Commercial Instruments Research Articles

Echelle Grating Spectroscopic Technology for High-Resolution and Broadband Spectral Measurement

Echelle grating provides high spectral resolving power and diffraction efficiency in a broadband wavelength range by the Littrow mode. The spectrometer with the cross-dispersed echelle scheme has seen remarkable growth in recent decades. Rather than the conventional approach with common blazed grating, the cross-dispersed echelle scheme achieves the two-dimensional spatial distribution of the spectrum by one exposure without scanning in the broadband spectral range. It is the fastest and most sensitive spectroscopic technology as of now, and it has been extensively applied in commercial and astronomical spectrometers. In this review, we first highlight the characteristics of the echelle and then present the optical layout, detection approach, and method of calibration. Finally, we discuss the state-of-the-art implementations and applications of commercial and astronomical instruments.

Cutting-edge mass spectrometry strategy based on imaged capillary isoelectric focusing (icIEF) technology for characterizing charge heterogeneity of monoclonal antibody

Imaging capillary isoelectric focusing (icIEF) technology has been becoming the gold criteria of monitoring monoclonal antibody (mAb) charge heterogeneity that is one of the major product-related variants in recombinant biopharmaceuticals, since the first commercial instrument developed twenty years ago. However, the protein identification in icIEF separation is just based on isoelectric point (pI) measurement of protein. Although high resolution mass spectrometry (HRMS) is currently the most powerful means of qualitative protein analysis, traditional icIEF cannot compatibly be used in conjunction with MS due to the use of less volatile reagents. In addition, protein heterogeneity characterization in depth such as peptide mapping by high performance liquid chromatography (HPLC) requires the focused protein bands to be collected as fractions after the icIEF separation, which is a great challenge in biopharmaceutical discovery. In this work, pembrolizumab was employed as targeting mAb (a highly selective anti-PD-1 humanized mAb), an integrated icIEF platform was developed including analytical profiling, MS coupling and fraction collections for charged variant preparation. Multiple operation modes can be rapidly and flexibly switched just by changing customized capillary separation cartridges without more configurations. Main component, four acidic variants (A1-A4) and three basic variants (B1–B3) were baseline separated then directly detected by icIEF-HRMS online coupling for rapid screening of intact protein heterogeneity where reliable and accurate molecular weight of protein charged variants were obtained. Next, by installing preparative capillary separation cartridge, fractions of major charge variants (A2-3 and B1-2) and main component were collected for following LC-MS peptide mapping characterization. The whole workflow of icIEF-based MS strategy for protein heterogeneity is straight forward, reliable and accurate, which provides a comprehensive and revolutionary technology for protein drug quality control (QC) monitoring, MS coupling for fingerprinting intact protein and HPLC-MS peptide mapping in depth.

Using multiple heart-cutting valves and stop-flow operation to enable variable second dimension gradient times in online comprehensive two-dimensional liquid chromatography

Online comprehensive two-dimensional liquid chromatography (LC×LC) is the preferred method currently for the separation of highly complex mixtures of non-volatile compounds. With fully automated commercial instrumentation and software making the method appealing to both researchers and industry, the demand for systems with improved separation capabilities for highly complex mixtures such as those found in natural products or proteomics has increased. In this study we report an approach that enables variable second dimension analysis times based on the use of multiple heart-cutting valves and stop-flow operation to circumvent the requirement for very fast second dimension (2D) analyses in online LC×LC. As application, the HILIC×RP-LC analysis of condensed tannins (proanthocyanidins) in cocoa, grape seed and quebracho extracts is used to demonstrate the performance on the proposed methodology. The method offers increased flexibility compared to conventional online LC×LC separations in that longer 2D gradients can be used to accommodate more complex portions of the chromatogram, while shorter 2D gradients can be used in sections containing fewer peaks, while largely maintaining the benefits of comprehensive separation. We present an evaluation of the performance of the variable gradient time stop-flow HILIC×RP-LC method compared to a comparable, conventional online HILIC×RP-LC system in terms of practical peak capacities using established 2D-LC theory. The improved separation of especially low-level intermediate molecular weight proanthocyanidin oligomers by the former method demonstrates the benefits of the developed approach.

