Moderate Signal Enhancement in Electrospray Ionization Mass Spectrometry by Focusing Electrospray Plume with a Dielectric Layer around the Mass Spectrometer's Orifice.

Electrospray ionization (ESI) is among the commonly used atmospheric pressure ionization techniques in mass spectrometry (MS). One of the drawbacks of ESI is the formation of divergent plumes composed of polydisperse microdroplets, which lead to low transmission efficiency. Here, we propose a new method to potentially improve the transmission efficiency of ESI, which does not require additional electrical components and complex interface modification. A dielectric plate-made of ceramic-was used in place of a regular metallic sampling cone. Due to the charge accumulation on the dielectric surface, the dielectric layer around the MS orifice distorts the electric field, focusing the charged electrospray cloud towards the MS inlet. The concept was first verified using charge measurement on the dielectric material surface and computational simulation; then, online experiments were carried out to demonstrate the potential of this method in MS applications. In the online experiment, signal enhancements were observed for dielectric plates with different geometries, distances of the electrospray needle axis from the MS inlet, and various compounds. For example, in the case of acetaminophen (15 µM), the signal enhancement was up to 1.82 times (plate B) using the default distance of the electrospray needle axis from the MS inlet (d = 1.5 mm) and 12.18 times (plate C) using a longer distance (d = 7 mm).

Mass Specthoscope: A Hand-held Extendable Probe for Localized Noninvasive Sampling of Skin Volatome for Online Analysis.

Human skin emits a unique set of volatile organic compounds (VOCs). These VOCs can be probed in order to obtain physiological information about the individuals. However, extracting the VOCs that emanate from human skin for analysis is troublesome and time-consuming. Therefore, we have developed "Mass Specthoscope"─a convenient tool for rapid sampling and detecting VOCs emitted by human skin. The hand-held probe with a pressurized tip and wireless button enables sampling VOCs from surfaces and their transfer to the atmospheric pressure chemical ionization source of quadrupole time-of-flight mass spectrometer. The system was characterized using chemical standards (acetone, benzaldehyde, sulcatone, α-pinene, and decanal). The limits of detection are in the range from 2.25 × 10-5 to 3.79 × 10-5 mol m-2. The system was initially tested by detecting VOCs emanating from porcine skin spiked with VOCs as well as unspiked fresh and spoiled ham. In the main test, the skin of nine healthy participants was probed with the Mass Specthoscope. The sampling regions included the armpit, forearm, and forehead. Numerous skin-related VOC signals were detected. In the final test, one participant ingested a fenugreek drink, and the participant's skin surface was probed using the Mass Specthoscope hourly during the 8 h period. The result revealed a gradual release of fenugreek-related VOCs from the skin. We believe that this analytical approach has the potential to be used in metabolomic studies and following further identification of disease biomarkers─also in noninvasive diagnostics.

Are Most Micrometer Droplets (>10 µm) Wasted in Electrospray Ionization? An Insight from Real-Time High-Speed Imaging.

Electrospray ionization (ESI) is one of the main techniques used in mass spectrometry (MS) of nonvolatile compounds. ESI is a disordered process, in which a large number of polydisperse droplets are projected from a fluctuating Taylor cone and jet protruding ESI emitter. Here, we disclose a system for sectioning electrospray plumes to discrete packets with millisecond and submillisecond lifetime, which are introduced to the MS orifice, one at a time. A high-speed camera was triggered at 10,000 frames per second to capture consecutive images of the electrospray packets transmitted to the mass spectrometer. We further correlated the high-speed images of electrospray packets with MS signals of a test analyte (acetaminophen). Following computational treatment of the images, we determined the number of droplet observations (<300), average diameter of droplets (∼10-20 µm), and average volume of droplets (few tens of picoliters) in the individual electrospray packets. The result shows that most micrometer droplets (>10 µm) do not have any significant contribution to the MS signals. This finding is in agreement with the prior conjecture that most of the MS signals are mainly attributed to nanodroplets. Based on this finding, one can deduce that only a small number of the initial microdroplets effectively carry analyte molecules that undergo ionization. We discuss that, in future, one may propose a way to "recharge" the emitted initial micrometer droplets to increase the efficiency of conventional ESI setups.

