Optimization of the design and operation of FAIMS analyzers

Field asymmetric waveform ion mobility spectrometry (FAIMS) holds significant promise for post-ionization separations in conjunction with mass-spectrometric analyses. However, a limited understanding of fundamentals of FAIMS analyzers has made their design and operation largely an empirical exercise. Recently, we developed an a priori simulation of FAIMS that accounts for both ion diffusion (including anisotropic components) and Coulomb repulsion, and validated it by extensive comparisons with FAIMS/MS data. Here it is corroborated further by FAIMS-only measurements, and applied to explore how key instrumental parameters (analytical gap width and length, waveform frequency and profile, the identity and flow speed of buffer gas) affect FAIMS response. We find that the trade-off between resolution and sensitivity can be managed by varying gap width, RF frequency, and (in certain cases) buffer gas, with equivalent outcome. In particular, the resolving power can be approximately doubled compared to "typical" conditions. Throughput may be increased by either accelerating the gas flow (preferable) or shortening the device, but below certain minimum residence times performance deteriorates. Bisinusoidal and clipped-sinusoidal waveforms have comparable merit, but switching to rectangular waveforms would improve resolution and/or sensitivity. For any waveform profile, the ratio of two between voltages in high and low portions of the cycle produces the best performance.     

Modeling the resolution and sensitivity of FAIMS analyses

Field asymmetric waveform ion mobility spectrometry (FAIMS) is rapidly gaining acceptance as a robust, versatile tool for post-ionization separations prior to mass-spectrometric analyses. The separation is based on differences between ion mobilities at high and low electric fields, and proceeds at atmospheric pressure. Two major advantages of FAIMS over condensed-phase separations are its high speed and an ion focusing effect that often improves sensitivity. While selected aspects of FAIMS performance are understood empirically, no physical model rationalizing the resolving power and sensitivity of the method and revealing their dependence on instrumental variables has existed. Here we present a first-principles computational treatment capable of simulating the FAIMS analyzer for virtually any geometry (including the known cylindrical and planar designs) and arbitrary operational parameters. The approach involves propagating an ensemble of ion trajectories through the device in real time under the influence of applied asymmetric potential, diffusional motion incorporating the high-field and anisotropic phenomena, and mutual Coulomb repulsion of ionic charges. Calculations for both resolution and sensitivity are validated by excellent agreement with measurements in different FAIMS modes for ions representing diverse types and analyte classes.     

Expanded theory for the resolving power of a linear ion mobility spectrometer

An expanded theory for the resolving power of a linear ion mobility spectrometer (IMS) is derived. By definition, the resolving power is directly proportional to the total drift time for the ion through the drift tube divided by the full-width-at-half-height (FWHH) of the observed ion mobility peak. Two approaches to theoretically estimating these two parameters are possible, depending on the operating parameters of the IMS cell. The drift time is given by the first moment of the IMS response. If the electric fields (assumed uniform) are equal in both the shutter/aperture and aperture/collector region, the FWHH is given by a difference in error functions. If the electric fields (again assumed uniform) are not equal, the FWHH is given by the second central moment of the IMS response and can only be known to within a multiplicative factor. The effectiveness of these two approaches is demonstrated using IMS data from the published literature.The additional peak broadening often observed in a linear IMS has several possible sources. One depends on the construction of the cell and the parallelism (or lack thereof) that might exist between the aperture grid and ion collector. Another depends on electric fields used to bias the cell. If the electric field in the aperture/collector region is less than in the shutter/aperture region, peak broadening occurs. Induction effects in the aperture/collector region not only shorten drift times, but also create diffusion-like broadening of the peak. Shortening the distance between the aperture grid and ion collector, or using a higher electric field in that region, minimizes induction effects. Drift time calibration requires adjustments for induction effects.     

