection systems.Often before analysis a complex sample preparation is required which might include cleaning steps, dilution or even derivatization procedures.Existing systems are not ¯exible automated solutions and do not have ¯exible material and information interfaces.Thus, the objective of our investigation was the development of an automated system for combined sample preparation and analysis being used for organic synthesis.The system used is a commercial liquid handling station (CTC CombiPal), which has been equipped with diå erent trays for heating, ® ltration, mixing, etc. and has been adapted to diå erent analytical devices such as gas or liquid chromatographi c systems or mass spectrometers.Technically, the system is controlled by a digital computer operating both chromatograp h and sampler as diå erent tasks in the WINDOWS-NT 4.0 operating system.The sample preparation task allows the application of user-speci® c methods.Sampler and chromatograph communicate for transmitting remote START/STOP-information via hardware handshake lines.Because of the ¯exible system strategy, the system can be set up easily for diå erent applications.The system developed allows the fully automatic analysis of samples to run in parallel with the sample preparation.It can be used as a stand-alone system or can be integrated in complex robotic systems.Motivation for such a combination is to reduce further testing costs by increasing throughput and by reducing manual intervention.Other advantages include, for example, reducing the delay between the analysis and the sample preparation.The presentation will outline the technical details of the system developed and will show the use of the system for high-throughpu t screening applications in the ® eld of combinatorial catalysis, chiral determinations and synthesis control.

Development of a high-throughpu t analysis infrastructure for combinatoria l materials science applications
Radislav A. Potyrailo, General Electric, Corporate Research and Development, PO Box 8, Schenectady, NY 12301-0008, USA High-throughput methods for materials science combine a parallel or combinatorial materials synthesis and processing with an automated screening and datamanagement tools.They can rapidly optimize molecular properties and process conditions that are diae cult to predict using existing knowledge.From an analytical chemistry perspective this leads to major challenges in developing rapid analysis techniques that can deal with large numbers of small samples required to construct combinatorial libraries.To meet these analytical challenges in the discovery of new materials at General Electric, a high-throughpu t analysis infrastructure was established at GE' s Corporate Research Center.This presentation will describe the strategy taken in the development of this infrastructure .For high-throughpu t analysis of materials properties from a wide variety of projects, analytical instrumentation consists of interchangeable modular subsystems coupled with the new data processing capabilities.Examples from several projects will illustrate the broad applicability of the developed high-throughpu t analysis instrumentation .

High-throughpu t analytics employing automated sample preparation and the integrated use of HPLC-DAD, HPLC-DAD-MS, GC-MS and ow injection NMR
Winfried Etzel The establishment of automated and parallelized synthesis and microscale reactions in the chemical synthesis laboratories of the Agrochemicals Division at Bayer created an increasing demand for analytical information (purity, structure, physicochemical properties).With the availability of ¯ow injection NMR systems (BEST-NMR) the use of the same sample format on diå erent analytical instruments was possible.This fact encouraged us to realize an integration of diå erent analytical techniques (NMR, HPLC-DAD, HPLC-DAD-MS, GC-MS) in one service unit.The optimization of the ¯ow NMR system for the use of diå erent organic solvents will be demonstrated and the quality of the spectra and the throughput of samples will be discussed.In addition, the results and advantages of an integration of diå erent analytical techniques will be presented.Workings with barcodes and microtitreplate (MTP) technology were introduced.Electronic laboratory journal, sample preparation robotic system and analytical instruments were connected together via a laboratory information and management system (LIMS).This resulted in a simpli-® ed work¯ow in the chemical research laboratories and in the analytical department.The throughput of samples and measurements was doubled without raising the number of technicians.