Scanning pH-metry for Observing Reversibility in Protein Folding.

One of the main factors affecting protein structure in solution is pH. Traditionally, to study pH-dependent conformational changes in proteins, the concentration of the H+ ions is adjusted manually, complicating real-time analyses, hampering dynamic pH regulation, and consequently leading to a limited number of tested pH levels. Here, we present a programmable device, a scanning pH-meter, that can automatically generate different types of pH ramps and waveforms in a solution. A feedback loop algorithm calculates the required flow rates of the acid/base titrants, allowing one, for example, to generate periodic pH sine waveforms to study the reversibility of protein folding by fluorescence spectroscopy. Interestingly, for some proteins, the fluorescence intensity profiles recorded in such a periodically oscillating pH environment display hysteretic behavior indicating an asymmetry in the sequence of the protein unfolding/refolding events, which can most likely be attributed to their distinct kinetics. Another useful application of the scanning pH-meter concerns coupling it with an electrospray ionization mass spectrometer to observe pH-induced structural changes in proteins as revealed by their varying charge-state distributions. We anticipate a broad range of applications of the scanning pH-meter developed here, including protein folding studies, determination of the optimum pH for achieving maximum fluorescence intensity, and characterization of fluorescent dyes and other synthetic materials.

Elevating Chemistry Research with a Modern Electronics Toolkit.

With the rapid development of high technology, chemical science is not as it used to be a century ago. Many chemists acquire and utilize skills that are well beyond the traditional definition of chemistry. The digital age has transformed chemistry laboratories. One aspect of this transformation is the progressing implementation of electronics and computer science in chemistry research. In the past decade, numerous chemistry-oriented studies have benefited from the implementation of electronic modules, including microcontroller boards (MCBs), single-board computers (SBCs), professional grade control and data acquisition systems, as well as field-programmable gate arrays (FPGAs). In particular, MCBs and SBCs provide good value for money. The application areas for electronic modules in chemistry research include construction of simple detection systems based on spectrophotometry and spectrofluorometry principles, customizing laboratory devices for automation of common laboratory practices, control of reaction systems (batch- and flow-based), extraction systems, chromatographic and electrophoretic systems, microfluidic systems (classical and nonclassical), custom-built polymerase chain reaction devices, gas-phase analyte detection systems, chemical robots and drones, construction of FPGA-based imaging systems, and the Internet-of-Chemical-Things. The technology is easy to handle, and many chemists have managed to train themselves in its implementation. The only major obstacle in its implementation is probably one's imagination.

Robotized Noncontact Open-Space Mapping of Volatile Organic Compounds Emanating from Solid Specimens.

Analysis of volatile organic compounds (VOCs) is normally preceded by sample homogenization and solvent extraction. This methodology does not provide spatial resolution of the analyzed VOCs in the examined matrix. Here, we present a robotized pen-shaped probe for open-space sampling and mapping of VOCs emanating from solid specimens (dubbed "PENVOC"). The system combines vacuum-assisted suction probe, mass spectrometry, and robotic handling of the probe. The VOCs are scavenged from the sample surface by a gentle hydrodynamic flow of air sustained by a vacuum pump. The sampled gas is transferred to the proximity of corona discharge in an atmospheric pressure chemical ionization source of a tandem mass spectrometer. The PENVOC has been attached to a robotic arm to enable unattended scanning of flat surfaces. The specimens can be placed away from the mass spectrometer during the scan. The robotized PENVOC has been characterized using chemical standards (benzaldehyde, limonene, 2-nonanone, and ethyl octanoate). The limits of detection are in the range from 2.33 × 10-5 to 2.68 × 10-4 mol m-2. The platform has further been used for mapping of VOCs emanating from a variety of specimens: flowers, glove exposed to smoke, fuel stains, worn medical face mask, worn clothing, cheese, ham, and fruits. The chemical maps show unique distributions of the VOCs on the scanned surfaces. Obtaining comparable results (VOC maps) using other techniques (e.g., repetitive headspace sampling prior to offline analysis) would be time-consuming. The presented mapping technique may find applications in environmental, forensic, and food science.

