Linear ion traps offer a wealth of experimental options for tandem MS and provide a platform for conducting foundational ion chemistry experiments. Despite an extensive history of contributions to mass spectrometry fundamentals and applications, the commercial availability of such instruments has decreased from historical highs, and the cost and maintenance of new instrumentation have become increasingly prohibitive. In domains where a broad degree of functionality is ideally paired with low-cost maintenance (e.g., scientific education and experiments not requiring high throughput), few options exist. To address this growing concern in the mass spectrometry community, this work demonstrates a tractable, proof-of-principle approach to digitally revitalize an end-of-life linear quadrupole ion trap. The factory electronics and drivers were removed and replaced with lower-cost and readily available components. The analyzing quadrupole was operated and controlled entirely digitally using the Astraea digital waveform generation platform. The digital driver is largely hardware-agnostic as the waveforms are software-defined (e.g., not resonantly tuned), making it applicable to most quadrupole designs. This work presents an initial characterization of the digitally driven ion trap. The ion trap was able to isotopically resolve singly and doubly charged ions when a low-voltage auxiliary waveform was used in conjunction with the duty cycle during mass scans. The auxiliary waveform can match the secular frequency of any ion in the trap regardless of the AC frequency and voltage. This permitted mass-selective elimination of ions during accumulation or afterward. The tunable auxiliary frequency facilitated activation and fragmentation of a small peptide ion. The duty cycle was used to isolate a fragment as the precursor ion for a second activation step. The second activation step proceeded without altering the AC conditions of the trap.

Linear ion traps offer a wealth of experimental options for tandem MS and provide a platform for conducting foundational ion chemistry experiments. Despite an extensive history of contributions to mass spectrometry fundamentals and applications, the commercial availability of such instruments has decreased from historical highs, and the cost and maintenance of new instrumentation have become increasingly prohibitive. In domains where a broad degree of functionality is ideally paired with low-cost maintenance (e.g., scientific education and experiments not requiring high throughput), few options exist. To address this growing concern in the mass spectrometry community, this work demonstrates a tractable, proof-of-principle approach to digitally revitalize an end-of-life linear quadrupole ion trap. The factory electronics and drivers were removed and replaced with lower-cost and readily available components. The analyzing quadrupole was operated and controlled entirely digitally using the Astraea digital waveform generation platform. The digital driver is largely hardware-agnostic as the waveforms are software-defined (e.g., not resonantly tuned), making it applicable to most quadrupole designs. This work presents an initial characterization of the digitally driven ion trap. The ion trap was able to isotopically resolve singly and doubly charged ions when a low-voltage auxiliary waveform was used in conjunction with the duty cycle during mass scans. The auxiliary waveform can match the secular frequency of any ion in the trap regardless of the AC frequency and voltage. This permitted mass-selective elimination of ions during accumulation or afterward. The tunable auxiliary frequency facilitated activation and fragmentation of a small peptide ion. The duty cycle was used to isolate a fragment as the precursor ion for a second activation step. The second activation step proceeded without altering the AC conditions of the trap.

Digital quadrupoles are driven with rectangular RF waveforms and, through duty cycle control, can access higher-order Mathieu space stability zones (HZs) using comparatively low voltages. Analytically, accessing these zones remains attractive as HZs exhibit higher resolving powers ((m/z)/(Δm/z)) compared to the conventional quadrupole operation in zone (1,1). Presented here is a modification of a commercial quadrupole time-of-flight (Q-TOF) equipped with a low-voltage digital waveform driver to navigate the filtering quadrupole in the HZs. We demonstrate that a digital quadrupole operating in zones (3,1) and (3,2) achieves a narrowed isolation window for analytes up to 690 and 1543 m/z, respectively, compared to conventional operational modes. Experimental trends suggest this performance to continue to approximately 900 m/z in zone (3,1) and 2500 m/z in zone (3,2). These narrowed isolation windows were utilized to generate MS/MS data from a mixture of m-hydroxybenzoylecgonine (306 m/z) and cocaine-d3 (307 m/z), which exhibited minimal chimeric nature without the use of deconvolution software. The development of hybrid systems capable of both sine and digital operation provides a mechanism to maximize selectivity of the HZs while maintaining the benefits of traditional modes of operation. Minimizing chimeric interferences is of particular importance, and the narrow isolation widths afforded by HZs are readily accessible using tandem instruments for analytes <2500 m/z.