Scaling factors for activated corrosion products in low-level-wastes from power reactors

Long-lived isotopes of corrosion products are very important for the disposal of low-level radioactive wastes. These nuclides serve for risk calculations of accidents. Their determination needs the radiochemical separation from high active main nuclides. Supervision of waste vessels is done by direct non-destructive -spectrometry of the key nuclide⁶⁰Co for corrosion products. The activity ratios of the long-lived nuclides to the key nuclides are called scaling factors. We have determined such factors radiochemically in evaporation residues of power water and cooling water. They are used for activity calculations of long-lived nuclides in the waste vessels. In case of⁵⁹Ni the obtained scaling factor was compared with a literature value and values calculated on the basis of nuclear data. Our value was in a good agreement with the calculated one. Due to the fact, that we have used low-level measurement techniques, we could perform the necessary chemical separations in a laboratory not exceeding the 10-fold free-level limit.     

Are we using the full resolving power of capillary GC?

Precise evaluation of the chromatographic data by means of the relative retention will allow the full use of the resolution achieved by capillary columns. In practice, retention-index determination is best suited for this purpose. The reproducibility of retention-index determination strongly depends, among other factors, on the accuracy of retention time measurement. A reproducibility of ±0,05 i.u. can be easily achieved with modern gas chromatographic equipment, provided the retention times can be measured with an accuracy of at least ±0.01%. This can be shown by error propagation analysis of retention-index calculation performed with the aid of a programmable pocket calculator.     

Probing the Complementarity of FAIMS and Strong Cation Exchange Chromatography in Shotgun Proteomics

High field asymmetric waveform ion mobility spectrometry (FAIMS), also known as differential ion mobility spectrometry, coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) offers benefits for the analysis of complex proteomics samples. Advantages include increased dynamic range, increased signal-to-noise, and reduced interference from ions of similar m/z. FAIMS also separates isomers and positional variants. An alternative, and more established, method of reducing sample complexity is prefractionation by use of strong cation exchange chromatography. Here, we have compared SCX-LC-MS/MS with LC-FAIMS-MS/MS for the identification of peptides and proteins from whole cell lysates from the breast carcinoma SUM52 cell line. Two FAIMS approaches are considered: (1) multiple compensation voltages within a single LC-MS/MS analysis (internal stepping) and (2) repeat LC-MS/MS analyses at different and fixed compensation voltages (external stepping). We also consider the consequence of the fragmentation method (electron transfer dissociation or collision-induced dissociation) on the workflow performance. The external stepping approach resulted in a greater number of protein and peptide identifications than the internal stepping approach for both ETD and CID MS/MS, suggesting that this should be the method of choice for FAIMS proteomics experiments. The overlap in protein identifications from the SCX method and the external FAIMS method was ~25 % for both ETD and CID, and for peptides was less than 20 %. The lack of overlap between FAIMS and SCX highlights the complementarity of the two techniques. Charge state analysis of the peptide assignments showed that the FAIMS approach identified a much greater proportion of triply-charged ions.      

High mass accuracy and high mass resolving power FT-ICR secondary ion mass spectrometry for biological tissue imaging

Biological tissue imaging by secondary ion mass spectrometry has seen rapid development with the commercial availability of polyatomic primary ion sources. Endogenous lipids and other small bio-molecules can now be routinely mapped on the sub-micrometer scale. Such experiments are typically performed on time-of-flight mass spectrometers for high sensitivity and high repetition rate imaging. However, such mass analyzers lack the mass resolving power to ensure separation of isobaric ions and the mass accuracy for elemental formula assignment based on exact mass measurement. We have recently reported a secondary ion mass spectrometer with the combination of a C₆₀ primary ion gun with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for high mass resolving power, high mass measurement accuracy, and tandem mass spectrometry capabilities. In this work, high specificity and high sensitivity secondary ion FT-ICR MS was applied to chemical imaging of biological tissue. An entire rat brain tissue was measured with 150 μm spatial resolution (75 μm primary ion spot size) with mass resolving power (m/Δm _50%) of 67,500 (at m/z 750) and root-mean-square measurement accuracy less than two parts-per-million for intact phospholipids, small molecules and fragments. For the first time, ultra-high mass resolving power SIMS has been demonstrated, with m/Δm _50%?>?3,000,000. Higher spatial resolution capabilities of the platform were tested at a spatial resolution of 20 μm. The results represent order of magnitude improvements in mass resolving power and mass measurement accuracy for SIMS imaging and the promise of the platform for ultra-high mass resolving power and high spatial resolution imaging.