Jon D. Thompson and Peter W. Carr, University of Minnesota, Department of Chemistry, 207 Pleasant Street SE, Minneapolis, MN 55455-0431, USA
It is very important that the speed of HPLC be increased.Improving throughput for stability analysis, quality control assays, and dissolution testing are all key motivations for improvement in separation speed in the pharmaceutical industry.Unlike CE, HPLC does not lend itself well to multiparallel analysis because the cost is prohibitive.Recent work in this lab has focused on the theory of high temperature on analysis time in LC.Disregarding selectivity considerations, we have shown that the ® ve to 10-fold decrease in e ¥ uent viscosity that comes from working at very high temperature (180± 200 8 C), and the concomitant increase in analyte diå usitivity, combine to dramatically decrease the time needed to generate a theoretical plate.The lower viscosity at elevated temperature decreases the pressure drop across the column and allows higher linear velocities to be reached as the pressure limit of the pump is approached.Simultaneously, faster analyte diå usion at higher temperature improves eae ciency at high linear velocity conditions compared to the eae ciency at lower temperatures at the same velocity.In this presentation, we will show how the theory of speed guides column selection and system design.We will show that at high temperatures, 10 gradient runs can be done in 40 min on columns of conventional geometry.We will also show that dramatic improvements in stability indicating assay throughput can be achieved at elevated temperature using conventional equipment.

A new stand-alone autosampler for high-throughput HPLC and HPLC/MS
Curtis R. Campbell, Susan M. Steinike and Kara M. Merkle, Shimadzu Scienti¢c Instruments, LCMS Applications, 7102 Riverwood Drive, Columbia, MD 21046-1245, USA Fast LC and fast LCMS methods have been developed to meet the demands of combinatorial chemistry and drug discovery.Now more than ever, is there a corresponding requirement for ultrafast sample introduction in LC and LCMS instruments along with the most stringent requirements for zero carryover and optimum sample repeatability.In this communication we describe the performance of a newly designed, stand-alone autosampler for HPLC and HPLC/MS applications featuring a 15-s injection cycle for 10m l injections, excellent sample repeatability, and the lowest carryover currently attainable.The unit can function independently, allowing interface with a variety of instrument models in a variety of venues including combinatorial chemistry, drug discovery, and high-throughpu t screening.

Control of low-frequency noise in inductivel y coupled plasma optical emission spectroscop y by on-line periodic reference standard correction
Michelle E. Cree The understanding of ICP± OES instrumentation has attained a level where various engineering, chemical and mathematical techniques have been implemented to improve the precision of analytical results.The remaining sources of noise in an ICP-OES spectrophotometer can be broken into high-and low-frequency noise.High-frequency noise in a simultaneous ICP± OES can be corrected by the use of `real-time' internal standardization .The ability to control the eå ects of high-frequency noise has seen the eå ects of low-frequency noise now dominate the precision of results.Drift from lowfrequency noise can be de® ned as a slow varying change in the background or intensity of a signal while the instrument is performing analysis.Until now, the primary method used to produce `drift free' results was to compensate for an instruments slow, varying change during analysis by performing constant recalibrations.An alternative method is to perform data processing externally, using third party software.Both methods are time consuming, and decrease productivity.Now a faster more economical approach uses `real-time' drift correction.Varian has implemented a real-time drift correction technique that allows long-term collection of data, with little loss in precision due to drift.Just as internal standardization was ® rst implemented and precision was improved, now drift correction will further improve the precision of analytical results.To obtain the most precise results, two drift models are available, linear and polynomial.Both use de® ned drift correction points to complete mathematically a goodness of ® t correlation, with the polynomial using progressively higher orders of ® t until the threshold acceptance criteria is established.Here we will look at the performance of both models.

