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Opioids have become a serious public health concern over the last decade. These compounds are commonly found mixed, or cut, with safer compounds to make the opioids appear unadulterated while also enhancing the psychoactive effect on the user. Commercial benchtop and handheld IMS devices are capable of detection but published reduced ion mobility (K 0 ) values, used to identify the target analytes with IMS instrumentation, have shown variability. This lack of agreement, even for compounds used for calibration, is often due to the effects of drift tube temperature, drift gas water vapor levels and the use in-house built instrumentation rather than commercial equipment. Multiple reports exist on assessment of IMS reference standards but a single, consensus universal standard does not exist. Assessment of opioid cutting agents as internal standards is a worthwhile pursuit if precise and accurate K 0 values are obtained. The effects of drift gas water vapor content and drift tube temperature were used to evaluate the cutting agents. The K 0 values of papaverine, a representative opioid with a similar K 0 value to heroin and fentanyl, were calculated with respect to quinine and were in agreement with literature data. The use of quinine as an internal standard also improved precision relative to the instrument standard and shows promise in the application presented here., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Fluctuations in ultra high purity (UHP) helium supply has the potential to negatively impact critical research efforts. Disruptions have increased significantly with suppliers prioritizing delivery to medical facilities. Due to the greater demand for helium, supply issues are likely to continue through the coming years. Many gas chromatography (GC)-based analytical methods rely on the supply of UHP helium, including those developed for the quantification of trace explosives. Vapor validation is critical in establishing sensor performance, limits of detection, and instrument performance. An alternate carrier gas must be established to maintain these critical functionalities. To circumvent the UHP helium disruptions, UHP nitrogen was explored as a replacement carrier gas in negative mode chemical ionization-gas chromatography/mass spectrometry (CI-GC/MS). Although, hydrogen is considered an acceptable alternative to helium in most GC-based separations, its' use as a replacement was omitted due to reactivity resulting in degradation of the CI-MS detector and incompatibility with the programmable temperature vaporization inlet on the GC used in this work. Herein discusses the method development of nitrogen carrier gas in the separation of an explosives mixture. Adjustments in flow rate, initial oven temperature, and ramp rate were made to achieve comparable analysis to that of helium. By lowering the flow rate and initial oven temperature peak resolution and sensitivity increased when using nitrogen carrier gas. Development of this method allows for continual laboratory output in times of helium scarcity.

Amphiphilic copolymers of various-molecular-weight (MW) poly(ethylene glycol) (PEG) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The first PEG series, poly(ethylene glycol)monomethacrylate (PEGMA, average Mn 200 and 400 MW), contained an -OH terminal group, and the second series, poly(ethylene glycol) monomethyl ether monomethacrylate (PEGMMA, average Mn 200, 400, and 1000 MW), possessed an -OCH 3 terminal group. A total of five PEG-functionalized copolymers contained the same hydrophobic monomer, butyl acrylate (BA), and were successfully reproduced via a one-pot synthesis. The resulting PEG-functionalized copolymers provide a systematic trend of properties including surface tension, critical micelle concentration (CMC), cloud point (CP), and foam lifetime based on the average MW of the PEG monomer and final polymer properties. In general, the PEGMA series produced more stable foams with PEGMA200 demonstrating the least change in foam height with time over a 10 min period. The important exception is that at elevated temperatures, the PEGMMA1000 copolymer had longer foam lifetimes. The self-assembling copolymers were characterized by gel permeation chromatography (GPC), 1 H nuclear magnetic resonance (NMR), attenuated total reflection Fourier transform infrared (FTIR-ATR), CMC, surface tension, dynamic light scattering (DLS), as a foam using a dynamic foam analyzer (DFA), and foam lifetime at ambient and elevated temperatures. The copolymers described highlight the importance of the PEG monomer MW and terminal end group for surface interaction and final polymer properties for foam stabilization.

A series of amphiphilic statistical copolymers involving poly(ethylene glycol) monomethacrylate (PEGMA, -OH terminated, average Mn 200 molecular weight) and various hydrophobic acrylates were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The gradient copolymers were characterized by gel-permeation chromatography (GPC), 1 H nuclear magnetic resonance (NMR), and attenuated total reflection Fourier transform infrared spectroscopy (FTIR-ATR). Solution properties of the copolymers were investigated utilizing surface tension measurement, dynamic light-scattering (DLS), as well as foam analysis using a dynamic foam analyzer (DFA). The PEG-functionalized copolymers showed a systematic trend depending on the hydrophobic moiety in properties including surface tension, critical micelle concentration (CMC), foam lifetime, and liquid drainage from the foam. Copolymers with alkyl-acrylates exhibited the best foam lifetime, demonstrating that the choice of hydrophobic moiety is crucial for foam stability. The PEG-functionalized materials described are considered promising additives for foam-stability purposes.