Recent improvements to the comparison-based method of digital waveform generation increased the reproducibility of the waveforms so that the higher-order Mathieu stability zones can be accessed reliably. Digitally driven quadrupole mass filters access these zones using a fixed AC voltage and rectangular waveforms that are defined by a duty cycle. In this context, the duty cycle is the fraction of the waveform period where the waveform remains in the high state. Because digitally driven quadrupoles navigate stability using a duty cycle, there is no need to apply a resolving DC offset between electrode pairs. Accessing the higher stability zones using a conventional resonantly tuned RF requires the use of thousands of AC and DC voltages making the mode of operation less accessible with these devices. Stability zones higher than (1,1) and (2,1) have theoretical resolving powers that are on the order 1,140 and 3,447 at fwhm which drives efforts to practically access these operational conditions. Accessing these zones digitally requires the use of extremely precise waveforms. In a previous effort, waveform generation produced waveforms to reliably access the (1,1) and (2,1) zones without impacting performance. However, recent work found more improvement was needed to reliably access neighboring higher stability zones. Derived from that work, it was determined that a waveform resolution of ∼10 ppm or less was needed to reliably access the (3,1) and (3,2) zones. The present work utilized digital waveforms that achieve this level of precision to experimentally access and characterize attributes of the (3,1) and (3,2) zones. This work dives into the investigation of different beam energies to overcome the destabilizing fringing fields, improve transmission, and their overall effect on the experimental resolving power and signal-to-noise. In addition, the AC voltage of the driving RF was varied to understand the effects on the initial ion beam energy that is needed to achieve balanced separation and how the overall signal-to-noise is affected. Lastly, an assessment was made on the effects of the temporal parameters of a digital mass scan on peak sensitivity, peak fidelity, and overall duration for a scan.

Digital mass filters are advantageous for the analysis of large molecules due to the ability to perform ion isolation of high-m/z ions without the generation of very high radio frequency (RF) and DC voltages. Experimentally determined Mathieu stability diagrams of stability zone 1,1 for capacitively coupled digital waveforms show a voltage offset between the quadrupole rod pairs is introduced by the capacitors which is dependent on the voltage magnitude of the waveform and the duty cycle. This changes the ion's a value from a = 0 to a < 0. These effects are illustrated for isolation for single-charge states for various protein complexes up to 800 kDa (GroEL) for stability zone 1,1. Isolation resolving power (m/Δm) of approximately 280 was achieved for an ion of m/z 12,315 (z = 65+ for 800.5 kDa GroEL D398A), which corresponds to an m/z window of 44.

This work presents the experimental evaluation of a digital tandem mass filter that is composed of two digitally operated low-resolution mass filters in series whose mass windows are shifted with respect to each other. The overlap of the mass windows allows the resolution (Δm) of ions to be narrowed to provide better resolving power, while the acceptance of the tandem mass filter is defined by the acceptance of the first low-resolution quadrupole. Our experiments show that digital operation fulfills the promise of the tandem mass filter for providing better ion transmission at the same or better resolving power as a single quadrupole mass filter. It allows the user to continuously adjust the resolving power and sensitivity to meet current needs. Most importantly, the observed resolving power/sensitivity characteristics are the same at any mass and m/z.

