Mass Spectrometry Basics

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt

Technische Universität München
Mass Spectrometry - Basics
PD Dr. J. Graßmann; PD Dr. T. Letzel


Parts of a Mass Spectrometer
vacuum
pump
sample
mass
introduction ion source detector computer
analyzer
/inlet
vaccum or atmospheric
pressure vaccum chamber

A MS separates and detects gas-phase ions according to their mass-to-charge

ratio by means of an magnetic and/or electric field.
- MS provides information about the molecular weight of molecules

 Calculation of possible molecular/elemental composition
- Possibility to elucidate the structure of unknown molecules (via fragmentation

patterns („fingerprint“) and obtained highly accurate masses)
- Analysis of complex mixtures by coupling of MS to chromatographic

separation (e.g. High performance liquid or gas chromatography)
- MS provides accurracy, resolution and sensitivity


Mass-to-charge-ratio (m/z), e.g. protonation of lysine
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)


Mass-to-charge-ratio (m/z), e.g. protonation of lysine
Uncharged lysine Singly charged Doubly charged

M lysine [M+H]+ lysine [M+2H]2+
Molecular weight 146.10552 Da 146.10552 Da + 146.10552 Da +

(lysine charged and mass of one mass of two
uncharged)
proton (1x protons (2x
1.0078Da) 1.0078Da)
- m= 147.11332 m= 148.12112
z= 1 z= 2
𝑚 - 147.11332 148.12112
Detection as
𝑧 1 2
- 147.11332 74.06056


Mass spectrometry terminology
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 spectrometry modes
Full scan mode

A m/z target range is given and detected, e.g. m/z 50 to m/z 400.
The given range excludes ions lighter than 50 and heavier than 400 but
considers all peaks within the selected range 50 to 400. The m/z range is freely
selectable within the means of the respective mass spectrometer employed.
However large ranges may decrease the instruments sensitivity.
Selected Ion mode „SIM“

By focusing on only one or only few defined m/z of interest, the detection
limit can be decreased due to the enhanced sensitivity. SIM thus allows the
investigation of very low concentrations. However, the molecules must be
known to be able to select the respective m/z to be detected.


Mass spectrometry modes
MS/MS Scan mode

Some mass spectrometer (details can be found in slides „MS mass analyzer“)
provide the possibility of a so-called MS/MS scan mode, which allows the
fragmentation of a specific target ion(s) into product ion(s) to investigate the
molecular structure of (e.g. unknown) molecules.
Selected reaction monitoring (SRM)

Can be used for the sensitive and specific quantification of molecules.
SRM delivers a unique fragment ion after the fragmentation of the „parent ion“,
which can be monitored and quantified, despite the presence of a complicated
sample matrix. SRM can be considered as „SIM“ of a fragment ion.


Total ion chromatogram (TIC) of full scan mode – Sample introduction via ...
... Direct injection
mass analyzer
ion source
sample
detector
direct introduction of sample to mass spectrometric detection
sample introduction
the MS of sample components
via syringe pump
 Sample is continuously and consistently introduced to the MS without the implemen-

tation of an upstream chromatography
 Each MS scan therefore contains the same molecular composition
 Constant EIC intensities for sample molecules


... Direct injection
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
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)

... Direct injection 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
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)
10609C05(2) #217 RT: 3.98 AV: 1 NL: 1.34E4

Experimental time
T: + p Full ms [100.00-400.00] 10609C05(2) #765 RT: 14.05 AV: 1 NL: 1.25E4
181.93 T: + p Full ms [100.00-400.00]
100
181.93
100
95
95
90
90
85
85 136.00
80
80
75 136.00
75
70
70
65 164.93
65
60
Mass spectra
Relative Abundance
Relative Abundance 60
165.00
55
55
50
45 Approx. same composition 50
45
40
35 for each scan in direct injection 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


Extracted ion chromatogram (EIC) of full scan mode – Sample introduction via ...
... Direct injection 10609C05(2) #217 RT: 3.98 AV: 1 NL: 1.34E4
T: + p Full ms [100.00-400.00]
100
181.93
10609C05(2) #765 RT: 14.05 AV: 1 NL: 1.25E4
T: + p Full ms [100.00-400.00]
100
181.93
95
95
90
90
85
85 136.00
80
80
75 136.00
75
70
70
65 164.93
65
60
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
RT: 0.00 - 20.00 SM: 15G

