13
ions, is in direct relation to the turnaround time in the above equation. A similar resolution was then achieved by Waters using the W-mode, with multiple passes through grid-based refl ectors. However, each pass through the refl ectron grids leads to a loss in sensitivity. Agilent changed both the detector and ion optics, addressing opportunities in the detector peak width as well as the residual term. Several years ago, Sciex introduced an instrument using N-optics, resulting in an instrument balancing sensitivity loss and resolution increase.
Two main principles are used for detecting and converting the ions arriving at the detector: analogue-to-digital conversion (ADC) and time-to-digital conversion (TDC).
The TDC detector is an ion-counting detector. The basic principle is that they register the arrival of a single ion at discrete time bins, and the obtained counts are then summed together for all consecutive spectra. The pulse rate is typically in the kHz range, allowing thousands of spectra to be summed together.
Figure 2. Simulation showing the thermal profi le of the Agilent Jet Stream technology.
Note the creation of a thermal confi nement zone by introduction of a super-heated N2 sheath gas.
In modern day mass spectrometry instruments, two design options are implemented following the initial analyte ionisation stage: skimmer-based entrance or ion funnels. Of these, ion funnels offer the advantage that ions are more effectively captured and guided into the high-vacuum region of the mass spectrometer, leading to improvements in sensitivity for small molecules. There is, however, a bias against high m/z species.
The concept of ion funnels was prototyped by R. Smith [7]. Ion funnels operate best in the low Torr range, which is relatively high for the entrance region after the capillary. As the only available gas comes from the capillary source, several modifi cations are needed to accommodate this factor, including a shorter capillary as well as multiple capillary inlets. Alternatively, the gas pressure can be regulated by the supply of an external gas source.
The next opportunity to gain instrument sensitivity is the trapping of ions. For some instruments, this trapping is optional, i.e., it occurs before the pulser region via lenses. In others, trapping is essential to ensure best functionality; an automatic gain control is used to avoid an overfi ll of the electrostatic trap and consequent space-charge effects leading to resolution loss.
Ion mobility adds another dimension of separation, and, as a result of removing background from different regions, sensitivity. The main advantage of ion mobility is the ability to separate molecules based on their collision cross section (CCS) or ion structure. Molecules can have the same m/z value, but very different structures, so ion mobility can distinguish between isomeric molecules [8]. The main techniques include: differential (ion) mobility spectrometry (DMS, commercialised by Owlstone as FAIMS), travelling wave (Waters), drift tube-based IM (Agilent), and trapped ion mobility spectrometry (TIMS, Bruker Daltonics).
Another important aspect for gaining sensitivity relies on instrument tuning. Manually tuning an instrument may be optional for advanced users to obtain the best results for resolution or sensitivity. However, this strategy is not suitable for an ever-growing market size with a fi nite number of specialists.
Automatic tuning of the instrument is therefore a valid solution. Nevertheless, determining the right boundaries and tunable parameter space proves challenging today. Multiple parameters impact the resolution, including grids in the pulser region, grids at the refl ector, and voltages prior to entering the pulser. The complexity of these elements no longer permits multiple iterative cycles to determine the best combination, but rather requires tuning multiple elements simultaneously.
All TOF instruments increase in resolution as the m/z value increases. With a typical mass range of 3000 m/z for most TOF instruments, instruments are typically tuned for resolution where the impact is the highest, namely for high m/z species. However, metabolomics researchers and pesticide and environmental laboratories are often less interested in the best resolution at high m/z, preferring high sensitivity and good resolution at low m/z. An application tune needs to consider this performance requirement. Electrostatic traps, on the other hand, have excellent resolution at low mass, and is optimised for this range, but require different settings for larger molecules.
Resolution
As previously mentioned, the resolving power is defi ned as m/Δm between two peaks. In mass spectrometry, resolving power and resolution are frequently used interchangeably, and all instruments are specifi ed by their resolution, which is R=m/FWHM, assuming that the peak width at half maximum of a single peak corresponds to the ability to separate two neighbouring peaks. The resolution on TOF instruments, defi ned in the time domain, is approximately R=TOF/Δt. To defi ne Δt, the following equation is used: Δt2 τTA2
= + τPW2 + τR2 , with TA for the turnaround time, PW for the detector pulse width, and R for residual terms.