The relationships between resolution, stability, pseudopotential well depth, acceptance aperture, and transmission for sinusoidal quadrupole mass filters are examined graphically and mathematically. Simple linear or power relationships are revealed. Comparison of these quantities plotted against resolving power show that the pseudopotential well depth correlates well with the mass filter transmission. Pseudopotential well depth is directly proportional to the product of dimensionless stability well depth and the AC voltage. This relationship extends to all quadrupoles regardless of operational zone because it is rooted in stability. Ion transmission and sensitivity scale directly with the pseudopotential well depth. Resolving power and pseudopotential well depth increase when operating in higher stability zones for all types of mass filters. Unfortunately, for fixed frequency sine wave mass filters, the increased resolving power and pseudopotential well depth are accompanied by a significant reduction of the mass range and increased in fringe field effects. For these reasons, sine mass filter operation in higher stability zones has been reported but not commercially produced. In contrast, for rectangular wave mass filter operation, there is no mass range limitation in any stability zone. The fringe field does not increase because the AC voltage is constant and does not change within a single stability zone or between them. A DC voltage is also unnecessary to access any zone. The high resolution and sensitivity of rectangular wave mass filters that can be gained by operation in higher stability zones without mass limit and limited fringe field restrictions suggest a bright and expansive future for this technology.

Waveform reproducibility is a critical factor for performing high resolution mass analysis with digitally operated quadrupole mass filters and traps operating in higher stability zones. In this work, Hill equation-based stability calculations were used to define the effect of period jitter on mass analysis in higher stability zones. These calculations correlate well with experimental observations in higher stability zones. Comparison of experiment to theory supplies the basis for defining jitter-based expectations and limits for mass analysis in higher zones.

Even though sinusoidal quadrupole mass filters have been around for more than 50 years, the relationships defining resolution, resolving power, and transmission from the applied voltages have not been rigorously quantified or discussed. Traditional quadrupole mass filter theory implies that voltages are scanned at constant direct current (DC) to alternating current (AC) voltage ratios with the scanline passing through the origin of the voltage stability diagram. A prominent feature of constant voltage ratio scans is constant baseline theoretical resolving power (m/Δm) that is the same for all masses. Commercial quadrupole instruments rarely scan at constant resolving power because ion transmission increases with mass. Instead, they scan at constant resolution, meaning that the mass window width is fixed. Constant resolution mass scans are preferred because ion transmission does not change with mass. Commercial mass filter systems create constant resolution scans by linearly changing the DC and AC voltages at a fixed ratio in the presence of an additional negative DC voltage offset. This manuscript systematically quantifies the effects of the DC and AC voltages on resolution, resolving power, pseudopotential well depth, and transmission. To quantify these properties, recently developed spreadsheet tools that calculate the laboratory frame stability of ions from the matrix solutions of Hill's equation were used. Voltage scanning methods and their effects on theoretically determined transmission and sensitivity will be discussed.

Mass filter operation in higher stability zones is known to provide better resolution. Unfortunately, for sine driven instruments, higher stability zone operation reduces the accessible mass range and increases the degenerative effects of fringe fields. Conversely, digitally driven mass filters do not suffer from loss of mass range, and the fringe field effects do not increase significantly by switching stability zones because the AC voltage is always constant and the DC voltage is always zero. This work catalogues 12 stability zones that are accessible with the new digital waveform generation technology. These zones have theoretical baseline resolving powers that range from 22 to 1 300 000 with pseudopotential well depths that range from 3.5 to 43 V. Operation in higher stability zones also has the advantage of aligned axial stability wells. That alignment maximizes the pseudopotential well depth for each higher stability zone, making them more than an order of magnitude greater than the standard ∼0.2 V well of a sine filter operating in the first stability zone at unit resolution. Increased pseudopotential well depth correlates with better ion transmission and sensitivity. Our theoretical examination suggests that the digital mass filter can obtain both high resolution and high sensitivity with essentially unlimited mass range.