A Closed-Loop Autologous Erythrocyte-Mediated Delivery Platform for Diabetic Nephropathy Therapy.

Failure to control blood glucose level (BGL) may aggravate oxidative stress and contribute to the development of diabetic nephropathy (DN). Using erythrocytes (ERs) as the carriers, a smart self-regulatory insulin (INS) release system was constructed to release INS according to changes in BGLs to improve patients’ compliance and health. To overcome the limited sources of ERs and decrease the risk of transmitting infections, we developed an in vitro, closed-loop autologous ER-mediated delivery (CAER) platform, based on a commercial hemodialysis instrument modified with a glucose-responsive ER-based INS delivery system (GOx-INS@ER). After the blood was drained via a jugular vein cannula, some of the blood was pumped into the CAER platform. The INS was packed inside the autologous ERs in the INS reactor, and then their surface was modified with glucose oxidase (GOx), which acts as a glucose-activated switch. In vivo, the CAER platform showed that the BGL responsively controlled INS release in order to control hyperglycemia and maintain the BGL in the normal range for up to 3 days; plus, there was good glycemic control without the added burden of hemodialysis in DN rabbits. These results demonstrate that this closed-loop extracorporeal hemodialysis platform provides a practical approach for improving diabetes management in DN patients.

Quantitative rheo-microscopy of soft matter

Direct observation of the microscopic material structure and dynamics during rheological shear tests is the goal of rheo-microscopy experiments. Microscopically, they shed light on the many mechanisms and processes that determine the mechanical properties at the macroscopic scale. Moreover, they permit for the determination of the actual deformation field, which is particularly relevant to assess shear banding or wall slip. While microscopic observation of the sample during mechanical probing is achieved by a variety of custom and commercial instruments, the possibility of performing quantitative rheology is not commonly available. Here, we describe a flexible rheo-microscopy setup that is built around a parallel-sliding-plate, stress-controlled shear cell, optimized to be mounted horizontally on a commercial microscope. Mechanically, soft materials with moduli ranging from few tens of Pa up to tens of kPa can be subjected to a variety of waveforms, ranging from standard step stress and oscillatory stress to more peculiar signals, such as triangular waves or any other signal of interest. Optically, the shear cell is designed to be compatible with different imaging methods (e.g. bright field or confocal microscopy). Most of the components of the shear cell are commercially available, and those that are not can be reproduced by a standard machine shop, easing the implementation of the rheo-microscopy setup in interested laboratories.

LunaR: Overview of a versatile Raman spectrometer for lunar exploration

The LunaR concept study investigated the scientific value, feasibility, and deployment options for a Raman spectrometer on future lunar landed missions. It consists of a breadboard instrument that covers the 150–4000 cm−1 wavelength range with a resolution of ∼6 cm−1; Raman scattering is induced by a 532 nm continuous wave laser. The current conceptual design envisions the Raman spectrometer performing a downward-looking, 90-point one-dimensional across-track scan (±45°off nadir) of the lunar surface with the instrument mounted on the underside of a rover. A downward-looking context camera would provide information on the physical nature of targets interrogated by the Raman spectrometer and localization of the Raman spectra. Our laboratory investigations indicate that Raman spectroscopy is applicable to addressing a wide range of lunar surface exploration goals related to geology, in situ resource identification, and condensed volatile detection in diverse geological terrains, including permanently shadowed regions. Testing of a breadboard and commercial instrument on lunar samples and analogues indicates that a complete spectral scan of a target of interest can be completed in ∼90 min, permitting its use on even short-duration lunar landed missions. All of the major minerals present on the Moon can be detected, and in many cases their compositions can be quantified or constrained.