In 2008, Bruker Daltonics introduced an instrument with a resolution of 40,000 (mass 2,722), which more than doubled the resolution possible with instruments from other vendors [3]. This was achieved by increasing the length of the fl ight tube with a single refl ector. The three-meter-long fl ight tube, resulting in a nearly six-meter-long fl ight time of
Figure 3: Schematics of a wide band quadrupole experiment. Similar to AII Ions, but using the quadrupole to take sequential windows of the mass range to reduce complexity of the MS/MS spectra.
MSE
(Waters) and All Ions (Agilent) are instrument acquisition modes that are capable of fragmenting all analytes without any prior quadrupole isolation. Both wideband isolation and full-spectrum fragmentation are part of the data-independent acquisition (DIA) modes.
The most sophisticated acquisition mode is auto MS/MS, a DDA mode where several variables must be confi rmed prior to selecting the precursor. These include charge state determination, abundance threshold/ranking, isotope grouping, neighbouring peaks, chromatographic apex prediction, number of selected precursors per cycle, precursor exclusion, and abundance-dependent acquisition time to name a few, followed by prediction of the most suitable collision energy (typically in proteomics experiments).
On the other hand, ADC detectors digitise the pulsed ion current from the detector at discrete time intervals. The time intervals are in direct proportion to the sampling rate of the acquisition board. Whereas (for example) a 2 GHz acquisition board allows a maximum time interval of 500 picoseconds, this can be substantially reduced by higher acquisition boards. The 10 GHz acquisition boards of Agilent’s latest GC and LC/Q-TOF (7250 and 6546) allow for time intervals of 100 picoseconds. It is noteworthy that the higher acquisition rates are only useful if the detector pulse width is as narrow as possible; otherwise users are exposed to a risk of oversampling. The most prominent effect of the faster digitisation occurs at low m/z species, because of the lower arrival times. A peak at low arrival times has, in absolute value, much narrower peak width compared to later arrival times/higher m/z. Due to this narrow peak, slow digitisation would lead to undersampling of the peak, and therefore lower resolution. The impact of the fast 10 GHz acquisition board is that previously undersampled low m/z peaks now have enough datapoints over the peak, and therefore a resolution at full width at half maximum (FWHM) refl ective of their real peak shape. In general, based on the summation of ion currents, ADC detectors show a wider dynamic range and better isotope fi delity compared to TDC detectors, as the ion current is a better measure at high and low ion intensities.
In the electrostatic trap, a completely different principle for ion detection is used. The oscillation of ions is measured, converted into an m/z spectrum via Fourier transform (FT), and a single scan is used for the assembly of a spectrum. Here, the resolution is directly dependent upon the time allowed to measure the oscillation; the longer the measuring time, the better the resolution. An instrument can have a resolution at 1 Hz of 240,000, but at 3 Hz the resolution will drop to 70,000, and at 10 Hz to 10,000 (at m/z 200).
Acquisition modes
For a long time, mass spectrometry had two basic modes of operation: MS-only and tandem MS. The latter was split into two variants: auto MS/MS, which is a data- dependent acquisition (DDA) method, and targeted MS/MS. Targeted MS/MS is a mode similar to single reaction monitoring (SRM) or multiple reaction monitoring (MRM) on a triple quadrupole instrument. A variation of this mode, used on electrostatic traps, is called single ion monitoring (SIM), where a selected m/z species is isolated in the quadrupole and then accumulated in the C-trap. The ion of interest can then either be scanned directly, or fragmented and then analysed. In general, tandem MS follows the principle of precursor selection in the quadrupole followed by fragmentation (a collision cell in all TOF instruments, or ion trap-based fragmentation and/or collision cell). Over 10 years ago, Sciex introduced a new acquisition mode called SWATH - where instead of a distinct precursor isolation, a wide band of m/z species was isolated by the quadrupole (Figure 3). This new mode has the advantage that fragment information in intervals over the whole m/z range can be obtained. The most prominent application was proteomics; post-fragmentation, each peptide generates a suffi cient number of fragmented ions used for the subsequent identifi cation. Chromatography results typically showed broader peaks in nano-LC ranges, therefore this mode was widely accepted because users were able to identify and quantitate purer analytes. Versions of it are now adopted by all vendors.
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