A tandem mass filter consists of two low-resolution mass filters arranged in series that operate with a small offset between their mass windows. In principle, the overlap of the two individual mass windows defines the tandem window. Tandem operation provides improved resolution and transmission compared to a single mass filter operated with the same mass window. The improvement in transmission is owed to the larger acceptance of the low-resolution quadrupoles. The tandem filter resolution and transmission are adjusted by changing the amount of offset separating the mass windows of the individual filters. Sine wave systems create this offset through voltage changes. Digital tandem mass filters depart from convention because they do not change voltage. The tandem mass window is created when the individual filters are operated with two slightly different duty cycles. Both quadrupoles operate at the same frequency, phase, and voltage. When the frequency, phase, and voltage of each quadrupole are identical, there theoretically are no changes to the Mathieu parameters to cause ion excitation and loss during transition between the quadrupole pair. The work presented here shows that a fixed AC voltage digital tandem mass filter can only operate in higher stability zones. However, unlike sine mass filters, the mass range of a digital system is not limited. This makes the digital tandem mass filter feasible as a commercial product. For the tandem digital mode to be successful, the duty cycles of each quadrupole must be precisely controlled because the duty cycle differences required to shift the mass windows are small. The creation of these mass window offsets requires waveform generation that can obtain high duty cycle resolution. Our method of generating waveforms can meet this demand; however, modifications to our current printed circuit board must be made. These modifications are minor and will be discussed.

Here, we describe a digital-waveform dual-quadrupole mass spectrometer that enhances the performance of our drift tube FT-IMS high-resolution Orbitrap mass spectrometer (MS). The dual-quadrupole analyzer enhances the instrument capabilities for studies of large protein and protein complexes. The first quadrupole (q) provides a means for performing low-energy collisional activation of ions to reduce or eliminate noncovalent adducts, viz., salts, buffers, detergents, and/or endogenous ligands. The second quadrupole (Q) is used to mass-select ions of interest for further interrogation by ion mobility spectrometry and/or collision-induced dissociation (CID). Q is operated using digital-waveform technology (DWT) to improve the mass selection compared to that achieved using traditional sinusoidal waveforms at floated DC potentials (>500 V DC). DWT allows for increased precision of the waveform for a fraction of the cost of conventional RF drivers and with readily programmable operation and precision (Hoffman, N. M. . A comparison-based digital-waveform generator for high-resolution duty cycle. Review of Scientific Instruments 2018, 89, 084101).

Mass analysis in a linear ion trap is traditionally performed using resonant ejection induced by auxiliary waveforms. For sinusoidally driven ion traps without resonant ejection, resolution and sensitivity are poor because mass-selected instability yields excitation along both the x and y axes simultaneously. Digital ion traps, on the other hand, have the advantage of duty cycle manipulation that can be used to change the ion excitation along the x and y axes. Consequently, the duty cycle can be used to enhance the resolution and sensitivity for mass-selected instability in a linear ion trap without the application of an auxiliary waveform. This work introduces and explores mass selected instability in a linear trap without the use of auxiliary waveforms.

The acceptance of quadrupole mass filters is improved when the alternating current (AC) and direct current (DC) fields are developed separately. Physically, this is achieved when a short RF only quadrupole (prefilter) is situated directly ahead of the mass filter. The acceptance gained by a system operating with a prefilter can be observed as an increase in sensitivity over conventional operation. Frequency dynamic duty cycle based rectangular waveform driven (rectangular wave) mass filters, a recent development, currently do not operate with prefilters. Little is known about the influence of duty cycle changes on the acceptance of rectangular wave mass filters. The sensitivity gain seen by conventional systems operating with prefilters indicates that the sensitivity of duty cycle based rectangular wave systems should increase comparably. The objective of this work was to determine prefilter efficacy for nonspecific rectangular wave mass filter systems. In this work, the plane method of acceptance was used to model the change to the acceptance and transmittance of sine and rectangular waveform driven mass filters under different modes of field development. Both systems indicated a fourfold increase in sensitivity when the mass filtering DC or duty cycle was delayed.