Quasi-continuou s monitoring of process and waste streams by on-line inductively coupled plasma-optical emission spectroscop y
Dirk Ardelt, Heinz Falk, Hendrik M. Smol and Hans-Joerg Waarlo, Spectro Analytical Instruments GmbH & Co. KG, Boschstrasse 10, Kleve D-47533, Germany Over the past decade, increasingly more sophisticated process analysers derived from `mature' laboratory methods of instrumental analysis have found widespread use in the chemical process industry to cope with increasing demands for more eae cient energy and material usage and better process control.Although many such applications still rely on rather simple sensors, an increasing amount of formerly laboratory based only methods, like InfraredÐ or Raman-Spectroscopy , X-rayÐ Fluorescence or magnetic resonance methods, are nowadays routinely applied as on-line systems.As diverse as these methods are their ® elds of application in the online process environment, which typically is divided among the distinction between the aggregate states of the medium to be analysed.Not surprisingly, one major ® eld is the analysis of liquid process-and waste streams.Considering laboratory use, inductively coupled optical emission spectroscopy, ICP-OES, probably does the most universal atomic spectroscopy method exist.Combining high ¯exibility, good sensitivity, wide dynamic range and fast measurement cycles, this method should ideally be suited for migrating into the process analytical ® eld, especially regarding its relative `maturity' .Despite of these ® ndings, ICP-OES up to now has not found the expected use as on-line analyser, even where it outperforms other methods under laboratory conditions.Using recently installed systems for wastewater and process stream monitoring as examples, we will discuss probable reasons for the rather low past development of on-line ICP-OES and examine, in how far these reasons have been or can be overcome.Relevant analytical and technical ® gures of merit for on-line applications shall be deduced.Furthermore, modern multichannel detection ICP-OES allows for using sophisticated algorithms (e.g.chemometrics) for data evaluation, in this case resulting in higher degrees of automation and system availability.Accounting for such advances, we will try de® ning a `state of the art' for ICP-OES as process analytical method and highlight future development areas.

Millisecond monitoring of fast transient signals by Hadamard transform time-of-ight mass spectrometry
Joel R. Kimmel Hadamard transform time-of-¯ight mass spectrometry (HT-TOFMS) involves the modulation of a continuous ion beam using a pseudo-random sequence of pulses [1].This sequence of pulses is applied using a charged particle modulator consisting of an interleaved comb of wires.Ions ¯ying from the electrospray ion source to the ¯ight chamber are `gated' at this modulator following the Hadamard-typ e binary sequence.The applied sequence has an approximately equal number of zeros and ones, thus the duty cycle of the spectrometer is close to 50%.TOFMS has become the preferred detector for separation techniques used in bioanalytical chemistry such as CE and LC.As research continues, the obtained separation eae ciency of these methods increases, making the eluting peaks sharper.To describe correctly the shape of these narrow peaks, detectors must acquire data at a rapid speed.The only means of increasing the spectral acquisition speed of a TOFMS without sacri® cing mass range is to use encoding methods that eliminate the lag time between successive ion packets.In this presentation, we describe the ability of HT-TOFMS to scan transient signals in the millisecond time-scale.A brief theoretical background on how TOFMS can be multiplexed is given.Alternative Hadamard transform algorithms that improve the performance of slow multichannel scalers are discussed in terms of their eå ect on the spectral storage rate.The eå ect of acquisition systems' end-of-pass dead time is examined.The ability of HT-TOFMS to scan transient peaks of decreasing width is investigated for CE and pulsed electrospray ionization peaks.For femtomole injections of biomolecules, full spectra in a mass range of 1500 Da are obtained in 3.6 ms.Owing to the extremely large amounts of data collected, only state-ofthe-art acquisition systems can be used for this task.Further increase in the spectral storage rate can only be achieved by further increasing the duty cycle of the instrument.The possibility of using two MCP detectors to produce a 100% duty cycle with no ion storage is discussed.lifecycles to provide close ® t solutions in a world of such rapidly changing businesses and requirements.Delivering laboratory centric solutions in `Internet time' creates new challenges for professional services organizations and requires new approaches.This paper will examine these challenges and the place of both software and services in providing Internet solutions in Internet timescales.

Sample management for DNA testing laboratorie s
Nick Townsend, Martin Waugh and Peter B. Mans¢eld, Autoscribe Ltd, 81 Technology Park Drvie, East Falmouth, MA 02536, USA DNA technology has revolutionized applications such as paternity testing, forensics, clinical diagnosis and the detection of Genetically Modi® ed Organisms (GMOs).This success has in-turn created a very large increase in the number of samples being processed by DNA testing laboratories and the associated logistical issues this can bring.In the last two decades LIMS have become established supporting the work of analytical and research laboratories but is it only recently that DNA testing laboratories been able to realize the bene® ts LIMS can deliver.In this presentation we will describe a LIMS con® gured to meet the needs of DNA contract testing laboratories.We will describe how, via web technology, clients submit samples for testing, specify the DNA testing pro® le and con® rm order details.Once samples are received, bar code technology is used to track sample location and process steps including extraction, puri® cation, ampli® cation and identi® cation.A very graphic and intuitive interface is provided for technicians allowing creation and editing of plate and electrophoresis gel plans using drag and drop techniques.The system supports the use of complex `plating rules' providing a large degree of automation as to how primers, samples and reference standards should be pipetted onto the plates.A comprehensive audit trail is provided recording details of how samples are analysed including the run conditions associated with processing the plates and the transfer of samples from plates to gels.Once analysis is complete the LIMS automates the production of reports for the client and will interface with order processing systems to deliver an invoice for the work.