The transport efficiency of 2,4-dinitrotoluene (2,4-DNT), 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitro-1,3,5-triazinane (RDX) trace vapors through tubing materials that commonly constitute vapor handling infrastructures have been determined for a variety of tubing dimensions and sampling conditions. Using a programmable temperature vaporization inlet coupled with a gas chromatography mass spectrometer (PTV-GC-MS), the explosive vapors were quantified both with and without a length of tubing of a specific material in the sampling flow path. At vapor temperatures of 30 °C and 66 °C, minimal attenuations were observed for 2,4-DNT and TNT vapor concentrations when the tubing material was in-line with the sampling flow path, indicating that the transport is largely unaffected by interactions with the surface of the tubing materials. In contrast, RDX vapors showed large attenuations as a function of both sampling conditions and tubing materials/dimensions. For those experiments where attenuated RDX vapor transport was observed, the mass sequestered by interactions between the flowing vapor and the internal tubing surface was determined to be in the range of tens to hundreds of picograms. Of all the materials examined for RDX transport, fluorinated ethylene propylene (FEP) tubing resulted in the least amount of mass loss to surface interactions, with vapor transport efficiencies (VTEs) between 95-100%. However, for some materials, the combination of tubing dimensions and sampling conditions resulted in no RDX transport, even after sampling more than 250.0 L of vapor through the tubing.

The Trace Vapor Generator for Explosives and Narcotics (TV-Gen) is a portable and compact instrument designed to deliver a continuous source of trace-level vapors and vapor mixtures. It provides a tool to assist in the independent validation and verification of new materials and sensors under development for the vapor detection of explosives and narcotics. The design was conceived for use with a broad range of analytes, detection systems, materials, and sensors and to switch easily between the clean and analyte vapor streams. The TV-Gen system utilizes nebulization of aqueous analyte solutions, an oven to promote efficient transport, and a control box that provides dedicated computer control with logging capabilities. Resultant vapor streams are stable over several hours, with the vapor concentration controlled by a combination of aqueous analyte solution concentration, liquid flow rate through the nebulizer, and volume flow rate of air through the TV-Gen manifold.

Secondary electrospray corona discharge ionization (SECDI) combines the principles of secondary electrospray ionization (SESI) and corona discharge (CD) to achieve higher sensitivity, which is demonstrated through the detection of 2,4,6-trinitrotoluene (TNT) and 2,6-dinitrotoluene (2,6-DNT) vapors using ion mobility spectrometry (IMS). Using SECDI, enhancements in the IMS signal for TNT and 2,6-DNT vapors at trace concentrations are as much as 2-26 times that observed with CD or SESI alone. The enhancement in sensitivity is hypothesized to result from an increase in ionization efficiency driven by a higher number of reactant ions associated with SECDI compared to either technique individually. The ability of SECDI to achieve higher sensitivity without the aid of dopant molecules demonstrates its merit as an alternative ionization technique., (Published by Elsevier B.V.)

The development of an online sampling method using programmable temperature vaporization gas chromatography with a mass spectrometer detector (PTV-GC/MS) for the analysis of trace pentaerythritol tetranitrate (PETN) vapor is presented. PETN degradation was minimized by optimizing the temperature and flow rates of the vapor sampling infrastructure. Validation of the online method was done using a previously published technique, vapor sampling with Tenax-TA thermal desorption tubes followed by analysis with a thermal desorption system coupled to a programmable temperature vaporization gas chromatograph with a micro-electron capture detector (TDS-PTV-GC/μECD). Trace PETN vapor was generated using state-of-the-art instrumentation known as the TESTbed, located at the US Naval Research Laboratory. For PETN vapor concentrations in the parts per trillion volume (ppt v ) range, quantitative results from the TDS-PTV-GC/μECD consistently showed concentrations approximately double that measured by the online PTV-GC/MS method, indicating that sample loss due to additional exposure to the vapor sampling infrastructure occurs when using online sampling. Further utilization of the online PTV-GC/MS system allowed for the quantitation/semi-quantitation of PETN vapor concentrations as low as 260 parts per quadrillion volume (ppq v ) with only 10 min of sampling time., (Published by Elsevier B.V.)