Advances and challenges in the detection of academic stress and anxiety in the classroom: A literature review and recommendations.

In recent years, stress and anxiety have been identified as two of the leading causes of academic underachievement and dropout. However, there is little work on the detection of stress and anxiety in academic settings and/or its impact on the performance of undergraduate students. Moreover, there is a gap in the literature in terms of identifying any computing, information technologies, or technological platforms that help educational institutions to identify students with mental health problems. This paper aims to systematically review the literature to identify the advances, limitations, challenges, and possible lines of research for detecting academic stress and anxiety in the classroom. Forty-four recent articles on the topic of detecting stress and anxiety in academic settings were analyzed. The results show that the main tools used for detecting anxiety and stress are psychological instruments such as self-questionnaires. The second most used method is acquiring and analyzing biological signals and biomarkers using commercial measurement instruments. Data analysis is mainly performed using descriptive statistical tools and pattern recognition techniques. Specifically, physiological signals are combined with classification algorithms. The results of this method for detecting anxiety and academic stress in students are encouraging. Using physiological signals reduces some of the limitations of psychological instruments, such as response time and self-report bias. Finally, the main challenge in the detection of academic anxiety and stress is to bring detection systems into the classroom. Doing so, requires the use of non-invasive sensors and wearable systems to reduce the intrinsic stress caused by instrumentation.

Improving Accuracy in Mass Spectrometry-Based Mass Determination of Intact Heterogeneous Protein Utilizing the Universal Benefits of Charge Reduction and Alternative Gas-Phase Reactions.

In mass analysis of proteins, mass spectrometry directly measures the mass to charge ratios of ionized proteins and promises higher accuracy than that of indirect approaches measuring other physicochemical properties, provided that the charge states of detected ions are determined. Accurate mass determination of heterogeneously glycosylated proteins is often hindered by unreliable charge determination due to the insufficient resolution of signals from different charge states and inconsistency among mass profiles of ions in individual charge states. Limited charge reduction of a subpopulation of proteoforms using electron transfer/capture reactions (ETnoD/ETnoD) solves this problem by narrowing the mass distribution of examined proteoforms and preserving the mass profile of the precursor charge state in the reduced charge states. However, the limited availability of ETnoD/ETnoD function in commercial instruments limits the application of this approach. Here, utilizing a range of charge-dependent and accuracy-affecting spectral features revealed by a systematic evaluation at levels of both the ensemble and subpopulation of proteoforms based on theoretical models and experiments, we developed a limited charge reduction workflow that enables using collision-induced dissociation and higher energy collisional dissociation, two widely available reactions, as alternatives to ETnoD/ETnoD while providing adequate accuracy. Alternatively, substituting proton transfer charge reduction for ETnoD/ETnoD provides higher accuracy of mass determination. Performing mass selection in a window-sliding manner improves the accuracy and allows profiling of the whole proteoform distribution. The proposed workflow may facilitate the development of universal characterization strategies for more complex and heterogeneous protein systems.

Design of a Soft Sensor for Monitoring Phosphorous Uptake in an EBPR Process

Phosphorus (P) is a key nutrient targeted for removal by wastewater treatment, increasingly being achieved using biological processes such as enhanced biological phosphate removal (EBPR). However, commercial instrumentation for automated measurement of P is costly and provides only limited temporal resolution, constraining implementation of real-time controls in EBPR processes. This study designs a soft sensor for real-time controls using a suite of relatively low-cost sensors (ion-selective electrodes) to monitor P removal by measuring (1) secondary cations tightly coupled to bioP metabolism and (2) process bulk chemistry. Data collected from a highly instrumented lab scale reactor are used to evaluate which and how many sensors are required to achieve this goal. Machine learning (ML) approaches (support vector machines, nonlinear logistic regression, random forest, and Bayesian classification) are evaluated for sensor data fusion and coupled with a decision metric to operationalize the algorithm for reactor controls. Two key results emerge: (1) use of the slope of sensor data (mV/min) rather than raw data (mV) as the predictor signals significantly improves accuracy and resilience of the soft sensor-based system and (2) the K+ ion-selective electrode, in combination with any of the four ML algorithms, is sufficient to detect completion of P removal (within study sampling granularity of 4 min) with 100% accuracy in real time. High accuracy is maintained even as process chemistry is varied to increase the interference experienced by the sensors, indicating that this soft sensor is viable for use in real wastewater applications.