Multivariate analysis in chromatography : peak tables versus pro les
L. Scott Ramos, Infometrix, Inc., PO Box 1528, Woodinville, WA 98072-1528, USA Chemometric software tools can detect the presence of meaningful information in chromatographi c data and create multivariate models able to interpret future chromatograms.Commonly, tables of integration results are processed using nearest neighbour or factor-base d classi-® cation algorithms.Complex applications bene® t from the increased prediction accuracy and sensitivity to outliers (e.g.unusual samples, process upsets).However, peak tables have limitations: peak assignments are prone to error when peaks elute closely, peaks that show only as shoulders are not integrated, and peaks not in the calibration table are ignored.Now that computationa l power is cheap and ubiquitous, processing of whole chromatographi c pro® les is attractive.Using whole pro-® les eliminates the disadvantage s cited for peak tables.Nevertheless, pro® les have distinct requirements for reliable use: chromatogram s must be aligned so that peaks of the same material are found at exactly the same time, the data collection rate must be consideredÐ 100 scans across a peak is unnecessary over sampling, and, ® nally, very large numbers of lengthy pro® les in a database can overwhelm even the most capable computers.This paper will contrast the two approaches, highlighting the advantages and disadvantage s of each.Examples from diå erent applications will demonstrate the relative merits of the two approaches.

Detection of heroin in drugs of abuse using multivariate curve resolution with two-dimensiona l wavelet compressio n
Guoxiang Chen A trend in contraband drug manufacturing has been to adulterate popular drugs such as cocaine and MDMA (ecstasy) with heroin.The adulterants are added to either increase the potential of addicting the user or as an introduction to harder drug use.Ion mobility spectrometry (IMS) is used for screening drug samples by law enforcement agencies.A chemometric procedure was developed for rapidly detecting heroin in other drug samples.Multivariate curve resolution techniques have been developed mathematically to separate the components in mixtures.The self-modelling curve resolution method SIMPLe-to-use Interactive Self-modeling Mixture Analysis (SIMPLISMA) decomposes a data set into a model that comprises concentration pro® les and spectra.SIMPLISMA has proven an eå ective tool for simplifying intricate ion chemistry that may occur in IMS data.In this work, a SIMPLISMA algorithm with two-dimensional (2D) wavelet compression was developed to analyse rapidly the data sets of drugs, including heroin, cocaine and MDMA (3,4-methylene-dioxymethamphetamine) .The 2D wavelet compression, which compresses both acquisition time and drift time dimensions of a measurement, was applied to the drug data sets before SIMPLISMA modelling.In comparison with the 1D compression, the multidimensional compression furnished a greater compression of data set size without losing important chemical information.This feature allows faster implementation of SIMPLISMA, improves the SIMPLISMA models by removing noise from the data, and lowers storage capacity that makes real-time modelling possible.The SIMPLISMA model obtained from the compressed data was wavelet transformed to the uncompressed representation.The eå ects of wavelet ® lter types and compression levels were investigated.The relative root-mean-squar e errors of SIMPLIMSA spectra, which calculate the relative diå erence between the extracted models with and without 2D compressions, were used to evaluate the eå ects of compression on self-modelling.The results showed that satisfactory models could be obtained at a compression ratio as high as 99.6%.