16.45 NL:
100 1.09E4 RT: 0.00 - 20.00 SM: 15G
4.33
Base Peak 11.40 NL:
1.48 4.88 13.73 14.05 100 1.68 6.22
95 6.55 1.42E4
2.20 3.56 4.07 6.92 m/z=
2.51 3.96
10.68 135.50- Base Peak
13.48 95 4.31
90 0.69 5.91 10.32 136.50 10.13 14.58 m/z=
2.77 8.61 10.94 11.71 17.15 1.24 2.80 4.82 9.29 9.44 14.34 14.96
9.10 17.98 18.67 MS 16.93 181.40-
7.60
7.86 12.85 10609C05( 90 13.97 182.40
85 0.97 13.48 15.42
2) 18.05 MS
19.32 85 18.55 10609C05(
80 2)
80 19.63
75
75
70
70
65
65
EIC 60
of m/z 136 of m/z 181.93

Relative Abundance
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)


... Chromatographic separation
mass analyzer
ion source
detector
sample
e.g. HPLC sample chromatographic mass spectrometric detection

introduction separation of sample components
„autosampler“
 The sample molecules are chromatographically separated depending on their

chemical properties (e.g. polarity) and introduced to the mass spectrometric source
one after another.
 EIC intensity of sample molecules changes over time, due to the molecule reaching
the MS at a defined time point.


Abundance/Intensity
TIC
Experimental time
Abundance/Intensity
Scans
.....
Experimental time
Data analysis software: MassHunter Quanlitative Analysis B.03.01 (Agilent Technologies)

TIC
Abundance/Intensity
Experimental time
Mass spectrum
Different composition


Extracted ion chromatogram (EIC) of full scan mode – Sample introduction via ...
Mass spectrum
TIC
TIC
EIC of m/z 329.2011 of 282.2785
EIC
EIC


Resolution (R):
The ability of a MS to distinguish between two peaks with only slightly different
mass-to-charge-ratio, i.e. the larger the resolution, the better the separation
of peaks.
𝑚 𝑚
𝑅= =
𝑚 𝑚2−𝑚1
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


Resolution (R) m
10% valley resolution 50% valley resolution

m1 m2
m m
50
10
m1 m2 m1 m2

Resolution (R)
Increasing resolution


Accurracy
Isotopes
Natural abundance of isotopes and their effect on molecular weight, e.g. Carbon C:
Stable carbon isotopes 12C and 13C naturally occur with an abundance of 98.9:1.1, i.e.
approximately 1% of C are 13C, whereas approx. 99% are 12C.
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
(98.9% ∗ 12.0 + 1.1% ∗ 13.0)

= 12.011
100%


Accurracy
The average mass of lysine is 146.18944 Da, taking into account all possible isotopic
exchanges, whereas the most abundant lysine (with all carbons being 12C isotopes)
has a mass of 146.10552 Da. The latter is called the monoisotopic mass of lysine.
+1 Da +1 Da
146.10552 Da ~147.10552 Da ~148.10552 Da


Accurracy
Mass spectrum of lysine & its isotopic peaks
1x 13C
12C
146.1 147.1
Abundance: 98.9% 1.1%


Accurracy
Other isotopic patterns
1x 81Br
1x 37Cl
79Br
35Cl
Due to the specific isotopic

patterns of molecules containing
e.g. Cl, MS provides the possibility
to conclude on the presence and
number of certain elements
mass mass+2 mass mass+2

abundance: 75.7% 24.3% 50.7% 49.3%


Isotopic peak of charged molecules
e.g. singly protonated lysine
[M+H]+
1x 13C
12C
Difference between
m/z = 147.11332 12C and 1x13C isotope peak
= ~1
with singly charged molecules
m/z isotopic peak (one 12C to 13C)

= 147.11332 + 1
= 148.11332


Isotopic peak of multiply charged molecules
e.g. doubly protonated lysine
[M+2H]2+
1x 13C
12C
Difference between
𝑚 148.12112 12C and 1x13C isotope peak
= = approx. 0.5
𝑧 2
= 74.06056 with doubly charged molecules
m/z isotopic peak (one 12C to 13C)

148.12112 + 1
= 2
= 74.56056


Accurracy
Depending on the type of mass analyzer employed, the mass accuracy is sufficient
to calculate the elemental composition of a molecule
Time-of-Flight mass spectrometer


Accurracy
to calculate the elemental composition of a molecule
Time-of-Flight mass spectrometer
Isotopic peak =
Isotopic peak = Monoisotopic m/z
Monoisotopic m/z 229.0857 + ~1
611.1611 + ~1 = [M+H]+
= [M+H]+
Mass spectra
Molecular weight of uncharged molecules [M]

= m/z – mass of one proton (1.0078Da)
- 610.1533Da
- 228.0779Da

Accurracy
Calculation of elemental composition
m/z 611.1611 229.0857

observed
Neutral mass 610.1533 228.0779
(calculated)
Elemental ppm Elemental composition ppm
composition
C25H19N14O6 -0.78 C7H12N6OS 3.24
(calc m/z 611.1607) (calc m/z 229.0866)
C29H31N4O7S2 2.87 C6H16N2O5S -2.58
(calc m/z 611.1629) (calc m/z 229.0853)
C27H30O16 -0.72 C14H12O3 0.57
(calc m/z 611.1607) (calc m/z 299.0859)
etc. .... .... .... ...