A high-throughput microplate toxicity screening platform based on Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), an established model organism, has been widely used in environmental toxicology research. However, most of the current toxicity testing methods based on worms are time-consuming. In this study we aimed to develop an automated and highly-integrated platform for high-throughput and in situ toxicity testing. Considering the superiority of C. elegans as a neurotoxicological model, this platform mainly evaluates general toxicology and neurotoxicology endpoints, which are usually induced by metals and pesticides, the major environmental contaminants. Microplates were used as a worm culturing system, which have good compatibility with any commercial microplate applicable instruments. We developed a microfluidic-based module for worm dispensing, and an image acquisition/analysis module for monitoring worms and detecting toxicity endpoints in bright filed. These were collectively incorporated with a commercial pipetting workstation for automated food/drug delivery and a high-content analysis system for fluorescence detection. The integrated platform achieved an efficient on-demand worm dispensing, long-term maintenance, regular monitoring and imaging, survival assay and behavioral analyses, and visualized gene reporter assay. Moreover, “Lab on Web” was achieved by connecting the platform to the web for remote operation, worm monitoring, and phenotype calculation. To demonstrate the ability of the platform for automated toxicity testing assays; worms were treated with cadmium and longevity, neurotoxicity, developmental toxicity and gst-4 expression were evaluated. We determined its feasibility and proposed the potential application in high-throughput toxicity screening for environmental risk assessment in the nearest future.

A miniaturized and integrated dual-channel fluorescence module for multiplex real-time PCR in the portable nucleic acid detection system.

A portable nucleic acid detection (PNAD) system based on real-time polymerase chain reaction (real-time PCR) has been developed for point-of-care testing (POCT) of infectious disease pathogens. In order to achieve “sample-in, result-out” while keeping the system compact, the hardware system integrates optical, thermal and motion control modules in a limited space for nucleic acid extraction, purification, amplification and detection. Among these hardware modules, the fluorescence module is one of the most important modules, because its performance directly affects the accuracy and sensitivity of the testing results. In this paper, a miniaturized, high-sensitivity and integrated dual-channel fluorescence module have been proposed for the homemade PNAD system. Based on the principle of confocal optical path, two group of excitation-emission optical paths of different wavelengths are integrated in a small space. In terms of circuitry, a current-light dual negative feedback light emitting diode (LED) drive circuit is applied to improve the stability of the excited light source. All optical and electronic components are integrated in a metal box of 55 mm × 45 mm × 15 mm, that helps miniaturize the detection system. Two different modules have been assembled to fit various fluorescent dyes or probes with the set of excitation and emission as follow: module 1#: 470 nm/525 nm, 570 nm/630 nm; module 2#: 520 nm/570 nm, 630 nm/690 nm. Finally, hepatitis B virus (HBV) concentration gradient detection and multiplex detection of different gene targets of SARS-CoV-2 are carried out on the PNAD system equipped with these two fluorescence modules for evaluating their performances. Compared with the commercial real-time PCR instrument, our fluorescence module has good stability and detection sensitivity.

A Low-Power IoT Device for Measuring Water Table Levels and Soil Moisture to Ease Increased Crop Yields.