Enzymatic assays on microchip platforms
Joseph Wang, New Mexico State University, Department of Chemistry and Biochemistry, PO Box 30001, Las Cruces, NM 88003-8001, USA Microfabricated ¯uidic devices, integrating the sample preparation process and the measurement step onto microchip platforms, are of considerable recent interest.Such technology oå ers great promise for developing versatile and miniaturized analytical microsystems with high degree of automation, rapid analysis times and negligible consumption of reagents.Separation microchips have been combined with chemical derivatization reactions, but rarely to enzymatic processes.In this presentation, we will report on the versatility of micro-¯uidic devices for carrying out novel multi-enzyme assay protocols and for tailoring the ¯uidics to suit speci® c bioassays.These will include the use of mixed oxidase/ dehydrogenas e preseparatio n reactions, or post-capillary derivatizations using multiple oxidases or dehydrogenases.Such on-chip enzymatic assays have been combined with fast electrophoretic separation and amperometric detection of the liberated NADH and hydrogen peroxide.We are currently exploring a multichannel operation, involving diå erent combinations of enzymes, for high-throughpu t measurements of numerous substrates.An inductively coupled plasma mass spectrometer operated in low rf power (700 W) was demonstrated to be suitable for ultratrace determination of elements with high ionization potential.Hydride generation was used for sample introduction.Selenium analysis free from ArAr + interference was achieved by operating the plasma at a so-called critical power condition where a warm (as opposed to traditionally de® ned hot and cool plasma) plasma was believed to exist.In this warm plasma, there is suae cient population of energetic Ar + to ensure ionization of Se while the ArAr + (interfering with the Se isotope) population is at the minimum due to its bimodal pro® le.Detection limit of 1 ng l ¡1 was achieved for 78 Se without substantial interference from 78 ArAr + after optimizing the operating parameters.Background spectra of warm and hot plasma were compared.Two types of hydride generation gas± liquid separators were compared.This technique was applied to measure trace amounts of Se in water extracted aerosol samples with typical concentrations between 50 and 100 ng l ¡1 .Recoveries of the independent 50 ng l ¡1 QC and the SRM1640 (Natural Water) were 98 and 101%, respectively.It is conceivable that this technique can be used on other high ionization elements (e.g.As and Sb) if the critical power condition can be found with these elements.

High productivity of environmenta l sample analysis using ICP-MS
Jenny Godfrey and Phil Shaw, Thermo Elemental, Ion Path, Road Three, Winsford CW7 3BX, UK Elemental analysis of environmental samples in survey and monitoring programmes, must meet various criteria.These include cost-eå ectiveness, robustness, highthroughput, and ® t-for-purpose accuracy and precision.The characteristics of environmental samples collected in these programmes are dominated by the variability of the sample matrix.Samples with widely diå ering major element composition and total dissolved solids content are necessarily analysed within the same batch.This makes the possibility of using matrix-matched standards very diae cult.The technique of choice needs to be multielement, matrix tolerant, have wide dynamic range, rapid data acquisition and good washout characteristics with appropriate detection limits.To be cost-eå ective, an instrument needs to be analysing samples for > 12 h a day whilst retaining the desired characteristics.This requires reliable hardware and versatile software needing minimal user intervention.An ideal system would be able to run environmental samples with little daily maintenance, e.g.cone cleaning, and allow direct transfer of data into a LIMS without further processing.The current generation of ICP-MS instrumentation and software is capable of performing drift corrections, monitoring QCs and recalibrating accordingly, monitoring washout of sample and applying corrections for internal standards, dilution, blanks and interferences on-line.This paper will describe new strategies to obtain high productivity and describe methodologies for running high matrix environmental samples to achieve world-wide environmental protocols by ICP-MS.With data shown on improved detection limits and recoveries, handling of interferences and maintaining high-throughpu t of multielement samples.

Martin Pumera
1 and Joseph Wang Because of the upsurge in terrorist activity, there are urgent needs for fast-responding ultrasensitive portable devices for on-site detection of major explosives.A chipbased analytical system is presented that allows rapid ¯ow-injection measurements of total nitro-aromatic s explosives, as well as micellar chromatographi c identi® cation of the individual ones in the same microchannel.Fast-respondin g ® eld deployable explosive analysers, providing a timely warning and alarm (in case of sudden concentration change), are being developed in our laboratory.A unique and rapid `switching' between the `non-separation ' (ultrafast screening, analysis time.