Accurracy
Calculation of mass deviation/accurracy - ppm (parts per million)
𝑐𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒 −𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒

Mass deviation (ppm) = 𝑥 1 000 000
𝑐𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒
e.g. C14H12O3 (resveratrol)
observed monoisotpic mass [M+H]+ = 229.0857

calculated monoisotopic mass [M+H]+ = 229.08591
229.08591 −229.0857
Mass deviation (ppm) = 𝑥 1 000 000 = 0.92ppm
229.08591


Accurracy
to calculate the elemental composition of a molecule. However there are
generally various possibilities for a structural formula with only one molecular formula!
Possible structural formulas for C27H30O16 (calc m/z 611.1607)
or and many more

....
 Definite identification only via additional mass spectrometric fragmentation pattern (fingerprint),
e.g. in comparison to purchased standard compounds for verification

Accurracy
The same molecule (calc m/z of 137.046336) detected with a
Time-of-Flight MS and a Single Quadrupole MS
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.


Sensitivity is generally determined using a calibration curve with decreasing concentrations
of the molecule of interest, i.e. sensitivity reflects the assessment of the systems response to small
changes in analyte concentrations.
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.

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Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt

Mass Spectrometry - Basics

PD Dr. J. Graßmann; PD Dr. T. Letzel

Mass Spectrometry - Basics

PD Dr. J. Graßmann; PD Dr. T. Letzel

Mass Spectrometry - Basics

A MS separates and detects gas-phase ions according to their mass-to-charge

- MS provides information about the molecular weight of molecules

- Possibility to elucidate the structure of unknown molecules (via fragmentation

- Analysis of complex mixtures by coupling of MS to chromatographic

- MS provides accurracy, resolution and sensitivity

PD Dr. J. Graßmann; PD Dr. T. Letzel

Mass Spectrometry - Basics

PD Dr. J. Graßmann; PD Dr. T. Letzel

Mass Spectrometry - Basics

Uncharged lysine Singly charged Doubly charged

Molecular weight 146.10552 Da 146.10552 Da + 146.10552 Da +

PD Dr. J. Graßmann; PD Dr. T. Letzel

Mass Spectrometry - Basics

PD Dr. J. Graßmann; PD Dr. T. Letzel

Mass Spectrometry - Basics

Full scan mode

Selected Ion mode „SIM“

PD Dr. J. Graßmann; PD Dr. T. Letzel

Mass Spectrometry - Basics

MS/MS Scan mode

Selected reaction monitoring (SRM)

PD Dr. J. Graßmann; PD Dr. T. Letzel

Mass Spectrometry - Basics

 Sample is continuously and consistently introduced to the MS without the implemen-

PD Dr. J. Graßmann; PD Dr. T. Letzel

Mass Spectrometry - Basics

Mass Spectrometry - Basics

10609C05(2) #217 RT: 3.98 AV: 1 NL: 1.34E4

45 Approx. same composition 50

35 for each scan in direct injection 40

Data analysis software: Xcalibur 2.1 software (Thermo scientific)

Mass Spectrometry - Basics

RT: 0.00 - 20.00 SM: 15G

of m/z 136 of m/z 181.93

Data analysis software: Xcalibur 2.1 software (Thermo scientific)

Mass Spectrometry - Basics

e.g. HPLC sample chromatographic mass spectrometric detection

 The sample molecules are chromatographically separated depending on their

PD Dr. J. Graßmann; PD Dr. T. Letzel

Mass Spectrometry - Basics

Mass Spectrometry - Basics

Data analysis software: MassHunter Quanlitative Analysis B.03.01 (Agilent Technologies)

Mass Spectrometry - Basics

Data analysis software: MassHunter Quanlitative Analysis B.03.01 (Agilent Technologies)

Mass Spectrometry - Basics

PD Dr. J. Graßmann; PD Dr. T. Letzel

Mass Spectrometry - Basics

10% valley resolution 50% valley resolution

Mass Spectrometry - Basics

PD Dr. J. Graßmann; PD Dr. T. Letzel

Mass Spectrometry - Basics

(98.9% ∗ 12.0 + 1.1% ∗ 13.0)

PD Dr. J. Graßmann; PD Dr. T. Letzel

Mass Spectrometry - Basics