Flow-through optical chromatography (FT-OC), an advanced mode of optical chromatography, achieved baseline separation of a mixture of silica microparticles (SiO 2 , 1.00 and 2.50 μm) and a mixture of polystyrene microparticles (PS, 1.00, 2.00, and 3.00 μm) based on particle size. Comparisons made between experimentally determined velocities for the microparticles and theoretically derived velocities from Mie theory and Stokes' law validated the data collection setup and the data analysis for FT-OC. A population shift in live macrophages (cell line IC-21, ATCC TIB-186) responding to environmental stimuli was sensitively detected by FT-OC. The average velocity of macrophages stressed by nutritional deprivation was decreased considerably together with a small but statistically significant increase in cell size. Mie scattering calculations demonstrated that the small increase in cell size of macrophages stressed by nutritional deprivation was not entirely responsible for this decrease. Confocal fluorescence microscopy and atomic force microscopy (AFM) studies revealed morphological changes of macrophages induced by nutritional deprivation, and these changes were more likely responsible for the decrease in average velocity detected by FT-OC. Confocal Raman microspectroscopy was used to shed light upon biochemical transformations of macrophages suffering from nutritional deprivation., Competing Interests: The authors declare no competing financial interest.

Silicon nanowire (SiNW) arrays are demonstrated as a suitable platform for the preconcentration of trace nitroaromatic compounds and subsequent desorption via Joule heating of the array. Arrays are fabricated from Si wafers containing an epitaxially grown layer of low conductivity intrinsic Si sandwiched between layers of high conductivity p-type Si. Passage of current through the nanowires results in nanowire temperatures in excess of 200 °C during heating of the arrays as verified by using the temperature-dependent shift of the Si Raman band at ˜520 cm -1 . Analyte vapor preconcentration and partial separation is achieved on the array at analyte concentrations nearly two orders-of-magnitude below saturated vapor concentrations at room temperature. The effects of desorption carrier gas flow rate and temperature on the ability to preconcentrate and resolve the analytes of interest are determined. 2,6-dinitrotoluene (2,6-DNT) and 2,4-dinitrotoluene (2,4-DNT) were detected at nominal vapor concentrations of 800 ppt v with a 1 min sample time (1.1 ng nominal mass load) and trinitrotoluene (TNT) was detected at a nominal vapor concentration of 65 ppt v with a 10 min sample time (1.1 ng nominal mass load)., (Published by Elsevier B.V.)

Micellar electrokinetic chromatography (MEKC) is a mode of capillary electrophoresis that allows for the separation of neutral molecules in an electric field. Typically, neutral molecules move with electroosmotic flow (EOF) or bulk flow during electrophoretic separations resulting in no temporal resolution between mixtures of neutral analytes. Inclusion of surfactant micelles in the separation buffer allows for the separation of neutral analytes from one another through association with the micelle. Here we outline the implementation of MEKC for the separation of neutral molecules using a mixture of nitroaromatic explosives and their degradation products serving as a test analyte mixture.

Typical trace vapor analysis involves sorbent trapping, followed by desorption and chromatographic separation. This communication describes a method for streamlining this process by combining sorbent sampling/preconcentration with partial separation achieved through temperature-programmed thermal desorption. A novel sorbent trap was formulated in which tubular glass liners for a programmable-temperature gas chromatograph inlet were coated with a sol-gel based polymer stationary phase synthesized from methyltrimethoxysilane precursor and installed into the inlet, which was directly connected to a mass-selective detector by a fused silica capillary transfer line. This method is shown to achieve partial separation of two nitroaromatic vapors in a total 3-5min analysis time, which represents a tenfold improvement in speed in terms of the overall cycle time compared to an analogous conventional vapor analysis method. Both analytes proved to have a high dynamic range and loading capacity, with nitrobenzene achieving both high and low sampling extremes (0.32ng-4μg sampling concentration) with only a slight compromise in peak broadening. The multivariate curve resolution by alternating least squares algorithm (MCR-ALS) was shown to successfully resolve the overlapped elution profiles of the two nitroaromatic test vapors examined in this study., (Published by Elsevier B.V.)

A novel vapor delivery testbed, referred to as the Trace Explosives Sensor Testbed, or TESTbed, is demonstrated that is amenable to both high- and low-volatility explosives vapors including nitromethane, nitroglycerine, ethylene glycol dinitrate, triacetone triperoxide, 2,4,6-trinitrotoluene, pentaerythritol tetranitrate, and hexahydro-1,3,5-trinitro-1,3,5-triazine. The TESTbed incorporates a six-port dual-line manifold system allowing for rapid actuation between a dedicated clean air source and a trace explosives vapor source. Explosives and explosives-related vapors can be sourced through a number of means including gas cylinders, permeation tube ovens, dynamic headspace chambers, and a Pneumatically Modulated Liquid Delivery System coupled to a perfluoroalkoxy total-consumption microflow nebulizer. Key features of the TESTbed include continuous and pulseless control of trace vapor concentrations with wide dynamic range of concentration generation, six sampling ports with reproducible vapor profile outputs, limited low-volatility explosives adsorption to the manifold surface, temperature and humidity control of the vapor stream, and a graphical user interface for system operation and testing protocol implementation.