Portable Pen-Probe Analyzer Based on Ion Mobility Spectrometry for in Situ Analysis of Volatile Organic Compounds Emanating from Surfaces and Wireless Transmission of the Acquired Spectra.

The analysis of volatile organic compounds (VOCs) normally involves sample collection, sample transfer to laboratory, sample preparation, and the chromatographic separation of analytes. However, in some cases, it is impractical or impossible to collect samples prior to the analysis, while the analysis time has to be minimized. Ion mobility spectrometry (IMS) is an ideal technique for a rapid in situ chemical analysis. Here, we present a portable cloud-integrated pen-probe analyzer based on IMS and demonstrate its applications in the analysis of VOCs emanating from surfaces. The user approaches the pen-probe to a sampled surface and presses a button on the pen-probe. The analysis is then executed automatically. The VOCs are scavenged from the surface by a suction force and directed to a corona discharge atmospheric pressure chemical ionization source. The ions are separated in a drift tube according to their size and charge and then detected by a Faraday plate detector. The detector signal is amplified and digitized. The spectral data are deposited in the Internet cloud along with time and location data for further retrieval and processing. The platform incorporates a mobile Wi-Fi router for easy connectivity and a global positioning system module for geolocation. The prototype was developed using low-cost electronic modules (Arduino, Tinker Board S). It was further characterized using chemical standards. The limits of detection for pyrrolidine, 2,4-lutidine, and (-)-nicotine are 48.9, 2.30, and 416 nmol, respectively (amounts of substances placed on the sampling surface). The selected real specimens (nicotine patch, skin exposed to nicotine, fish sauce, and fried chicken) were also subjected to analysis yielding the characteristic ion mobility spectra.

Catalytic Oxygenation-Mediated Extraction as a Facile and Green Way to Analyze Volatile Solutes.

Sparging-based methods have long been used to liberate volatile organic compounds (VOCs) from liquid sample matrices prior to analysis. In these methods, a carrier gas is delivered from an external source. Here, we demonstrate "catalytic oxygenation-mediated extraction" (COME), which relies on biocatalytic production of oxygen occurring directly in the sample matrix. The newly formed oxygen (micro)bubbles extract the dissolved VOCs. The gaseous extract is immediately transferred to a separation or detection system for analysis. To start COME, dilute hydrogen peroxide is injected into the sample supplemented with catalase enzyme. The entire procedure is performed automatically-after pressing a "start" button, making a clapping sound, or triggering from a smartphone. The pump, valves, and detection system are controlled by a microcontroller board. For quality control and safety purposes, the reaction chamber is monitored by a camera linked to a single-board computer, which follows the enzymatic reaction progress by analyzing images of foam in real time. The data are instantly uploaded to the internet cloud for retrieval. The COME apparatus has been coupled on-line with the gas chromatography electron ionization mass spectrometry (MS) system, atmospheric pressure chemical ionization (APCI) MS system, and APCI ion-mobility spectrometry system. The three hyphenated variants have been tested in analyses of complex matrices (e.g., fruit-based drinks, whiskey, urine, and stored wastewater). In addition to the use of catalase, COME variants using crude potato pulp or manganese(IV) dioxide have been demonstrated. The technique is inexpensive, fast, reliable, and green: it uses low-toxicity chemicals and emits oxygen.

Please Avoid Plotting Analytical Response against Logarithm of Concentration.

This perspective highlights a malpractice in handling calibration data sets. It refers to relating analytical response with logarithm of concentration, fitting such a data set with linear function, and deriving method characteristics from the slope of that function. A distinction is made between this malpractice and the proper use of logarithmic plots in representation of calibration data sets.