Estimation of the optimal sampling frequency for process analyzers

The sampling theorem states that the sampling frequency should be twice the highest frequency of the studied signal. This sets limits to the lowest allowable sampling frequency of a process analyzer. Economic criteria together with the instrument analysis time define the highest sampling frequency. The stability of the control loop requires that the roots of the characteristic equation of the control loop in the z plane should lie inside the unit circle. An example is discussed. In order to minimize the error in the estimation of parameters of a system using an impulse excitation signal, the sampling time should be equal to the dominant time constant of the system.     

Directing power of cyclobutenoid annelations on the double bonds of planar cyclooctatetraenes

Ab initio and hybrid density functional quantum mechanical computations are applied to the structure and energetics of a series of two-atom-bridge annelated cyclooctatetraenes. The contribution of each annelation to the exo/endo relative energy is estimated. Key directing factors for a given type of annelation, such as strain, electronegativity, or cyclic electron count, can be sorted out by comparison of various bridge compositions. Overall, electron count and the essential components of the Clar/Robinson rule work well to predict the exo/endo preferences. Specifically, three 4-e(-) Hückel systems (CH-CH, NH-BH and NH-C(O)) display dominant exo forms whereas the three 4n + 2 Hückel counterparts (C(O)-C(O), BH-BH, and planar NH-NH) display a common preference for endo. These endo systems act like four independent four-membered "aromatic" rings linked by "single" bonds. An analysis based on the effective hybridization of carbon atoms in the annulene (Bent's rule) provides a rationale for subtle trends in their specific annulene geometry.     

Influence of the molecular-weight distribution of the polymer and of the forming conditions on the resolving power of ultrafiltration membranes

The possibility of enhancing the resolving power of ultrafiltration membranes by using forming polymers with narrow molecular-weight distribution and by optimizing the forming conditions was examined.     

Influence of the length and position of the collision cell on the resolving power attainable from collisionally activated processes in sector instruments

The energy resolution in a high resolution tandem mass spectrometer was investigated in terms of the length and position of the central collision cell. Both these parameters have to be chosen carefully to eliminate collisional broadening phenomena that can severely limit the energy resolution in such instruments. A theory that describes collisional broadening, to first order, has been derived that accounts for the observed phenomena. It was found experimentally that collisional broadening can be reduced to less than 0.001 eV per kiloelectronvolt collision energy. Experimental data confirmed that only the size of the resolving slits and thermal broadening, due to the target gas, limited the ultimate energy resolution available.     

Measurements of reduced mobility of standard compounds by high resolving power ion mobility spectrometer in remote laboratories

An atmosphere pressure ion mobility spectrometer is described. Both electrospray and corona discharge ion sources were used. Increased up to 100 resolving power allows obtaining higher selectivity and potentially widens application area. Satisfactory reproducibility of measured reduced mobility is required for improving reliability of identification including interlaboratory reproducibility of measured reduced mobility. Results of measurement were shown for the samples containing compounds proposed earlier as standards, those were measured in remote laboratories. Measured reduced mobility for 2,6-di(tert-butyl)pyridine amounted 1.48 cm²/V · sec for the measurements where corona discharge was used, and 1.46 cm²/V · sec for the measurements, where electrospray ionization was used. Both values are in a good agreement with a value 1.48 cm²/V · sec obtained earlier on the previous version of the instrument. Values of reduced mobility calculated by results of measurements accomplished in Munich for tetraethylammonium iodide, tetra-pentylammonium iodide and tetraoctylammonium bromide amounted correspondingly 1.77, 1.10, and 0.64 cm²/V · sec when electrospray ionization was used. The data are in a very good agreement with the values obtained earlier on the previous version of the instrument in remote laboratory.     