As a reagent gas for positive- and negative-mode chemical ionization mass spectrometry (CI-MS), isobutane (i-C 4 H 10 ) produces superior analyte signal abundance to methane. Isobutane has never been widely adopted for CI-MS because it fouls the ion source more rapidly and produces positive CI spectra that are more strongly dependent on reagent gas pressure compared with methane. Isobutane was diluted to various concentrations in argon for use as a reagent gas with an unmodified commercial gas chromatograph-mass spectrometer. Analyte spectra were directly compared using methane, isobutane, and isobutane/argon mixtures. A mixture of 10% i-C 4 H 10 in argon produced twice the positive-mode analyte signal of methane, equal to pure isobutane, and reduced spectral dependence on reagent gas pressure. Electron capture negative chemical ionization using 1% i-C 4 H 10 in argon tripled analyte signal compared with methane and was reproducible, unlike pure isobutane. The operative lifetime of the ion source using isobutane/argon mixtures was extended exponentially compared with pure isobutane, producing stable and reproducible CI signal throughout. By diluting the reagent gas in an inert buffer gas, isobutane CI-MS experiments were made as practical to use as methane CI-MS experiments but with superior analytical performance. Graphical Abstract ᅟ.

The generation of trace 2,4,6-trinitrotoluene (TNT), cyclotrimethylenetrinitramine (RDX), and pentaerythritol tetranitrate (PETN) vapors using a pneumatically modulated liquid delivery system (PMLDS) coupled to a polytetrafluoroethylene (PTFE) total-consumption micronebulizer is presented. The vapor generator operates in a continuous manner with final vapor concentrations proportional to the explosive concentration in aqueous solution delivered through the nebulizer and the diluent air flow rate. For quantitation of concentrations in the parts per billionvolume (ppbv) to parts per trillionvolume (pptrv) range, Tenax-TA thermal desorption tubes were used for vapor collection with subsequent analysis on a thermal-desorption system programmable-temperature vaporization gas chromatograph (TDS-PTV-GC) with a μ-ECD detector. With 30 min sample times and an average sampling rate of 100 mL min(-1), vapor concentrations of 38 pptrv for TNT, 25 pptrv for RDX, and 26 pptrv for PETN were determined. For parts per quadrillionvolume (ppqv) vapor quantitation of TNT and RDX, an online PTV-GC system with a negative-ion chemical ionization mass spectrometer (methane reagent gas) was used for direct sampling and capture of the vapor on the PTV inlet. Vapor concentrations as low as 160 ppqv and 710 ppqv for TNT and RDX were quantified, respectively, with an instrument duty cycle as low as 4 min.

An analytical method for establishing calibration curves for the quantitation of pentaerythriol tetranitrate (PETN) from sorbent-filled thermal desorption tubes by gas chromatography with electron capture detection (TDS-GC-ECD) was developed. As PETN has been demonstrated to thermally degrade under typical GC instrument conditions, peaks corresponding to both PETN degradants and molecular PETN are observed. The retention time corresponding to intact PETN was verified by high-resolution mass spectrometry with a flowing atmospheric pressure afterglow (FAPA) ionization source, which enabled soft ionization of intact PETN eluting the GC and subsequent accurate-mass identification. The GC separation parameters were transferred to a conventional GC-ECD instrument where analytical method-induced PETN degradation was further characterized and minimized. A method calibration curve was established by direct liquid deposition of PETN standard solutions onto the glass frit at the head of sorbent-filled thermal desorption tubes. Two local, linear relationships between detector response and PETN concentration were observed, with a total dynamic range of 0.25-25ng., (Published by Elsevier B.V.)

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.

A novel liquid injection vapor generator (LIVG) is demonstrated that is amenable to low vapor pressure explosives, 2,4,6-trinitrotoluene and hexahydro-1,3,5-trinitro-1,3,5-triazine. The LIVG operates in a continuous manner, providing a constant and stable vapor output over a period of days and whose concentration can be extended over as much as three orders of magnitude. In addition, a large test atmosphere chamber attached to the LIVG is described, which enables the generation of a stable test atmosphere with controllable humidity and temperature. The size of the chamber allows for the complete insertion of testing instruments or arrays of materials into a uniform test atmosphere, and various electrical feedthroughs, insertion ports, and sealed doors permit simple and effective access to the sample chamber and its vapor.