Rapid Extraction and Analysis of Volatile Solutes with an Effervescent Tablet.

Extraction of volatile compounds from complex liquid matrices is a critical step in volatile compound analysis workflows. Recently, green chemistry principles are increasingly implemented in extraction processes. Some of the available approaches are solvent-free but still require concentration or trapping of analytes. Here, we propose effervescent tablet-induced extraction (ETIE) as a method of transferring volatile/semivolatile compounds from liquid matrices to the gas phase for analysis. This technique relies on the release of carbon dioxide produced in situ during a neutralization reaction, which occurs when a tablet is inserted into an aqueous sample matrix. In this process, many bubbles of carbon dioxide are instantly formed in the sample matrix. The bubbles rapidly extract and liberate volatile compounds from the sample. The gaseous effluent is then immediately transferred to a detector (atmospheric pressure chemical ionization mass spectrometry (MS) or gas chromatography (GC) hyphenated with MS). ETIE-GC-MS can be used for analysis of volatile compounds present in real samples. The method was validated for analysis of selected ethyl esters present in a yogurt drink. The calibration data set was linear over a range from 5 × 10-7 to 1 × 10-5 M. The limits of detection ranged from 1.51 × 10-7 to 6.82 × 10-7 M, while the recoveries ranged from 71 to 118%. Inter- and intraday precision of selected ethyl esters in aqueous solution was satisfactory (relative standard deviation, 3.6-18.3%). Furthermore, it is shown that ETIE improves the performance of headspace solid-phase microextraction while eliminating the need for heating and shaking samples.

On-Line Coupling of Simultaneous Distillation-Extraction Using the Likens-Nickerson Apparatus with Gas Chromatography.

Simultaneous distillation-extraction (SDE) using the Likens-Nickerson apparatus is a convenient technique used to isolate volatile organic compounds (VOCs) from complex liquid matrices. The technique combines steam distillation with solvent extraction. While analytical extractions are normally followed by off-line separation/detection, it is advantageous to couple extractions on-line with separation and detection systems that are employed in the same analytical workflow. Here, we have coupled the Likens-Nickerson apparatus on-line with a gas chromatograph hyphenated with a mass spectrometer. For that purpose, we have devised an automated liquid transfer setup comprising a peristaltic pump, control unit, customized transfer vial with a drain port, and an autosampler arm to deliver liquid extract aliquots at defined time points. The on-line SDE-GC/MS system enables one to record real-time extraction profiles. These profiles reveal extraction kinetics of various VOCs present in the extracted samples. The data sets were fitted with the first order kinetic equation to obtain numeric values characterizing the extraction process (rate constants ranging from 0.21 to 0.01 min-1 for the ethyl esters from C6 to C19). A comparison of on-line and off-line results reveals that the on-line system is more dependable, while the off-line analysis leads to artifacts. To demonstrate the operation of the on-line SDE-GC/MS system, we performed analyses of selected real samples (beer). The real-time data sets revealed extraction kinetics for VOCs present in these samples. The devised extraction-analysis system allows the analysts to make an evidence-based decision on the extraction time for different groups of analytes in order to maximize extraction yield and minimize analyte losses.

Sample Flow Rate Scan in Electrospray Ionization Mass Spectrometry Reveals Alterations in Protein Charge State Distribution.