An experimental study of sampling time effects on the resolving power of on-line two-dimensional high performance liquid chromatography

The experimental effects of sampling time on the resolving power of on-line LC×LC were investigated. The first dimension gradient time ((1)t(g)) and sampling time (t(s)) were systematically varied ((1)t(g)=5, 12, 24 and 49 min; t(s)=6, 12, 21 and 40s). The resolving power of on-line LC×LC was evaluated in terms of two metrics namely the numbers of observed peaks and the effective 2D peak capacities obtained in separations of extracts of maize seeds. The maximum effective peak capacity and number of observed peaks of LC×LC were achieved at sampling times between 12 and 21s, at all first dimension gradient times. In addition, both metrics showed that the "crossover" time at which fully optimized 1DLC and LC×LC have equal resolving power varied somewhat with sampling time but is only about 5 min for sampling times of 12 and 21s. The longest crossover time was obtained when the sampling time was 6s. Furthermore, increasing the first dimension gradient time gave large improvements in the resolving power of LC×LC relative to 1DLC. Finally, comparisons of the corrected and effective 2D peak capacities as well as the number of peaks observed showed that the impact of the coverage factor is quite significant.     

Comparison of thick film capillaries with packed columns from the point of view of loadability in combination with high resolving power

The present paper compares the potential of high pressure packed column gas chromatography, with a particle size in the range of 30–80 μm and conventional packed column GC. with that of thick film capillaries for obtaining the maximum loadability at a given performance in efficiency and speed of separation. An alternative treatment, discussing the maximum efficiency of the three column types at normalized loadability and speed of separation is given. Known and established relationships describing plate height, loadability, and linear velocity are used to arrive at the said comparisons. The conclusion of the paper indicates a reconsideration of packed column GC for particular types of analyses where large amounts or high concentrations are required in the detection step.     

On an Aerodynamic Mechanism to Enhance Ion Transmission and Sensitivity of FAIMS for Nano-Electrospray Ionization-Mass Spectrometry

Simulations show that significant ion losses occur within the commercial electrospray ionization-field asymmetric waveform ion mobility spectrometer (ESI-FAIMS) interface owing to an angular desolvation gas flow and because of the impact of the FAIMS carrier gas onto the inner rf (radio frequency) electrode. The angular desolvation gas flow diverts ions away from the entrance plate orifice while the carrier gas annihilates ions onto the inner rf electrode. A novel ESI-FAIMS interface is described that optimizes FAIMS gas flows resulting in large improvements in transmission. Simulations with the bromochloroacetate anion showed an improvement of ~9-fold to give ~70% overall transmission). Comparable transmission improvements were attained experimentally for six peptides (2+) in the range of m/z 404.2 to 653.4 at a chromatographic flow rate of 300 nL/min. Selected ion chromatograms (SIC) from nano-LC-FAIMS-MS analyses showed 71% (HLVDEPQNLIK, m/z 653.4, 2+) to 95% (LVNELTEFAK, m/z 582.3, 2+) of ion signal compared with ion signal in the SIC from LC-MS analysis. IGSEVYHNLK (580.3, 2+) showed 24% more ion signal compared with LC-MS and is explained by enhanced desolvation in FAIMS. A 3–10 times lower limits of quantitation (LOQ) (<15% RSD) was achieved for chemical noise limited peaks with FAIMS. Peaks limited by ion statistics showed subtle improvement in RSD and yielded comparable LOQ to that attained with nano-LC-MS (without FAIMS). These improvements were obtained using a reduced FAIMS separation gap (from 2.5 to 1.5 mm) that results in a shorter residence time (13.2 ms?±?3.9 ms) and enables the use of a helium free transport gas (100% nitrogen). Graphical Abstract

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Precise determination of nonlinear function of ion mobility for explosives and drugs at high electric fields for microchip FAIMS

High‐field asymmetric waveform ion mobility spectrometry (FAIMS) separates ions by utilizing the characteristics of nonlinear ion mobility at high and low electric fields. Accurate ion discrimination depends on the precise solution of nonlinear relationships and is essential for accurate identification of ion species for applications. So far, all the nonlinear relationships of ion mobility obtained are based at low electric fields (E/N <65 Td). Microchip FAIMS (μ‐FAIMS) with small dimensions has high electric field up to E/N = 250 Td, making the approximation methods and conclusions for nonlinear relationships inappropriate for these systems. In this paper, we deduced nonlinear functions based on the first principle and a general model. Furthermore we considered the hydrodynamics of gas flow through microchannels. We then calculated the specific alpha coefficients for cocaine, morphine, HMX, TNT and RDX, respectively, based on their FAIMS spectra measured by μ‐FAIMS system at ultra‐high fields up to 250 Td. The results show that there is no difference in nonlinear alpha functions obtained by the approximation and new method at low field (<120 Td), but the error induced by using approximation method increases monotonically with the increase in field, and could be as much as 30% at a field of 250 Td. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparison of experimental and calculated peak shapes for three cylindrical geometry FAIMS prototypes of differing electrode diameters