Two methods for quantitation of triacetone triperoxide (TATP) vapor using a programmable temperature vaporization (PTV) inlet coupled to a gas chromatography/mass spectrometer (GC/MS) have been demonstrated. The dynamic headspace of bulk TATP was mixed with clean humid air to produce a TATP vapor stream. Sampling via a heated transfer line to a PTV inlet with a Tenax-TA™ filled liner allowed for direct injection of the vapor stream to a GC/MS for vapor quantitation. TATP was extracted from the vapor stream and subsequently desorbed from the PTV liner for splitless injection on the GC column. Calibration curves were prepared using solution standards with a standard split/splitless GC inlet for quantitation of the TATP vapor. Alternatively, vapor was sampled onto a Tenax-TA™ sample tube and placed into a thermal desorption system. In this instance, vapor was desorbed from the tube and subsequently trapped on a liquid nitrogen cooled PTV inlet. Calibration curves for this method were prepared from direct liquid injection of standards onto samples tube with the caveat that a vacuum is applied to the tube during deposition to ensure that the volatile TATP penetrates into the tube. Vapor concentration measurements, as determined by either GC/MS analysis or mass gravimetry of the bulk TATP, were statistically indistinguishable. Different approaches to broaden the TATP vapor dynamic range, including diluent air flow, sample chamber temperature, sample vial orifice size, and sample size are discussed. Vapor concentrations between 50 and 5400ngL(-1) are reported, with stable vapor generation observed for as long as 60 consecutive hours., (Published by Elsevier B.V.)

A simple method for establishing calibration curves with sorbent-filled thermal desorption tubes has been demonstrated for nitroaromatic and nitramine vapor samples using a thermal desorption system with a cooled inlet system (TDS-CIS), which was coupled to a gas chromatograph (GC) with an electron capture detector (ECD). The method relies upon the direct liquid deposition of standard solutions onto the glass frit at the head of sorbent-filled thermal desorption tubes. Linear calibration results and ideal system conditions for the TDS-CIS-GC-ECD were established for mixtures containing both cyclotrimethylenetrinitramine, a.k.a. RDX, and 2,4,6-trinitrotoluene (TNT). Because of the chemical characteristics of RDX, a higher TDS-CIS flow rate relative to the optimized approach for TNT was required for efficient RDX desorption. Simultaneous quantitation of TNT and RDX using the direct liquid deposition method with optimized instrumentation parameters for RDX were compared to results from a standard split/splitless GC inlet and a CIS., (Published by Elsevier B.V.)

On-line sample preconcentration is an essential tool in the development of microfluidic-based separation platforms. In order to become more competitive with traditional separation techniques, the community must continue to develop newer and more novel methods to improve detection limits, remove unwanted sample matrix components that disrupt separation performance, and enrich/purify analytes for other chip-based actions. Our goal in this review is to familiarize the reader with many of the options available for on-chip concentration enhancement with a focus on those manuscripts that, in our assessment, best describe the fundamental principles that govern those enhancements. Sections discussing both electrophoretic and nonelectrophoretic modes of preconcentration are included with a focus on device design and mechanisms of preconcentration. This review is not meant to be a comprehensive collection of every available example, but our hope is that by learning how on-line sample concentration techniques are being applied today, the reader will be inspired to apply these techniques to further enhance their own programs., (Published by Elsevier B.V.)

The use of thermal desorption systems for the analysis of trace vapors typically requires establishing a calibration curve from vapors generated with a permeation tube. The slow equilibration time of permeation tubes causes such an approach to become laborious when covering a wide dynamic range. Furthermore, many analytes of interest, such as explosives, are not available as permeation tubes. A method for easily and effectively establishing calibration curves for explosive vapor samples via direct deposition of standard solutions on thermal desorption tubes was investigated. The various components of the thermal desorption system were compared to a standard split/splitless inlet. Calibration curves using the direct liquid deposition method with a thermal desorption unit coupled to a cryo-focusing inlet were compared to a standard split/splitless inlet, and a statistical difference was observed but does not eliminate or deter the use of the direct liquid deposition method for obtaining quantitative results for explosive vapors., (Published by Elsevier B.V.)

Practical considerations for the injection and separation of nitroaromatic explosives in seawater sample matrices are discussed. The use of high surfactant concentrations and long electrokinetic injections allows for improved detection limits. Sensitivity was enhanced by two mechanisms, improved stacking at the detector-side of the sample plug and desorption of analyte from the capillary wall by surfactant-containing BGE from the inlet side of the sample plug. Calculated limits of detection (S/N=3) for analytes prepared in pure seawater were 70-800 ppb with injection times varying from 5 to 100 s., (Published by Elsevier B.V.)

An electroosmotic flow (EOF)-based pump, integrated with a sol-gel stationary phase located in the electric field-free region of a microchip, enabled the separation of six nitroaromatic and nitramine explosives and their degradation products via liquid chromatography (LC). The integrated pump and LC system were fabricated within a single quartz substrate. The pump region consisted of a straight channel (3.0 cm x 230 microm x 100 microm) packed with 5-microm porous silica beads. The sol-gel stationary phase was derived from a precursor mixture of methyltrimethoxy- and phenethyltrimethoxysilanes and was synthesized in the downstream, field-free region of the microchip, resulting in a stationary-phase monolith with dimensions of 2.6 cm x 230 microm x 100 microm. Fluid dynamic design considerations are discussed, especially as they relate to integrating the EOF pump with the LC system. Pump and separation performance, as characterized by flow rate measurements, injection, elution, separation, and detection, point to a viable analytical chemistry platform that encompasses all of the benefits expected of portable, laboratory-on-chip systems, including reduced sample requirements and small packaging.

