GC/LC-MS: data acquisition rate and peak reconstruction

Transcription

1 GC/LC-MS: data acquisition rate and peak reconstruction Nyquist (Shannon-Kotelnikov-Whittaker) theorem Signal sampling does not involve any loss of information as long as the sampling frequency is at least twice as high as the highest frequency in the signal. Six points (at least) are required for an unbiased sampling of a perfect Gaussian peak, representing an ideal chromatographic peak. In a real chromatographic trace: the signal is noisy (noise must be adequately sampled) peak tailing/fronting occur (difficult detection of start/end points of the peak)

2 Overlaid fast GC-MS extracted ion chromatograms of chlorpyrifos-methyl (m/z 125), measured at different acquisition rates/number of data points obtained, show the dramatic effects of an inadequate signal sampling: Scanned m/z range: Hz 3.18 Hz 0.84 Hz 5.98 Hz Hz Note that the peak width at the baseline is about 0.02 min!

3 As shown by the following data, obtained by a GC-MS instrument equipped with a quadrupole analyzer, 7-10 points across a peak represent a good value in terms of peak area/height reproducibility: peak height peak area

4 Data acquisition rate: quadrupole MS vs. other GC detectors detector FID data acquisition rate Up to 200 Hz (very low dead volume; no extracolumn band broadening) Micro ECD Up to Hz (some peak broadening due to a still too large cell volume) Quadrupole MS From few Hz (old, slow scanning instruments) up to Hz (new, fast scanning analyzers) depending on the mass range scanned

5 Signal acquisition in MS full scan mode During a MS scan cycle multiple readings are taken at each m/z step and then averaged/summed to give one data point. For instance 2 N (N = 0,1,2,., 7, selectable by the operator) readings are performed by the Agilent 5973N MSD; the number of readings and the scanned mass range determine the acquisition rate: N N Acquisition rate (scans/s) ( mass range) On a quadrupole instrument (with unit resolution) MS peaks are usually 1 u wide and 7-8 points are typically needed per peak in order to characterize it well enough for a detector to consistently determine peak centroid value and position.

6 The following parameters are commonly considered: data acquisition rate = number of spectra acquired per second scan time = time spent on scanning the specified mass range inter-scan delay = time elapsing between one scan ending and the next scan starting scan speed: up to u/s for a new, fast scanning quadrupole ca u/s for an old, slow scanning quadrupole

7 In these tables, acquisition parameters are calculated under different operating conditions. The minimum detectable peak width is based on a 7 point peak definition.

8 Scan speed limitations A systematic investigation performed by P. Korytar and coworkers in 2005 (J. Chromatogr. A 1067 (2005) ) has led to the following conclusions about the effect of scan speed: the quality of the averaged mass spectra is found to be essentially constant up to 7000 u/s; increasing scan speed reduces resolution, which results in a significant loss (nominal resolution > 1 u) above 9000 u/s; at scan speeds higher than u/s the loss of resolution is so significant that the averaged mass spectra become distorted.

9 Comparison of averaged mass spectra (with no background subtraction) obtained for 3,3,4,4,5- Pentachlorobiphenyl at different scan speeds. Scanned mass range: 350 u (m/z ); inter-scan delay: 0.1 s. Spectral details are clearly lost at higher scan speed (although the effect is not so evident if peak centroids are reported).

10 Effect of inter-scan delay time If the delay time is too short proper processing and recording of the acquired spectrum cannot be achieved: The comparison of averaged mass spectra of octachloro-undecane acquired at different inter-scan delays shows loss of information at the low m/z range for shorter delays when the mass scan occurs from high to low m/z values.

11 Effect of mass resolving power As mass resolving power increases the transmission of ions through the analyzer and then towards the ion detector decreases, resulting in a loss of sensitivity, i.e. a lower signal-to-noise response: S/N response for the GC- MS analysis of 0.02 g/ml fenbuconazole performed with a quadrupole mass spectrometer operated at variable mass resolving power. Mass resolving power (log scale)

12 Signal acquisition in Selected Ion Monitoring (SIM) mode Pros Higher duty cycle (duty cycle decreases as the number of monitored ions increases) Excellent sensitivity Cons No Mass Spectrum available; the information obtained is limited only to target compounds

13 When multiple SIM is adopted, i.e. the signal from several m/z windows is acquired consecutively, the data acquisition rate is given by the following equation: where n is the number of monitored ions.

14 Increasing the number of monitored ions (i.e. the amount of information available) leads to a decrease in data acquisition rate, thus limiting the minimum detectable width for a chromatographic peak: Nonetheless, even for baseline widths of 300 ms, up to four different ions can be still monitored without sacrificing the seven-data pointsper-peak rule.

15 Ion traps: effect of scan speed on sampling rate A single ion trap microscan consists in an AGC prescan and an analytical scan: RF microscan Note that ion injection time is the most relevant part of the total scan time in this case.