High-field asymmetric waveform ion mobility spectrometry (FAIMS) separates ions at atmospheric pressure and room temperature based on the difference of the mobility of ions in strong electric fields and weak electric fields. This field-dependent mobility of an ion is reflected in the compensation voltage (CV) at which the ion is transmitted through FAIMS, at a given asymmetric waveform dispersion voltage (DV). Experimental CV, relative peak ion intensity, and peak width data were compared for three FAIMS prototypes with concentric cylindrical electrodes having inner/outer electrode radii of: (1) 0.4/0.6 cm, (2) 0.8/1.0 cm, and (3) 1.2/1.4 cm. The annular analyzer space was 0.2 cm wide in each case. A finite-difference numerical computation method is described for evaluation of peak shapes and widths in a CV spectrum collected using cylindrical geometry FAIMS devices. Simulation of the radial distribution of the ion density in the FAIMS analyzer is based upon calculation of diffusion, electric fields, and the electric fields introduced by coulombic ion-ion repulsion. Excellent agreement between experimental and calculated peak shapes were obtained for electrodes of wide diameter and for ions transmitted at low CV.     

Assigning product ions from complex MS/MS spectra: The importance of mass uncertainty and resolving power

This study offers a unique insight into the mass accuracy and resolving power requirements in MS/MS analyses of complex product ion spectra. In the examples presented here, accurate mass assignments were often difficult because of multiple isobaric interferences and centroid mass shifts. The question then arose whether the resolving power of a medium-resolution quadrupole time-of flight (QqTOF) is sufficient or high-resolution Fourier-transform ion cyclotron resonance (FT-ICR) is required for unambiguous assignments of elemental compositions. For the comparison, two paralytic shellfish poisons (PSP), saxitoxin (STX) and neosaxitoxin (NEO), with molecular weights of 299 and 315 g x mol(-1), respectively, were chosen because of the high peak density in their MS/MS spectra. The assessment of QqTOF collision-induced dissociation spectra and FT-ICR infrared multiphoton dissociation spectra revealed that several intrinsic dissociation pathways leading to isobaric fragment ions could not be resolved with the QqTOF instrument and required FT-ICR to distinguish very close mass differences. The second major source of interferences was M + 1 species originating from coactivated 13C12Cc-1 ion contributions of the protonated molecules of the PSPs. The problem in QqTOF MS results from internal mass calibration when the MH+ ions of analyte and mass calibrant are activated at the same time in the collision or trapping cell. Although FT-ICR MS readily resolved these interfering species, the QqTOF did not provide resolving power >20,000 (full width at half maximum) required to separate most isobaric species. We were able to develop a semi-internal QqTOF calibration technique that activated only the isolated 12C isotope species of the protonated molecules, thus reducing the M + 1 interferences significantly. In terms of overall automated elemental formulas assignment, FT-ICR MS achieved the first formula hit for 100% of the product ions, whereas the QqTOF MS hit rate was only 56 and 65% for STX and NEO product ions, respectively. External mass calibration from commercial FT-ICR and QqTOF instruments gave similar results.     

First derivative of the ratio spectra method for resolving iodide and thiocyanate in binary mixtures

A new method is described to analyse the binary mixture of iodide (I(-)) and thiocyanate (SCN(-)) ions, using the first derivative of the ratio spectra obtained by mathematical treatment of the data. The method is based on the formation of mixed ligand complexes between benzohydroxamic acid (BHA), vanadium (V) and I(-) or SCN(-) and their extraction in ammonium quaternary salt dissolved in toluene. Calibration graphs for 2-9 mug/ml of I(-) and for 2-6 mug ml of SCN(-) were established by measuring the analytical signals at 376 nm for I(-) and at 400.6 nm for SCN(-). The method has been applied for determining both ions in waste water of an power station at ng/ml levels after a preconcentration step with C(18), without any separation step.