A microchip device is demonstrated for the electrophoretic separation and UV-absorbance spectral detection of four toxic alkaloids: colchicine, aconitine, strychnine, and nicotine. A fused-silica (quartz) microchip containing a simple cross geometry is utilized to perform the separations, and a miniature, fiber-optic CCD spectrometer is coupled to the microchip for detection. Sensitive UV-absorbance detection is achieved via the application of online preconcentration techniques in combination with the quartz microchip substrate which contains an etched bubble-cell for increased pathlength. The miniature CCD spectrometer is configured to detect light between 190 and 645 nm and LabView programming written in-house enables absorbance spectra as well as separations to be monitored from 210 to 400 nm. Consequently, the configuration of this microchip device facilitates qualitative and quantitative separations via simultaneous spatial and spectral resolution of solutes. UV-absorbance limits of quantification for colchicine, 20 microM (8 mg/L); strychnine, 50 microM (17 mg/L); aconitine, 50 microM (32 mg/L); and nicotine, 100 microM (16 mg/L) are demonstrated on the microchip. With the exception of aconitine, these concentrations are > or =20-times more sensitive than lethal dose monitoring requirements. Finally, this device is demonstrated to successfully detect each toxin in water, skim milk, and apple juice samples spiked at sublethal dose concentrations after a simple, SPE procedure.

In order to understand the role of stacked micelles in sample preconcentration, it is necessary to understand the factors that contribute to the micelle stacking phenomenon. Various MEKC background electrolyte (BGE) solutions were prepared in the presence of Sudan III in order to monitor the micelle stacking phenomenon in the anionic sodium dodecyl sulfate and sodium cholate micelle systems. The data show that micelle stacking is a dynamic process that is strongly dependent upon the relative conductivities of the sample matrix and BGE, the relative column length of the sample plug, and the mobilities of the ions involved in the stacking process regardless of electric field conditions (i.e., field-amplified stacking, sweeping, or high-salt stacking). Conditions under which micelle stacking can be expected to occur are presented, and the extent of micelle stacking is quantified. The micelle stacking phenomenon is correlated to the separation performance of a series of neutral alkaloids. It is shown that neutral analytes migrate rapidly through the evolving stacked micelle region in the initial moments of the separation. As a consequence of this transient interaction, analytes with small retention factors spend less time in the stacked micelle region and experience lower stacked micelle concentrations than analytes with large retention factors that spend more time in the growing stacked micelle region. It is also demonstrated that the extent of analyte enrichment generally increases with injection length, by facilitating greater interaction time with stacked micelles; however, enrichment will eventually plateau with increasing injection length as a function of an analyte's affinity for the micelle. Finally, it is shown that, in contrast to conventional wisdom, a range of long injection plugs exist where separation efficiency can be dramatically improved due to analyte interaction with an actively growing stacked micelle region.

Microchip-based CEC of nitroaromatic and nitramine explosives with UV absorbance detection is described. The stationary phase was deposited in the microchip using the sol-gel process. Unique to this work, is the exclusive use of alkylated-trimethoxysilane precursors in the gel solution. Using alkylated precursors allows for the synthesis of a hydrophobic stationary phase in a single step. Three sol-gel formulations of increasing hydrophobicity and suitable for the separation of explosives are established from methyl- and ethyl-trimethoxysilane precursors. Increasing the alkyl-chain length improved the resolution significantly, allowing for the separation of up to seven analytes. Direct injection onto the head of the stationary phase for long injection times, results in sub-mg/L detection limits with little effect on separation efficiency.

Effective microchip extraction of deoxyribonucleic acid (DNA) from crude biological matrixes has been demonstrated using silica beads or hybrid phases composed of beads and sol-gel. However, the use of monolithic sol-gels alone for extraction of human genomic DNA has been more difficult to define. Here we describe, for the first time, the successful use of monolithic tetramethyl orthosilicate-based sol-gels for effective micro-solid-phase extraction (muSPE) of DNA in a glass microchip format. A functional monolithic silica phase with micrometer-scale pores in the silica matrix resulted from addition of poly(ethylene glycol), a poragen, to the precursor mixture. This allowed a monolithic sol-gel bed to be established in a microchip channel that provided large surface area for DNA extraction with little flow-induced back pressure. DNA extraction efficiencies for simple systems (lambda-phage DNA) were approximately 85%, while efficiencies for the reproducible extraction of human genomic DNA from complex biological matrixes (human blood) were approximately 70%. Blockage of the sol-gel pores by components in the lysed blood was observed in repeat extraction on a single device as a decrease in the extraction efficiency. The developed muSPE protocol was further evaluated to show applicability to clinical samples and bacterial cultures, through extraction of PCR-amplifiable DNA.