The simultaneous measurement of soil water content and water table levels is of great agronomic and hydrological interest. Not only does soil moisture represent the water available for plant growth but also water table levels can affect crop productivity. Furthermore, monitoring soil saturation and water table levels is essential for an early warning of extreme rainfall situations. However, the measurement of these parameters employing commercial instruments has certain disadvantages, with a high cost of purchase and maintenance. In addition, the handling of commercial devices makes it difficult to adapt them to the specific requirements of farmers or decision-makers. Open-source IoT hardware platforms are emerging as an attractive alternative to developing flexible and low-cost devices. This paper describes the design of a datalogger device based on open-source hardware platforms to register water table levels and soil moisture data for agronomic applications. The paper begins by describing energy-saving and wireless transmission techniques. Then, it summarizes the linear calibration of the phreatimeter sensor obtained with laboratory and field data. Finally, it shows how non-linear machine-learning techniques improve predictions over classical tools for the moisture sensor (SKU: SEN0193).

Measurement of the molecular diffusion coefficient and the effective longitudinal diffusion under supercritical fluid chromatography conditions in packed bed columns

The improvement of supercritical fluid chromatography (SFC) instrumentation enhanced its reliability and utility over the past decade. The further development of high speed and high resolution separations is however obstructed by the lack of accurate models for axial dispersion in SFC. This work is a first step to tackle this by developing more reliable methods to measure molecular (Dmol) and longitudinal diffusion (Deff) in SFC, as these affect all aspects of separation efficiency. In the present contribution, we report on an improved method, to enable more flexible, reliable and accurate measurements of Dmol in SFC using commercial instrumentation. A two-column variant of the stopped-flow experiment is proposed as an adapted set-up for measuring the effective longitudinal diffusion coefficient Deff in SFC-conditions. Using the set-ups for a number of test-compounds, it has been found that Deff, and the coefficients describing its constituent sub-processes (cf. particle diffusion Dpart and surface diffusion γsDs), all vary in a linearly proportional way with the bulk diffusion coefficient Dmol within a high degree of accuracy. It has also been found that Deff decreases much more sharply with increasing retention factor compared to LC. By applying the effective medium theory, it was found that the relative surface diffusion coefficient γsDs/Dmol decreases strongly with retention factor for the investigated solutes and column, in contrary to what is typically observed in reversed phase liquid chromatography. Results indicate that this might be related to a change in retention behavior of the analytes. Obviously, more analytes and conditions need to be explored to complete this picture and the extend range of applicability of these observations.

Comparability of Steroid Collision Cross Sections Using Three Different IM-HRMS Technologies: An Interplatform Study

Steroids play key roles in various biological processes and are characterized by many isomeric variants, which makes their unambiguous identification challenging. Ion mobility-mass spectrometry (IM-MS) has been proposed as a suitable platform for this application, particularly using collision cross section (CCS) databases obtained from different commercial IM-MS instruments. CCS is seen as an ideal additional identification parameter for steroids as long-term repeatability and interlaboratory reproducibility of this measurand are excellent and matrix effects are negligible. While excellent results were demonstrated for individual IM-MS technologies, a systematic comparison of CCS derived from all major commercial IM-MS technologies has not been performed. To address this gap, a comprehensive interlaboratory comparison of 142 CCS values derived from drift tube (DTIM-MS), traveling wave (TWIM-MS), and trapped ion mobility (TIM-MS) platforms using a set of 87 steroids was undertaken. Besides delivering three instrument-specific CCS databases, systematic comparisons revealed excellent interlaboratory performance for 95% of the ions with CCS biases within ±1% for TIM-MS and within ±2% for TWIM-MS with respect to DTIM-MS values. However, a small fraction of ions (<1.5%) showed larger biases of up to 7% indicating that differences in the ion conformation sampled on different instrument types need to be further investigated. Systematic differences between CCS derived from different IM-MS analyzers and implications on the applicability for nontargeted analysis are critically discussed. To the best of our knowledge, this is the most comprehensive interlaboratory study comparing CCS from three different IM-MS technologies for analysis of steroids and small molecules in general.