The first completely digital quadrupole mass filter was recently introduced. There is now a need to understand and demonstrate the benefits of digital operation and compare them to the commercial standards. Our work to date has demonstrated that sine and square wave operation are very similar because of their similar stability diagrams and because they use a direct current (DC) potential between the electrode pairs to narrow and limit the mass range. In contrast, rectangular wave-operated digital mass filters and ion traps narrow and limit the stable mass range with the waveform duty cycle without the need for a DC potential. To understand and compare the differences between rectangular and sinusoidal modes of operation, our group has developed new spreadsheet tools that permit calculation of the m/z versus frequency space stability diagrams with the application of a DC potential between the electrode sets for rectangular and sine waveforms, and plots of the pseudopotential well depth against the entire range of stable m/zs for rectangular and sine waveforms. Our spreadsheets were used to make comparisons between fixed-frequency variable-voltage and fixed-voltage variable-frequency modes of operation. They provide a comprehensive companion tool for operating in the laboratory frame and are tunable to the instrument. This manuscript introduces these tools as it compares sine and rectangular wave modes of operation and provides a basis for understanding the advantages and disadvantages of digital operation relative to conventional technology.

The quadrupole acceptance of sinusoidal mass filters has been an area of interest for over fifty years. More recently, frequency dynamic rectangular waveform driven mass filters (colloquially digital mass filters) have been demonstrated. The acceptance of these devices has not been investigated. Commonly the acceptance of a quadrupole mass filter is calculated using the method of ellipses. Earlier this year, our group introduced an alternative method of calculating acceptance planes. The plane method is a simple and fast approach compared to ellipses. In this work, sinusoidal and digital RF driven mass filters modeled in stability zones A and B are explored using the plane method. The acceptance behavior of systems in zone B differ greatly from their zone A counterparts. There are considerable differences between the mass filter types operated in zone B. The type of RF drive potential plays an important role in the behavior of filters modeled in zone B.

A quadrupoles acceptance is a measure of its ability to catch ions with certain trajectories. One way to calculate acceptance is the method of ellipses. The method arose partly from a simplification that trajectories could be calculated for an electrode axis independently of others. It has been used to calculate the acceptance and transmission of sine-driven quadrupole mass filters for over 50 years. Although the method is straightforward, it is generally described with little detail or presented as a confusing string of equations. As such, it may not be decipherable by all practitioners. For this reason, the first half of this paper presents a practical explanation of the method of ellipses and the concepts that make it work. Only equations necessary to describe the method are introduced. The tutorial also prepares the reader for the second half, which presents an alternative approach for calculating acceptance based on an array of initial trajectories. The alternative approach is used to compare the acceptance of simplified sinusoidal and digital ion guides. The method of ellipses was applied to validate results of the new approach for calculation of acceptance.

Digital waveform technology allows the waveform frequency and duty cycle to be instantly changed. These sudden changes affect ion motion and hence ion energy. Mass spectrometrists can take advantage of these sudden changes to isolate or excite trapped ions. To understand these processes on the microsecond time scale, a theoretical treatment of ion motion during the first repetitions of the newly applied waveform is needed. Fortunately, the matrix solution to the Hill equation allows the observation of ion trajectories before, during, and after the waveform transition process. This study begins the investigation of the short time scale effects of changing waveforms on ion motion. (C) 2019 Elsevier B.V. All rights reserved.

Digitally driven mass filter analysis is an advancing field. This work presents a tutorial of digital waveforms, stability diagrams, and pseudopotential well plots. Experimental results on digitally driven mass filter analysis in stability zones A and B are also shown. This work explains duty cycle manipulation of the waveforms to axially trap and eject ions from linear quadrupoles and how to change and distort the stability diagrams to create mass filters and their effects on the pseudopotential well depth. It discusses the sensitivity and resolution that can be obtained and what limits these benchmarks. It reveals the advantages of mass filter operation without any added direct current potential between the quadrupole electrodes (a=0).