The ability to detect sub-nanomolar concentrations of ricin using fluorescently tagged RNA aptamers is demonstrated. Aptamers rival the specificity of antibodies and have the power to simplify immunoassays using capillary electrophoresis. Under nonequilibrium conditions, a dissociation constant, Kd, of 134 nM has been monitored between the RNA aptamer and ricin A-chain. With use of this free-solution assay, the detection of 500 pM (approximately 14 ng/mL) or 7.1 amol of ricin is demonstrated. The presence of interfering proteins such as bovine serum albumin and casein do not inhibit this interaction at sub-nanomolar concentrations. When spiked with RNAse A, ricin can still be detected down to 1 nM concentrations despite severe aptamer degradation. This approach offers a promising method for the rapid, selective, and sensitive detection of biowarfare agents.

A fundamental premise in CE relies heavily on the assumption that temperature within the capillary is accurately known and controlled. Theoretical calculations for sample zone and BGE temperature during voltage application are presented. We propose that transient elevation of the sample zone temperature allowed for denaturing and renaturing of proteins in the presence of a fluorescent dynamic labeling reagent. Comparison with the extent of labeling possible with standard on-column dynamic labeling in the absence of elevated temperatures showed order-of-magnitude increases in the fluorescence detection sensitivity of proteins with low surface hydrophobicity. As a result, this represents an example where excess heating in the sample zone during electrophoresis can be exploited advantageously.

The ability to separate nitroaromatic and nitramine explosives in seawater sample matrices is demonstrated using both MEKC and CEC. While several capillary-based separations exist for explosives, none address direct sampling from seawater, a sample matrix of particular interest in the detection of undersea mines. Direct comparisons are made between MEKC and CEC in terms of sensitivity and separation efficiency for the analysis of 14 explosives and explosive degradation products in seawater and diluted seawater. The use of high-salt stacking with MEKC results, on average, in a three-fold increase in the number of theoretical plates, and nearly double resolution for samples prepared in 25% seawater. By taking advantage of long injection times in conjunction with stacking, detection limits down to sub mg/L levels are attainable; however, resolution is sacrificed. CEC of explosive mixtures using sol-gels prepared from methyltrimethoxysilane does not perform as well as MEKC in terms of resolving power, but does permit extended injection times for concentrating analyte onto the head of the separation column with little or no subsequent loss in resolution. Electrokinetic injections of 8 min at high voltage allow for detection limits of explosives below 100 microg/L.

We have previously demonstrated on-column dynamic labeling of protein-SDS complexes on capillaries and microchips for laser-induced fluorescence (LIF) detection using both a commercially available fluor and a protein separation buffer. Upon binding to hydrophobic moieties (of the analyte or separation buffer), the fluor undergoes a conformational change allowing fluorescence detection at 590 nm following excitation with 488-nm light. Our original work showed on-chip limits of detection (LOD) comparable with those using UV detection (1 x 10(-5) M) on capillaries-falling significantly short of the detection limits expected for LIF. This was largely a function of the physicochemical characteristics of the separation buffer components, which provided significant background fluorescence. Having defined the contributing factors involved, a new separation buffer was produced which reduced the background fluorescence and, consequently, increased the available dye for binding to protein-SDS complexes, improving the sensitivity in both capillaries and microchips by at least 2 orders of magnitude. The outcome is a rapid, sensitive method for protein sizing and quantitation applicable to both capillary and microchip separations with a LOD of 500 ng/mL for bovine serum albumin. Interestingly, sensitivity on microdevices was improved by inclusion of the dye in the sample matrix, while addition of dye to samples in conventional CE resulted in a drastic reduction in sensitivity and resolution. This can be explained by the differences in the injection schemes used in the two systems. The linear range for protein quantitation covered at least 2 orders of magnitude in microchip applications. On-chip analysis of human sera allowed abnormalities, specifically the presence of elevated levels of gamma-globulins, to be determined.