Monitoring cellular release using electrochemica l and uorescence detection on a microfabricate d electrophoresi s chip
Drew P. Manica As microchip analytical systems continue to advance, their use as separations-base d sensors for monitoring biological processes is increasing.One biological process that has received much attention for its role in neurological disorders is exocytosis, the process of neuronal communication.A method for monitoring exocytosis with microchip electrophoresis and electrochemical detection is demonstrated.Classical methods of monitoring cellular release involve massive dilution, or whole cell lysates.Microchip analysis systems provide the advantages of high speed and throughput, portability and low cost, while providing a convenient way to load lowvolume samples, including cellular contents, directly onto the device.Release of neurotransmitter s from small numbers of invertebrate neurons is performed in a total volume of a few microlitres, thereby reducing the eå ects of dilution.Then the extracellular ¯uid is injected, and the electro-active neurotransmitter s are detected at a microfabricated platinum electrode.Initial experiments suggest the feasibility of the system to monitor vesicular neurotransmitters .Future directions will include employing dual electrochemical and ¯uorescence detection on a microchip in order to broaden the selectivity of detectable neurotransmitter s and to aid in monitoring the extracellular environment.

Multichannel separations with optically gated uorescence injection on a chip
Hongwei Xu With the advantage s of low analytical sample volumes needed and short separation times, microfabricated capillary electrophoresis chips have provided high-eaeciency separations of DNA, proteins and other biological molecules.Also, the ability of fabricating multiple lanes on a single chip make it possible to parallel process many samples on one chip and reduce the total analysis time.The multiplexing of multiple channels on one microchip is complicated by the need for multiple injections.We have developed a technique that uses optical gating injection with multiple channels leading to high-throughput separations.Current work in our group has shown the ability of optical gating for sample introduction on a single channel chip.In optical gating, one laser beam is used to photobleach the ¯uorescent-labelled sample and injection is carried out by blocking this beam.The other beam detects the ¯uorescence from the injected sample after separation.Optical gating provides fast, serial injections and easy automation.With this technique, massive parallel separations in multiple channels can be carried out on a single chip.Combing optical gating and multiplexed channels will allow high speed, highthroughput separations for biological analysis, including genetic polymorphisms, proteins and others.In this technique, the chip is ® xed on a stage and a voice coil actuator is used to move the stage back and forth so that the two stationary beams (gating beam and detection beam) can scan all the channels on the chip.Initial separations of amino acids and oligonucleotides have been used to determine the promise of the technique.

