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vacuum
pump
sample
mass
introduction ion source detector computer
analyzer
/inlet
vaccum or atmospheric
pressure vaccum chamber
Uncharged lysine Singly charged lysine Doubly charged lysine Singly charged lysine
M [M+H]+ [M+2H]2+ [M+H]+
Negative charge neutralizes
one positive charge.
Drawing of chemical structures was performed with „MarvinSketch 14.8.25.0“, ChemAxon (www.chemaxon.com)
TIC
A TIC is a chromatogram created by the summed intensities of all mass spectral
peaks belonging to the same scan/time point of analysis across the entire range
of experimental time.
Scan
The sum of all mass spectral peaks per detected time point
Mass spectrum
Represents the entirety of ions detected per scan or over a selected
experimental time range.
EIC/XIC
EIC represents the abundance of one single or more molecules plotted against
the time. It is a small „extract“ of the entire date set (TIC).
mass analyzer
ion source
sample
detector
direct introduction of sample to mass spectrometric detection
sample introduction
the MS of sample components
via syringe pump
90
85
80
75
70
65
60
Relative Abundance
55
TIC 50
45
40
35
30
25
20
15
10
0
0 2 4 6 8 10 12 14 16 18
Time (min)
Experimental time
RT: 0.00 - 20.00
2.47 NL:
100 2.01 2.56 4.07 4.40 6.26 6.79 6.88 7.91 9.23 10.87 6.40E5
0.49 14.39 16.71
1.63 11.53 13.40 17.33 TIC MS
95 16.43 19.54
0.71 12.01 10609C05(
2)
Abundance/Intensity
90
Scans:
85
80
75
70
65
60
- dependending on mass spectrometric setting e.g. 1
Relative Abundance
Scans 55
50
45
scan per second
40
35
- Sum of the intensity of all ions in each individual scan
30
25
20
- TIC and scans include sample molecules as well as
15
10
0
..... background signals
0 2 4 6 8 10 12 14 16 18
Time (min)
Experimental time
Data analysis software: Xcalibur 2.1 software (Thermo scientific)
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
90
85
80
75
70
65
TIC 60
Relative Abundance
55
50
45 Underlying each scan is the respective mass spectrum, which contains the
40
35
30
detected ions of the scan
25
20
15
10
0
0 2 4 6 8 10 12 14 16 18
Time (min)
Mass spectra
Relative Abundance
Relative Abundance 60
165.00
55
55
50
45
40
35
282.13
282.13
30
25
sample introduction mode 30
25
122.93 280.00
122.93
20
20
15
15
146.93 183.00
10
182.93 283.13 10 118.87 361.00
136.87 214.00 283.20 297.87
119.00 204.13 226.00 255.87 299.00 361.00 380.73 183.93
5 319.13 352.00 397.07 5 116.67
213.87 226.07
255.73 315.20 343.20 362.87 387.20
399.13
0 0
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
m/z m/z
Mass spectrum
Relative Abundance
60
Relative Abundance
165.00
55
55
50
50
45
45
40
40
35
35
282.13
282.13
30
30
25 122.93 280.00
25
122.93
20 20
15 15
146.93 183.00
10 10 361.00
182.93 283.13 118.87
136.87 214.00 283.20 297.87
119.00 204.13 226.00 255.87 361.00 183.93
5 299.00 319.13 352.00
380.73
5 116.67
213.87 226.07
255.73 315.20 343.20 362.87 387.20
397.07 399.13
0 0
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
m/z m/z
EIC 60
60
Relative Abundance
55
55
50
50
45
45
40
40
35
35
30
25
20
EICs constant, due to direct injection of the sample 30
25
20
15
10
(each scan with the same ion composition) 15
10
5 5
0 0
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18
Time (min) Time (min)
ion source
detector
sample
TIC
Experimental time
Abundance/Intensity
Scans
.....
Experimental time
Data analysis software: MassHunter Quanlitative Analysis B.03.01 (Agilent Technologies)
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
TIC
Abundance/Intensity
Experimental time
Mass spectrum
Different composition
Mass spectrum
TIC
TIC
EIC of m/z 329.2011 of 282.2785
EIC
EIC
e.g. assuming a resolution (R) of 5000, one can differentitate between m/z 50.00 (m1)
and m/z 50.01 (m2) or m/z 100.00 and m/z 100.02
50 100
5000 = 5000 =
𝑚2−50 𝑚2−100
or
m2 = 50.01 m2 = 100.02
50
10
m1 m2 m1 m2
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
Increasing resolution
Average mass of a molecule is defined as the weighted average of the masses of all its
naturally occuring stable isotopes, e.g. Carbon C
+1 Da +1 Da
Drawing of chemical structures was performed with „MarvinSketch 14.8.25.0“, ChemAxon (www.chemaxon.com)
1x 13C
12C
146.1 147.1
Abundance: 98.9% 1.1%
1x 81Br
1x 37Cl
79Br
35Cl
Difference between
m/z = 147.11332 12C and 1x13C isotope peak
= ~1
with singly charged molecules
Difference between
𝑚 148.12112 12C and 1x13C isotope peak
= = approx. 0.5
𝑧 2
= 74.06056 with doubly charged molecules
Isotopic peak =
Isotopic peak = Monoisotopic m/z
Monoisotopic m/z 229.0857 + ~1
611.1611 + ~1 = [M+H]+
= [M+H]+
Mass spectra
229.08591 −229.0857
Mass deviation (ppm) = 𝑥 1 000 000 = 0.92ppm
229.08591
Definite identification only via additional mass spectrometric fragmentation pattern (fingerprint),
e.g. in comparison to purchased standard compounds for verification
Drawing of chemical structures was performed with „MarvinSketch 14.8.25.0“, ChemAxon (www.chemaxon.com)
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
Depending on the issue to be addressed, different mass spectrometric techniques are available, which differ widely
e.g. regarding the provided accurracy. ToF-MS are generally employed if one needs highly accurate molecular
weights or wants to investigate unknown molecules, whereas single quadrupoles are more robust and can be used
for applications in which an inaccurat molecular weight is sufficient, e.g for the routine screening of known molecules.
Detection limit (limit of detection, LOD) is the lowest quantity of a molecule, which can be
reliably distinguished from the sample matrix/background signals/noise.
It is dependent on the sample matrix, the solvents, the mass spectrometer employed, the
ionization efficiency of the molecule(s) and the mass spectrometric settings. Detection limits can
range in picogram or femtogram concentrations.
The instrument background are the signals, which are detected when a blank (sample without the
molecule or molecules of interest) is measured.
Signal-to-Noise (S/N) is the ratio calculated out the of the analyte signal and the background noise.