Sample flow rate is one of the parameters that influence the sensitivity of electrospray ionization (ESI) mass spectrometry. By varying the sample flow rate, initial droplets of different sizes can be generated. Protein molecules in small droplets may form gas-phase ions earlier than the ones in large droplets. Here, we have systematically studied the influence of sample flow rate on the ESI charge state distributions (CSDs) of model proteins. A dedicated programmable sample flow rate scanner was used to infuse protein samples at different flow rates into a mass spectrometer. The synergistic influence of sample flow rate and various electrolytes (ammonium acetate, ammonium bicarbonate, ammonium formate, and piperidine) was studied. Significant alterations to the CSDs with increasing flow rate were observed. For example, in the presence of ammonium acetate, at low flow rates, lower charge states of proteins showed high intensities, while at high flow rates, ions related to higher charge states of proteins dominated the spectra. On the other hand, in the presence of piperidine, a significant reduction in the ion currents of all charge states was observed during the flow rate scan. Our observations suggest that at low flow rates the protein molecules follow a charged residue model of ionization mechanism, and at high flow rates-due to structural changes in protein molecules in large ESI droplets-the charged residue and chain ejection models can possibly coexist. We propose the use of sample flow rate scan as a way to reveal the influence of flow rate on the CSDs of the studied proteins.

Temporal Analysis of Conformers in the Course of pH Scan Directed by Urea-Urease Reaction-A "Protein Clock".

We demonstrate an analytical approach to monitor the effect of pH on protein tertiary structure. An autocatalytic enzyme reaction is used to stimulate refolding of proteins during real-time analysis. The method takes advantage of a nonlinear pH ramp generated by the urea-urease clock reaction. In this study, alterations to the structures of model proteins were monitored by mass spectrometry (charge pattern shift) and fluorometry (tryptophan fluorescence quenching). The pH measurements were conducted at different points of the sample flow line by different methods to minimize artifacts. Interestingly, different protein ions (corresponding to native and unfolded proteins) show distinct temporal mass spectral profiles, which reveal gradual refolding and concomitant deprotonation of higher charge state ions in the course of the clock reaction. Every multiply charged ion of a protein is characterized with its own "clock time". This approach does not require major modification of standard instrumentation. It enables determination of "high sensitivity" pH intervals for small and large molecules within a single experiment. Thus, it can be useful for characterizing the protein folding in response to pH change.

On-line coupling of fizzy extraction with gas chromatography.

Fizzy extraction (FE) is carried out by first dissolving a carrier gas (typically, carbon dioxide) in a liquid sample at a moderate pressure (typically, 150 kPa) and then rapidly depressurizing the sample. The depressurization leads to instant release of numerous microbubbles in the liquid matrix. The abruptly released gas extracts the volatile solutes and elutes them toward a detector in a short period of time. Here, we describe on-line coupling of FE with gas chromatography (GC). The two platforms are highly compatible and could be combined following several modifications of the interface and adjustments of the extraction sequence. The analytes are released within a short period of time (1.5 s). Thus, the chromatographic peaks are satisfactorily narrow. There is no need to trap the extracted analytes in a loop or on a sorbent, as it is done in standard headspace and microextraction methods. The approach requires only minor sample pretreatment. The main parameters of the FE-GC-mass spectrometry (MS) method were optimized. The results of FE were compared with those of headspace flushing (scavenging headspace vapors), and the enhancement factors were in the order of ~ 2 to 13 (for various analytes). The limits of detection for some of the tested analytes were lower in the proposed FE-GC-MS method than in FE combined with atmospheric pressure chemical ionization MS. The method was further tested in analyses of selected real samples (apple flavor milk, mixed fruit and vegetable juice drink, mango flavored drink, pineapple green tea, toothpaste, and yogurt). Graphical abstract.

Automated Dual-Chamber Sampling System to Follow Dynamics of Volatile Organic Compounds Emitted by Biological Specimens.