16 Several microscans are summed in a single scan cycle, averaged and saved as one data point of the chromatographic trace:

17 The sampling rate across a chromatographic peak when ion trap-ms is adopted for detection can be increased by: reducing the number of microscans per data point reducing the injection time, which can be achieved in two ways: 1 - setting a lower maximum injection time, which reduces the number of ions in the trap, hence sensitivity; 2 - increase the efficiency of the ion source and ion optics, to improve the transmission of ions, thus filling the trap to the same level in a shorter period of time.

18 Microscans Scan event time (s) Range Range ,28 0,42 4 0,51 0,75 6 0,73 1,09 8 0,96 1, ,19 1,76

19 Sampling rate issues with non scanning analyzers: ToF The Time-of-Flight is a typical example of fast, non scanning analyzer. Since ToF resolution is increased with time of flight, a clear contrast can be observed between speed and resolution in a ToF device: High speed TOF High Res TOF Spectral acquisition rate (spectra/s) Upper mass limit (u) Mass Resolution (FWHM) Mass accuracy (ppm) Typical application at m/z at m/z 614 < 5 Fast GC, GC GC Note that these data are referred to GC-MS applications, thus the upper mass limit is not much higher than 1000 u

20 An example of a GC-MS system based on a high resolution ToF is the Waters-Micromass GCT Premier spectrometer, in which orthogonal acceleration and a single reflectron are adopted: The main features of this instrument are: Resolution: 7000 (FWHM); Accuracy: < 5 ppm; ion beam sampling rate: up to 25 KHz; spectral acquisition rate: up to 20 Hz.

21 The following scheme is referred to a GC-MS system equipped with a high speed ToF (Pegasus HT TOFMS from Leco). The maximum acquisition rate in this case is 500 Hz :

22 Peak reconstruction: scanning vs non scanning analyzers When very fast GC separations are considered, the acquisition rate provided by a non scanning analyzer, like ToF, enables a huge improvement in peak reconstruction, compared to a scanning analyzer: Non scanning (ToF) Scanning (quadrupole)

23 Signal-to-noise ratio: scanning versus non scanning analyzers The increase of mass range has a negative effect on the S/N ratio of a scanning analyzer, like a quadrupole, since a greater portion of ion population is lost when longer scans are required. The phenomenon is absent in the case of non scanning analyzers, like ToF. The effect is apparent when a comparison is made between the S/N response obtained for 0.02 g/ml of fenbuconazole using a time-of-flight (non scanning) or a quadrupole (scanning) LC-MS instrument:

24 Spectral skewing Spectral skewing is an alteration of peak intensity ratios in the MS spectrum, resulting from a non symmetric variation of the analyte concentration and a relatively low spectral acquisition rate. The spectral intensity alteration will depend on the type of peak asymmetry: scan

25 Once all the spectra acquired under the chromatographic peak are averaged the skewing effect will be almost canceled. However, if the peak is not symmetric skewing will affect the final spectrum, as shown in the following case (scan occurring from high to low m/z ratios):

26 Skewing-free spectra are important to perform correctly deconvolution processes on complex chromatographic bands, i.e. to resolve several overlapping peaks on the basis of increasing/decreasing intensity of ions in mass spectra acquired during band elution. A powerful deconvolutive algorithm, based on the systematic extraction of ion currents from spectra underlying a complex band, is embedded in the PeakFind software (Leco):

27 The algorithm needs a high number of acquired spectra per second to work properly, as shown in the following example, related to the GC-MS separation of 9 pesticides in 4 s:

28 Comparison between GC-MS separations with different speed SEF is the Speed Enhancement Factor, a factor used to normalize GC separations to that of a standard capillary column (30 m length, 0.25 mm I.D., 0.25 m thickness of stationary phase), operated at typical gas flow rate (Helium, 1 ml/min = 34 cm/s): The collection frequencies are calculated to give 5 points across full peak width.

29 Comparison between different mass anayzers used in GC-MS in terms of acquisition rate Analyzer Upper mass limit (u) Spectral acquisition rate Resolution Note that the Sector (i.e. a electrostatic/magnetic analyzer) has, by far, the highest resolution but also the lowest acquisition rate.

30 Practical approaches (and compromises) to achieve speed, sensitivity or selectivity in GC-MS

31 Speed (more points per GC peak) How to achieve it Decreasing the number of raw spectra (microscans) to be averaged in full scan mode decreasing the time spent per ion in SIM mode (dwell time, isolation time) Reducing the scan range in full scan mode or the number of monitored ions in SIM Decreasing the number of transients to be summed Analyzer Q, IT Sector Q, IT Sector TOF What is sacrificed Reproducibility of spectra and/or ion ratios Selectivity (ability to identify /confirm) Sensitivity, Selectivity (resolution) Decreasing resolution Sector Selectivity

