The data system records the signal strength of all ions being released to an ion detector at a given time. A data system stores all the spectral information from the chromatographic run in a three-dimensional block of data. An ion current chromatogram ortotal ion chromatogram(TIC; Figure 7.7a) is a plot of the sum
DATA AND CONTROL SYSTEMS 67
(a)
(b) 25.16
21.36 24.25
30.81 26.53
17.00
29.47
37.66 33.71
19.41 15.96 12.50
14.84 100
90 80 70 60 50 40 30 20 10 0
Relative Abundance
5 10 15 20 25 30 35 40 45 50
Time (min)
100 90 80 70 60 50 40 30 20 10 0
Relative Abundance
200 400 600 800 1000 1200 1400 1600 1800
1821.9 1714.8 1621.9 1456.5 966.8
1138.1 1241.7 1322.0 803.8
730.7 680.9 545.2 531.2 490.0 473.2
429.3 415.4 275.2 229.2 130.4
FIGURE 7.7 (a) Total ion chromatogram and (b)m/z spectrum. (From Tiller et al., 1997.)
of the signal strengths of all ions present versus time. Aspectrum(Figure 7.7b), is a plot of signal strength versus mass/charge (m/z) at a given time. For an IS interface system, this will be a single mass for the molecular ion. In an ion trap system we can read the molecular ion, flood the chamber with a heavy gas, fragment the molecular ion, and then read the ion fragment masses. If we are displaying fragmentation data, the spectrum will be a bar chart of all the fragment masses and their signal strengths. The frequency of the dc/RF signal is calibrated with standards of known mass and charge to provide them/zvalue.
It is obviously advantageous to acquire as much information as we can about a fragmentation sample the first time we run a new chromatogram on unknown material. We can set the mass spectrometer in scan mode to acquire data from
68 LC/MS OVERVIEW
a range of dc/RF frequencies wide enough to cover the expected range of m/z values while excluding low mass values from air and solvent. We need to set a signal-sampling rate and allow for the length of time to make the chromatographic run, and all these data have to fit in the data storage space available. The greater the number of sample points we can average at a given m/zfrequency and time, the more confidence we will have in the data point. If we want to scan a wider m/zrange, we may have to use a lower sampling rate and reduce the accuracy of the signal.
The signal coming from the mass spectrometer’s detector is a continuous voltage changing with time, an analog signal. Data processing in a computer works with discrete bits of digital data. We must convert the analog signal from the detector into a digital data stream to use it in our computer system. The signal conversion process involves measuring the vertical displacement of a series of time slices of known duration using an A/D conversion microprocessor card (Figure 7.8).
The value of the time duration and the changing values for the signal intensity are stored as the data set. A useful analogy is the creation of a motion picture. The moving image projected on a screen is created by viewing a series of changing still photo frames equivalent to the time-slice data taken from the analog mass detector signal. The still photos represent digital data combined to produce the analog motion picture. Data from the mass controller signal selecting the m/z range’s change with time is combined with the detector voltage versus time database to provide the information the computer needs to produce a total ion chromatogram, and at any given time, the mass spectra made up of the voltage signal versus them/zvalue.
Analog output
Detector
Interface box
A/D converter
Digital Input
Analog signal +
+ −GRn −
NET
FIGURE 7.8 A/D data conversion.
PEAK DETECTION, ID, AND QUANTITATION 69 7.5 PEAK DETECTION, ID, AND QUANTITATION
When we are operating an LC/MS system, we usually set the data system so that we can monitor the total ion current as a chromatograph on a display screen as it is being generated. Each point we see on a chromatogram is usually the summa- tion of eight or more dc/RF scans. We may want to extract and display a spectrum as the peaks come off and we see a peak of interest, to check its molecular weight or its fragmentation pattern if we are running an MS/MS experiment. Qualitative information can be derived from the spectrum as we analyze the sample. Frag- mentation patterns of interest can be sent to the data system for comparison with libraries of known fragmentation patterns to provide definitive compound identifi- cation beyond the molecular weight. Quantitative information can be obtained for HPLC chromatographic peaks from either a total ion chromatogram or by using the data system to compare the information to a calibration curve of known amounts of the material of interest versus peak heights or peak areas.
In obtaining data for a known sample, increased signal sensitivity can be obtai- ned by using the single-ion monitoring (SIM) mode by selecting only a single m/z frequency and acquiring data only at that point to produce a single-ion chromatogram. The sampling rate no longer has to be spread over the entire m/zspectrum, and the number of measurements can be increased for this single- ion mass.
The combination of fast analyzer scanning, fast detector recovery, and high- capacity data systems allows acquisition of about 25,000 data points per second.
This means that a mass spectrum run in scan mode from 35 to 550m/z can average 8 to 10 scans in 1 second. Run in single-ion mode the same mass spec- trometer could analyze 10m/zmass regions in a step scan and gain a tremendous gain in sensitivity by averaging a much higher number of points at each of the 10 points.
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