Picomolar detection limits for glyphosate by two‐dimensional column‐coupled isotachophoresis/capillary zone electrophoresis‐mass spectrometry

Capillary electrophoresis-mass spectrometry often lacks sufficient limits of detection for trace substances in the environment due to its low loadability. To overcome this problem, we conducted a feasibility study for column-coupling isotachophoresis to capillary electrophoresis-mass spectrometry. The first dimension isotachophoresis preconcentrated the analytes. The column-coupling of both dimensions was achieved by a hybrid capillary microfluidic chip setup. Reliable analyte transfer by voltage switching was enabled by an in-chip capacitively coupled contactless conductivity detector placed around the channel of the common section between two T-shaped crossings in the chip connecting both dimensions. This eliminated the need to calculate the moment of analyte transfer. A commercial capillary electrophoresis-mass spectrometry instrument with easily installable adaptations operated the setup. Prior to coupling isotachophoresis with capillary zone electrophoresis-mass spectrometry, both dimensions were optimized individually by simulations and verified experimentally. Both dimensions were able to stack/separate all degradation products of glyphosate, the most important herbicide applied worldwide. The first dimension isotachophoresis also removed phosphate, which is a critical matrix component in many environmental samples. Enrichment and separation of glyphosate and its main degradation product aminomethylphosphonic acid by the two-dimensional setup provided an excellent limit of detection of 150pM (25ng/L) for glyphosate.

MINI: A high-throughput point-of-care device for performing hundreds of nucleic acid tests per day

There are a variety of infectious diseases with a high incidence and mortality in limited resource settings that could benefit from rapid point of care molecular diagnosis. Global health efforts have sought to implement mass-screening programs to provide earlier detection and subsequent treatment in an effort to control transmission and improve health outcomes. However, many of the current diagnostic technologies under development are limited to fewer than 10 samples per run, which inherently restricts the screening throughput of these devices. We have developed a high throughput device called “MINI” that is capable of testing hundreds of samples per day at the point-of-care. MINI can utilize multiple energy sources – electricity, flame, or solar – to perform loop-mediated isothermal amplification (LAMP) in a portable and robust device which is ideal for use in limited resource settings. The unique opto-electronic design of MINI minimizes the energy and space requirements of the device and maximizes the optical isolation and signal clarity, enabling point-of-care analysis of 96 unique samples at once. We show comparable performance to a commercial instrument using two different LAMP assays for Kaposi's sarcoma-associated herpesvirus and a common housekeeping gene, GAPDH. With a single device capable of running hundreds of samples per day, increased access to modern molecular diagnostics could improve health outcomes for a variety of diseases common in limited resource settings.

Skin Sensory Assessors Highly Agree on the Appraisal of Skin Smoothness and Elasticity but Fairly on Softness and Moisturization

We tested the reliability of sensory evaluations of tactile sensation on bare skin and investigated the reliability among evaluation attributes by trained and untrained assessors. Two trained professional panelists and two untrained researchers evaluated skin in terms of several attributes: smooth–rough, elastic–not elastic, soft–hard (surface), soft–hard (base), moisturized–dry. Twenty-two women aged 25–57 years were evaluated, and the sensory evaluation was repeated twice. Correlation coefficients and intraclass correlation coefficients (ICCs) were used to examine intra- and inter-assessor reliability. The sensory evaluation and physical quantities acquired by commercial and non-commercial instruments were moderately correlated. Smooth–rough and elastic–not elastic showed high or moderate inter-assessor reliabilities with mean correlation coefficients between panelists of 0.81 and 0.58, respectively. Further, the ICC (2,1) values were 0.64 and 0.51, respectively, and the ICC (2,2) values were 0.77 and 0.67, respectively. Conversely, the reliabilities of soft–hard (surface), soft–hard (base), and moisturized–dry were low; the mean correlation coefficients between the panelists were 0.36, 0.23, and 0.22; the ICC (2,1) values were 0.27, 0.23, and 0.17; and the ICC (2,2) values were 0.42, 0.29, and 0.26, respectively. Reliability differed between attributes. We found no meaningful differences between the trained and untrained panelists regarding intra- or inter-assessor reliability.