Investigation of uorescence polarization as a novel detection for improved DNA sequencin g on a microchip
Yuan Yan Current four-colour DNA sequencing with ¯uorescence dye-labelled primers or terminators is the method most commonly used.Labels are chosen on the ability of a ¯uorimeter or suitable band-pass ® lters to distinguish their colours.Current technology for the one-lane fourcolour approach limits the number of dyes that can be eå ectively distinguished under sequencing conditions.Recently, we have demonstrated that ¯uorescence polar-ization could be used to discriminate the isochromatic dyes in ¯uid solution [1,2].Fluorescence polarization will be further studied as a novel detection for increased selectivity of dyes while simultaneously maintaining sensitivity in this report.DNA sequencing is performed on a micro¯uidic device under a home-made confocal ¯uorescence research system.Some characteristics such as speed, accuracy and feasibility of this approach will be reported as well.The goal of this research is to develop a micro¯uidic chip using capillary electrophoresis and sinusoidal voltammetry for the separation and detection of small biomolecules.Biomolecules frequently require derivitization when detected by ¯uorescence or electrochemistry, the two most common detection schemes used on-chip.Derivitization is time consuming and can produce interfering compounds , which further complicate the analysis.However, it has been shown that under alkaline conditions, biomolecules such as carbohydrates, peptides, and DNA undergo electrocatalytic oxidation in their native state at the surface of copper electrodes, at potentials ranging from 0.4 to 0.8 V versus Ag/AgCl.This leads to high sensitivity for the detection of DNA [1].Copper electrodes are fabricated onto a glass substrate, which are sealed reversibly to a poly (dimethylsiloxane) (PDMS) slab which contains micron-sized channels.A problem in the fabrication and assembly of such chip-based devices is leakage from the separation channel along the electrode surface, which can lead to increased background and band broadening.To optimize the electrode geometry and reduce background, a variety of polymers have been used to de® ne the electrode area.Other parameters, such as thickness of deposited electrodes, techniques of assembling and ® lling small diameter channels, and composition of PDMS have also been explored.To evaluate the performance of the micro¯uidic chip, a simple, well-established system is used.The analysis of carbohydrate s in free solution is a model system for the optimization of the above mentioned parameters.Upon characterization, the separation and detection of native oligonucleotides will be demonstrated.Time-resolved ¯uorescence detection has been proved to be a viable technique for the applications in DNA sequencing.To achieve high-speed separations and miniaturization of the devices, PMMA-based microchips are used in this study.In the ® rst part of this research, a onecolour microscope system with a single channel PMMA chip (eå ective separation distance of 10 cm) is used.Cy5.5 and IRD700 dye-labelled primer and sequencing ladders will be separated and the lifetimes will be obtained using a 3%T/3%C polyacrylamide sieving medium, which is polymerized within the microchip channel (the replaceable linear polyacrylamide gel has been studied by another member in our group).Timecorrelated single photon counting device is used for lifetime measurements.The other part of this research is using a dual colour microscope system (680/780 nm excitation), in which ¯uorescence lifetimes of two diå erent dyes (IRD700/IRD800) will be simultaneously obtained.This microscope will be ® rst tested on PMMA single channel chips and then mounted on a translational stage to read ¯uorescence from a 16-channel microchip which is constructed in our laboratory.In addition, techniques for ® lling microfabricated channels with denaturing polyacrylamide matrices, loading DNA sequencing samples on a chip, injecting samples, and the optimization of separation conditions will be discussed.Results of this research will signi® cantly prove the feasibility of performing high-speed genomic sequencing using microfabricated electrophoretic devices and advantages of using lifetime detection.

Monitoring biomolecule interactions at a single molecule level
Gang Yao We have imaged the process of biomolecule interaction and reactions at the single molecule level.Immobilizing single molecular beacon DNA molecules onto a solid surface and monitoring their ¯uorescence signal during the hybridization to their complementary DNA achieve this.Fluorescence images are taken using a total internal re¯ection ¯uorescence microscopy equipped with an intensi® ed charged couple device (ICCD).It is found that many individual molecular beacon DNA probes have a gradual ¯uorescence increase within about 20 s.This indicates the conformationa l change of the hairpin shaped molecular beacon from stem close state to open state happens synchronously with the process of the loop hybridization.Our result also shows that the intermediates with partially base pairing between the two single strands may exist during the hybridization de-spite the well known all-or-none mode of DNA duplex formation.We found that the hybridization increases the photostability of the dye in a double-stranded DNA.We have also used the same technique for DNA/protein interaction studies on a single molecule level.

Charlotte Culley, Thermo LabSystems, 1 St George's Court, Hanover B, Altrincham WA14 5TP, UK
The majority of LIMS today have some kind of interface for directly reading results from instrument data systems, hence avoiding manual transcription errors, etc.However, there are problems associated with losing the link to the original analytical data ® les.This paper will discuss progress towards an integrated system where the LIMS reads results directly from the instrument system, while at the same time gathering the corresponding data ® les etc and storing them in the same secure LIMS environment.This approach has the two bene® ts.First, from an Electronic Record Keeping perspective it ensures that the actual data used to generate the results in the ® rst place has true referential integrity with the results.Secondly, from a Knowledge Management perspective, other information relating to the instrument data is available to the LIMS user, allowing big increases in productivity when it comes to sharing and mining information.