32 Sensitivity (increased S/N, i.e. decreased LOD if not limited by chemical noise) How to achieve it Using SIM Decreasing spectrum storage rate, i.e. increasing the number of transients to be summed Increasing ion injection time Decreasing resolution Analyzer Q Sector TOF IT Sector What is sacrificed Analytical scope (targeted analysis only) Speed Speed Selectivity (resolution)

33 Selectivity (decreased chemical noise, i.e. potentially decreased LOD, depending on the extent of sample interference) How to achieve it Analyzer What is sacrificed Increasing the resolution Sector Speed, sensitivity Using MS n Using high-resolution IT, triple Q Q-TOF TOF Speed, sensitivity Speed

34 High Resolution TOF-MS: selectivity of detection The following GC High Res TOF-MS traces, obtained for a wastewater extract (spiked with pesticides at 50 pg/ml concentration), show the difference in extracted ion chromatograms for atrazine (windows A and B; m/z ) and chlorpyriphos (windows C and D; m/z ) when changing the extraction window width from 1 to 0.02 m/z units: atrazine chlorpyriphos A fold LOD improvement is achieved using a narrower window

35 Advantages and limitations of High Resolution TOF MS Low spectral acquisition rates, typical of high resolution ToF-MS, provide higher sensitivity but result unavoidably in rather poor peak shapes: Phosalone (50 pg injected) Peak width: ca 1 s J. Chromatogr. A 1058 (2004)

36 Adapting MS detection to GC GC Comprehensive two-dimensional GC (GC GC) provides three-dimensional chromatographic information, characterized by remarkably higher resolution and capacity, compared to one dimensional GC:

37 Comprehensive two-dimensional GC (GC GC) is based on the selection and transfer of all the fractions arising from a conventional GC separation (10-30 m column) to a fast GC separation, accomplished on a shorter column (1-2 m) and at higher speed: a) injector b) first column c) connectors d) modulator e) second column f) detector g) second oven (optional) A fraction eluting from the first column is parked inside the modulator until the previous fraction has completed its further separation in the second column.

38 Possible approaches for fraction transfer A fraction eluting from the first column is alternatively condensed into the downstream (D) and upstream (U) section of the modulator using liquid CO 2 jets. When the jet is shut-off revaporization (followed by transfer downwards) occurs. A fraction eluting from the first column is loaded into a conventional 6-port injection valve. When the valve is switched to the inject position the fraction can be transferred to the second column by an auxiliary gas flow.

39 Combination of two columns having a non polar (D1) and a polar (D2) stationary phase, respectively, introduces a second degree of freedom in analyte separation, i.e. polarity, besides volatility: D2 ( polar column) Low BP, high polarity Low BP, Medium polarity Medium BP, high polarity Medium BP, medium polarity High BP, High polarity High BP, Medium polarity Low BP, low polarity Medium BP, low polarity High BP, low polarity Low BP Medium BP High BP D1 (non polar column)

40 Detection and data processing in GC GC-MS The high-speed conditions of the second column result in very sharp peaks (~200 ms basewidth on average) that require detectors with fast response times (~20 Hz minimum). Flame ionization detectors (FID) and more selective detectors (e.g., electron capture, nitrogen chemiluminescence) that have been developed for high-speed GC ( > 50 Hz data acquisition rates) have successfully been applied also to GC GC. Quadrupole and magnetic sector high-resolution mass spectrometers also have been interfaced, but the slow nature of their operation limits their use to the selective mode, in order to speed up detector response time. TOF MS has been recently introduced by Leco as detector for GC GC with an additional dimension of resolution, i.e. deconvolution of complex bands.

41 Total ion chromatogram contour plot of a congested region in a GC GC chromatogram. Deconvolved slice. chromatographic Each peak marker indicates the presence of a sample component with a unique mass spectrum, as identified by the MS deconvolution software. Each colored trace represents a unique product ion

42 In this example, the PeakFind algorithm, developed by Leco, was applied to a complex band resulting from the co-elution of 4 hydrocarbons, belonging to a Naphta sample: Note that product ion with m/z 43 (fragmentation is not negligible in this case, as EI is performed) is shared by two analytes, the other ones are unique for the three remaining species. The software is able to extract automatically interference-free mass spectra, in which signals from ions shared between co-eluting analytes are accurately proportioned.

43 Species related to Peaks 17 and 18 were detected and accurately identified by library search against the NIST (National Institute for Standards and Technology) database, even though they were separated by only 0.07 seconds. Peaks 19 and 20 were accurately identified despite the high number of shared signals (shown in red) observed in their mass spectra.

44 Advantages of GC GC TOF MS Capability to fulfil demanding analytical tasks Enhanced separation of target analytes from matrix co-extracts Improved detectability: LOD (at S/N = 5) as low as 0.2 to 30 pg injected, detectability enhancement (compared to onedimensional GC TOF-MS) typically fold Reliable identification: full mass spectral information by time-of-flight mass spectrometry, integrated with the deconvolution capability of special software, allows for reliable identification of trace analytes in complex matrices.