Optical time domain backscattering of antiresonant hollow core fibers.

Today's lowest-loss hollow core fibers are based on antiresonance guidance. They have been shown both theoretically and experimentally to have very low levels of backscattering arising from the fiber structure - 45 dB below that of traditional optical fibers with a solid silica glass core. This makes their longitudinal characterization using conventional reflectometric techniques very challenging. However, it was recently estimated that when filled with air, their backscattering coefficient increases to about 30 dB below that of standard solid core fibers. This level should be measurable with commercially available high performance optical time domain reflectometers (OTDR). Here we demonstrate - for the first time to the best of our knowledge - the measurement of backscattering from the air inside a hollow core fiber. We show that the characterization of multi-km long hollow core fibers with 15 m spatial resolution is possible using a commercial OTDR instrument. To benefit from its full dynamic range, we strongly suppress the 4% back-reflections that ordinarily occur at the OTDR's standard fiber output when directly-connected to a hollow core fiber. Furthermore, low coupling loss into the hollow core fiber (0.3 dB in our experiment) also helps to maximize the achievable OTDR signal-to-noise ratio. This approach enables distributed characterization and fault-finding in low-loss hollow core fibers, a topic of increasing importance as these fibers are now starting to be installed in commercial optical communication networks.

Identifying the physical mechanisms of polycrystalline lead salt photoconductors

Polycrystalline infrared lead salt detectors exhibit exceptionally high sensitivity near room temperature and, as such, are ubiquitous in commercial instruments such as spectrometers and flame detectors. The underlying physical mechanism behind this remarkable performance has been the subject of speculation for many years. In this treatise, data from work performed at BAE Systems and St. John's Optical Systems and from the published literature are analyzed to identify common mechanisms that contribute to the photoconductivity of polycrystalline lead salt detectors from a multitude of material deposition and process technologies, and it was concluded that much of the behavior can be attributed to surface-related phenomenon. Physical models are proposed to explain the observations. Finally, there is a brief description of measurements related to noise in these complex structures, indicating the importance of ohmic contacts, but the observed low frequency spectral noise density remains a mystery.

The First Online Capillary Electrophoresis-Microscale Thermophoresis (CE-MST) Method for the Analysis of Dynamic Equilibria-The Determination of the Acidity Constant of Fluorescein Isothiocyanate.

This article presents the first successful application of a capillary electrophoresis-microscale thermophoresis tandem technique (CE-MST) for determining the values of equilibrium constant, realized by connecting online the CE and MST instruments using a fused-silica capillary. The acid-base dissociation of fluorescein isothiocyanate, expressed by the acidity constant value (pKa), was used as a model. The measurement procedure consisted of introducing a mixture containing the analyte and a deliberately added interferent into the CE capillary, electrophoretic separation of the analyte from the interferent, the detection of the analyte with a CE-integrated detector, detection with a MST detector, and then stopping the flow temporarily by turning off the voltage source to conduct the thermophoretic measurement. The analysis of migration times, peak areas and MST responses obtained concurrently for the same sample allowed us to determine the pKa value using three independent methods integrated within one instrumentation. The analyte was effectively separated from the interferent, and the acidity values turned out to be consistent with each other. An attempt was also made to replace the standard commercial CE instrument with a home-made portable CE setup. As a result, the similar pKa value was obtained, at the same time proving the possibility of increasing cost efficiency and reducing energy consumption. Overall, the CE-MST technique has a number of limitations, but its unique analytical capabilities may be beneficial for some applications, especially when sample separation is needed prior to the thermophoretic measurement.