A microchip solid-phase extraction method for purification of DNA from biological samples, such as blood, is demonstrated. Silica beads were packed into glass microchips and the beads immobilized with sol-gel to provide a stable and reproducible solid phase onto which DNA could be adsorbed. Optimization of the DNA loading conditions established a higher DNA recovery at pH 6.1 than 7.6. This lower pH also allowed for the flow rate to be increased, resulting in a decrease in extraction time from 25 min to less than 15 min. Using this procedure, template genomic DNA from human whole blood was purified on the microchip platform with the only sample preparation being mixing of the blood with load buffer prior to loading on the microchip device. Comparison between the microchip SPE (microchipSPE) procedure and a commercial microcentrifuge method showed comparable amounts of PCR-amplifiable DNA could be isolated from cultures of Salmonella typhimurium. The greatest potential of the microchipSPE device was illustrated by purifying DNA from spores from the vaccine strain of Bacillus anthracis, where eventual integration of SPE, PCR, and separation on a single microdevice could potentially enable complete detection of the infectious agent in less than 30 min.

The analysis of proteins under denaturing conditions is routinely performed with SDS-polyacrylamide gel electrophoresis. The automated capabilities of CE, use of nongel sieving matrixes, and on-line optical detection by either ultraviolet (UV) absorption or laser-induced fluorescence (LF) promise to revolutionize this method. While direct on-line detection of proteins is possible as a result of their intrinsic ability to absorb light in the UV part of the spectrum (detection sensitivity comparable to Coomassie Blue staining of gels), LIF provides more powerful detection but requires pre- or postcolumn fluorescence labeling of the proteins. The development of a protocol analogous to that used for double-stranded DNA analysis, where fluorescent intercalating dyes are simply included in the separation medium, would simplify size-based protein analysis immensely. This would avoid the complications associated with covalent modification of the proteins but still exploit the sensitivity of LIF detection. We demonstrate that this is possible with CE and microchip detection by incorporating, into the run buffer, a fluorescent dye that interacts hydrophobically with protein-SDS complexes. Key to this is a dye that fluoresces significantly when bound to protein-SDS complexes but not when bound to SDS micelles. Comparison of electropherograms from CE-based denaturing protein analysis with UV and LIF detection indicates that the presence of the fluor does not alter separation of the proteins. Moreover, comparison with electropherograms generated from microchip electrophoresis with LIF detection shows that equivalent patterns can be obtained. Despite the unoptimized nature of this separation system, a dynamic labeling protocol that allows for LIF detection for proteins is attractive and has the potential to circumvent the tedious labeling steps typically required.

There is much interest in developing methods amenable to amplifying nucleic acids by the polymerase chain reaction (PCR) in small volumes in microfabricated devices. The use of infrared-mediated temperature control to accurately thermocycle microliter volumes in microchips fabricated from polyimide is demonstrated. Amplification of a 500-base-pair fragment of lambda-phage DNA was achieved in a 1.7-microl chamber containing a thermocouple that allowed for accurate control of temperature. While previous work showed that Taq polymerase was inactivated when in direct contact with the thermocouple, this was circumvented with the polyimide chip by the addition of polyethylene glycol as a buffer additive. This, consequently, allowed for adequate amounts of PCR product to be observed after only 15 cycles, with a total time for amplification of 240 s., (Copyright 2001 Academic Press.)

As microchip technology evolves to allow for the integration of more complex processes, particularly the polymerase chain reaction (PCR), it will become necessary to define simple approaches for minimizing the effects of surfaces on the chemistry/processes to be performed. We have explored alternatives to silanization of the glass surface with the use of additives that either dynamically coat or adsorb to the glass surface. Polyethylene glycol, polyvinylpyrrolidone (PVP), and hydroxyethylcellulose (HEC) have been explored as potential dynamic coatings and epoxy (poly)dimethylacrylamide (EPDMA) evaluated as an adsorbed coating. By carrying out analysis of the PCR products generated under different conditions via microchip electrophoresis, we demonstrate that these coating agents adequately passivate the glass surface in a manner that prevents interference with the subsequent PCR process. While several of the agents tested allowed for PCR amplification of DNA in glass, the EPDMA was clearly superior with respect to ease of preparation. However, more efficient PCR (larger mass of amplified product) could be obtained by silanizing the glass surface.

Transferrin sialoforms with fewer than three sialic acid residues (carbohydrate deficient transferrin; CDT) have been implicated as a marker of certain liver pathologies. Transferrin sialoforms in human sera from alcoholic and non-alcoholic patients was analyzed by capillary electrophoresis (CE) using diaminobutane (DAB) to dynamically-coat the capillary wall to minimize protein-wall interactions. Using a DAB concentration of 3 mM, transferrin sialoforms were adequately resolved to allow for direct detection of CDT without extensive treatment of the sera. Serum immunoglobulins, which migrated close to the CDT region, were removed via subtraction with protein A, enhancing the detection of CDT. The reproducibility of sialoform separation in dynamically-coated capillaries was found to be acceptable with run-to-run relative standard deviation values of 0.15% for a sample on a given day and 0.29+/-0.06% for four samples day-to-day. These results suggest that dynamic-coating approaches may provide a simple alternative to the use of covalently-coated capillaries for the CE separation of complex samples.