Making our LIMS your LIMS-quickly!
Peter B. Mans¢eld and Clive Seaton, Zumatrix, Inc., 81 Technology Park Drive, East Falmouth, MA 02536-4442, USA When a laboratory embarks on a LIMS implementation the process typically begins by referring to a user requirement document that was prepared many months earlier which may not always re¯ect current needs.As the vendor and customer begin detailed implementation work, both parties usually reach a new level of understanding of what the LIMS is required to do.As the implementation progresses, the LIMS is moulded to meet the user requirementsÐ a process which often requires a measure of compromise by both parties depending on the ¯exibility of the LIMS.Once the LIMS is in production it will be expected to have a lifetime extending over many years and, during this time there is likely to be numerous requests to modify or extend the capabilities of the system.In this presentation we will describe an elegant suite of LIMS con® guration tools designed to facilitate the smooth implementation of a LIMS to meet the current user requirements.We will describe how this suite of tools can also be used to modify the LIMS as business and/or laboratory needs evolve.A `wizard' -style interface and use of a LIMS con® guration knowledge base helps the vendor, and later the system administrator, create and/or modify end-user screens.Other tools facilitate documenting the con® guration and providing con® guration control.Successful LIMS do not stand still and the presentation will explain how a new generation of LIMS con® guration tools will deliver signi® cant business bene® ts throughout the lifetime of the system.

DNA pro ling and LIMS integration
Phil Goddard, CSols, Inc., 13 Bolton Road East, Port Sunlight CH62 4RU, UK The integration of instruments with LIMS is well established for traditional analytical techniques such as chromatography , spectroscopy, etc. Standard, commercial software products exist (such as Links for LIMS produced by CSols) which can handle this type of link in a straightforward manner.Such software clearly handles the mechanistic functions of reading and writing data from/to the instruments.However, it also carries out many speci® c, technique-dependent tasks, which are necessary to automate laboratory processes and make it usable in real world situations.There has been a recent and rapid growth in biotechnology techniques and instrumentation .These instruments provide few additional problems for linking software with respect to reading and writing data.However, their techniquedependent requirements are very diå erent from those demanded by traditional analytical instruments.This lecture examines what instrument integration software has to handle in one speci® c biotechnology example DNA pro® ling to make it a usable tool for transferring useful data to LIMS.The application is an important one as it is widely used in forensics, paternity testing and wide range of R&D environments.Speci® c automation issues are discussed including: acquiring peak data from the instruments, identifying true allelic peaks, recognizing and dealing with spurious peaks and known experimental artefacts (e.g.Stutter peaks, N-Peaks, Pull-up Peaks), checking standards, presenting and managing data in an eå ective manner, communication with LIMS.The example is used to highlight the types of additional issues that are important when linking biotech instruments to LIMS.There is also a discussion on what extra features it has been necessary to add to Links for LIMS to produce a Biotech Version.

1 .
Singhal, P. and Kuhr, W. G. Analytical Chemistry, 69 (1997), 4828.DNA sequencing using time-resolve d uorescence detection in a PMMA microchip Li Zhu and Steven A. Soper, Louisiana State University, Department of Chemistry, 232 Choppin Hall, Baton Rouge, LA 70803-0001, USA Vial contents and moisture are measured as they move on a belt, without stopping, or aligning the vials.Combination with colour code and barcode readers possible.Product is safer. .Tablets are measured as they move on a belt, without stopping.Placebo versus active tablets are validated.Rates can vary from 10 to 60 per min.
Applications of a portable FTIR analyser system in the pharmaceutica l industryJohn P. Coates, Coates Consulting, 12 N. Branch Road, Newtown, CT 06470-1858, USA

time direct analysis of mercury in air using portable atomic absorbtion spectromete r with Zeeman correction
with Zeeman correction.This instrument was originally designed as a portable instrument to pinpoint the sources of mercury spills and accumulations.The high sensitivity of the instrument (several ng m ¡3 Michael Markelov, Joseph Siperstein, Olga Bershevits, John Kroczek and David De Chant, OhioLumex Co., R&D, 5405 E. Schaaf Road, Independence, OH 44131-1337, USA This paper will demonstrate the direct analysis of mercury in air without any preconcentration , amalgamation, or trapping options generating real-time mercury values using the Atomic Absorption Spectrometer RA-915 +