Growth of microorganisms is often accompanied by the release of volatile organic compounds (VOCs). The released VOCs may qualitatively or quantitatively reflect the physiological states of microbial cultures. A number of VOCs are produced during microbial degradation of organic matter accompanying food spoilage. In order to characterize the dynamics of microbial VOC production, and enable time-dependent analysis, we have constructed a dual-chamber sampling system and coupled it online with mass spectrometry (MS). The biological specimen is placed in a gas-tight sampling chamber. A carrier gas is introduced to the chamber periodically to transfer the VOCs present in the specimen headspace to the atmospheric-pressure chemical-ionization interface of a triple-quadrupole mass spectrometer. A control or blank spectrum is recorded before the specimen spectrum is recorded at each time point, enabling signal comparison and subtraction. The custom-made electronic control unit, incorporating three Arduino microcontrollers, operates six pinch valves and a miniature air compressor and triggers MS-data acquisition. The automated dual-chamber sampling system was first tested using standard mixtures to verify its analytical performance. To demonstrate the usefulness of this system in the studies of microbial volatomes, we implemented it in real-time monitoring of the growth of baker's yeast ( Saccharomyces cerevisiae), fructification of golden oyster mushroom ( Pleurotus citrinopileatus), and microbial degradation of a food-related sample (Pacific white shrimp, Litopenaeus vannamei). The recorded VOC signals show characteristic temporal profiles with transient bands or plateaus corresponding to different stages of microbial growth and putrefaction processes.

Chemical clocks, oscillations, and other temporal effects in analytical chemistry: oddity or viable approach?

Most analytical methods are based on "analogue" inputs from sensors of light, electric potentials, or currents. The signals obtained by such sensors are processed using certain calibration functions to determine concentrations of the target analytes. The signal readouts are normally done after an optimised and fixed time period, during which an assay mixture is incubated. This minireview covers another-and somewhat unusual-analytical strategy, which relies on the measurement of time interval between the occurrences of two distinguishable states in the assay reaction. These states manifest themselves via abrupt changes in the properties of the assay mixture (e.g. change of colour, appearance or disappearance of luminescence, change in pH, variations in optical activity or mechanical properties). In some cases, a correlation between the time of appearance/disappearance of a given property and the analyte concentration can be also observed. An example of an assay based on time measurement is an oscillating reaction, in which the period of oscillations is linked to the concentration of the target analyte. A number of chemo-chronometric assays, relying on the existing (bio)transformations or artificially designed reactions, were disclosed in the past few years. They are very attractive from the fundamental point of view but-so far-only few of them have be validated and used to address real-world problems. Then, can chemo-chronometric assays become a practical tool for chemical analysis? Is there a need for further development of such assays? We are aiming to answer these questions.

Prototyping Instruments for the Chemical Laboratory Using Inexpensive Electronic Modules.

Open-source electronics and programming can augment chemical and biomedical research. Currently, chemists can choose from a broad range of low-cost universal electronic modules (microcontroller boards and single-board computers) and use them to assemble working prototypes of scientific tools to address specific experimental problems and to support daily research work. The learning time can be as short as a few hours, and the required budget is often as low as 50 USD. Prototyping instruments using low-cost electronic modules gives chemists enormous flexibility to design and construct customized instrumentation, which can reduce the delays caused by limited access to high-end commercial platforms.

Probing Skin for Metabolites and Topical Drugs with Hydrogel Micropatches.

Sampling the skin surface is a convenient way to obtain biological specimens bearing clinically relevant information. Hydrogel micropatches enable noninvasive collection of skin excretion specimens, which can subsequently be subjected to rapid mass spectrometric analysis providing insights into the skin metabolome.

Spontaneous luminescence color change in the firefly luciferase assay system.

The temporal effects of luciferase reaction luminescence have only been discussed in the context of light intensity (flash vs. glow). However, alterations in the color of the light emitted over the course of the luciferase reaction have not been reported. Here, we show a temporal change in the light color emitted during the reaction catalyzed by unmodified firefly luciferase when concentrations of one of the substrates, adenosine triphosphate (ATP), are gradually increased. The temporal color change from green to red occurs within the first few minutes of the luciferase reaction when an ATP-containing solution is either added or synthesized in situ with the aid of an autocatalytic reaction occurring simultaneously. This color change is not accompanied by pH changes. An analysis of the red and green channels demonstrates dissimilar kinetics, suggesting the co-existence of two or more temporally shifted luminescence pathways. The implications of these findings might improve dual-color biosensing/imaging protocols and influence the engineering of biophotonic systems.