Introduction to UCL Chemistry

Mass Spectrometry Facility

https://www.ucl.ac.uk/chemistry/mass-spec

A mass spectrometer (MS) is an analytical instrument that produces a

beam of gas phase ions from sample (analytes), sorts the resulting

mixture of ions according to m/z ratios using electrical or magnetic fields

(or combination), and provides analog or digital output signals (peaks)

from which the m/z ratio and the intensity (abundance) of each detected

ionic species may be determined.

A mass spectrometer can only analyse IONS (electrically charged

molecules) and they must be in the GASEOUS phase.

Mass spectrometers MEASURE m/z (mass-to-charge ratios); where m is

the total of the masses of the component atoms and z is the number of

charges on the ion (positive or negative) expressed in whole numbers

(1,2,3, etc.).

1. Vaporised sample enters the ion source

2. Unresolved ion beam is accelerated to

the analyser where the ions are

separated either “in space” or “in time”

3. Ions with resolved m/z travel to the

detector

Mass spectrometers are composed of a sample

inlet, ion source, mass analyser, detector,

vacuum system, and computer.

While multiple types of each components can be

mixed and matched, e.g., different ion sources

with different analysers, the ionisation, analysis,

detection sequence must be maintained.

Analysers can be used in single, e.g., Q or TOF,

or in multi-analysers formats, e.g., Q-TOF and

TOF/TOF, with a collision cell incorporated

between the two analysers.

Computers control instrument operation, data

acquisition and data processing.

Block diagram of a mass spectrometer

1 2 3

Accela LC – LTQ mass spectrometer Waters LCT Premier Q-TOF mass spectrometer

The ion trap mass analyser The Q-TOF mass analyser

Waters UPLC – SQD mass spectrometer

The single quadrupole mass analyser

Aqilent LC – 6510 Q-TOF mass spectrometer

The Q-TOF mass analyser

Electron Ionisation (EI) source

The EI source located inside of the instrument’s vacuum chamber and held

at 130°C to maintain the analyte molecules in the vapor phase. Electrons

from the filament are collimated by a magnetic field and traverse the source

in a helical path (to increase efficiency) to the electron trap. The electrons

react with the analyte molecules to produce radical cations.

Reaction: M + e- → M+. + 2e-

Electrospray Ionisation (ESI)

As the droplets shrink the charge

density increases to the point at

which the droplets are no longer

stable and ions are released from the

liquid matrix into the vapor phase.

The choice of ionisation is determined by the polarity of the analyte molecules.

Mass ranges for the different ionisation methods. EI is selected for structural

determination, whereas ESI and MALDI are used for molecules above 1,000 Da.

Energy is added to molecules during ionisation. The distribution of the energy may result in the

breaking of chemical bonds and, consequently, in fragment ion formation. The fragmentation may

be so extensive that no molecular ion is observed.

The form of the molecular ion depends on the mode of ionisation and can include [M]+, [M+H]+ and

other ions, e.g., [M+Na]+.

The base peak represents the most stable ion resulting from the ionisation process and is the most

abundant peak in the spectrum. The intensities of all other are normalised with respect to the base

peak.

Ions, normally of lesser intensities and to the right of each molecular/fragment ion generally

represent isotopic species. Typically, but not always, isotope ions reflect the presence of carbon-

13 (13C).

A mass spectrum is a graphical representation of the m/z values (x-axis) of the ions together with

their intensities (y-axis), collected over a specified mass range.

The atoms of elements are composed of protons, electrons and neutrons. An element is

defined by the number of protons present in the nucleus. However, the number of

neutrons may vary over a small range, yielding products known as isotopes.

Carbon has three isotopes incorporating six, seven or eight neutrons yielding 12C, 13C

and 14C. The presence of the 13C isotope, which occurs with a natural abundance of

~1.1% per carbon atom results in the isotope peaks that are commonly found on the

right-hand side of all ions in mass spectra.

https://www.ucl.ac.uk/chemistry/mass-spec

(c)

(d)

EI mass spectra

[M]+˙

cr621_ei # 7-8 RT: 0.73-0.82 AV: 2 SB: 1 1.99 NL: 4.81E6 T: + c EI Full ms [59.50-800.50]

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

115.0

160.0

174.1

143.0

299.0 301.0

102.0

89.0

254.0

175.1

214.9

302.0 212.9

190.1

Instrument Resolution : 6120

Theoretical Mass (C11H10BrNO4) : 298.9788

Measured Mass : 298.9787

Error : 0.54 ppm

Ionisation mode and polarity

Mass range Electron Ionisation (+) mode

cr621_ei #7-8 RT: 0.73-0.82 AV: 2 SB: 1 1.99 NL: 4.81E6T: + c EI Full ms [59.50-800.50]

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

115.0

160.0

174.1

173.0

143.0

116.0

299.0301.0

144.0

102.0

89.062.8

254.0

130.0

175.1159.0

214.974.9

86.9 225.064.9 302.0212.9 226.9

145.0239.091.0 190.1

EXPERIMENTAL

THEORETICAL

288 290 292 294 296 298 300 302 304 306 308 310 312 314

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

299.0301.0

300.0 302.0

299.0301.0

300.0 302.0

NL:3.29E6

cr621_ei#7-8 RT: 0.73-0.82 AV: 2 SB: 1 1.99 T: + c EI Full ms [59.50-800.50]

NL:4.44E5

C 11 H10 BrNO4: C 11 H10 Br1 N1 O4

pa Chrg 1

4 x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

8

8.5

9

9.5

+ESI Scan (rt: 1.373 min) Frag=175.0V Tag_01.d

417.0363

319.0596

833.0650

Counts vs. Mass-to-Charge (m/z)

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

[M+H]+

[2M+H]+

OH

O

S

O

O

OH

OH

O

P

OH

OOH

NH

CH3

O

NH2

Exact mass: 417.03634 Da

Chemical formula: C11

H18

N2O

11PS

[M+H]+

Mass error: 0.09 ppm

H+

[M-H3PO4]+

Ionisation mode

and polarity Electrospray ionisation (+) mode

[M-3H]3-

[M-2H]2-

3 x10

0

0.025

0.05

0.075

0.1

0.125

0.15

0.175

0.2

0.225

0.25

0.275

0.3

0.325

0.35

0.375

0.4

0.425

0.45

0.475

0.5

0.525

0.55

0.575

0.6

0.625

0.65

0.675

0.7

0.725

0.75

0.775

0.8

0.825

0.85

0.875

0.9

0.925

0.95

0.975

1

1.025

1.05

1.075 -ESI Scan (rt: 7.115-7.264 min, 4 scans) Frag=20.0V AP1HP1_MS.d

1915.0

2873.0

Counts vs. Mass-to-Charge (m/z)

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200

m/z

100%

3200 1600 0

5' G A C A T T I C T T T I C A A T I T C 3'

[M-3H]3-

[M-2H]2-

Ionisation mode and polarity Electrospray ionisation (-) mode

S1494

m/z100 200 300 400 500 600 700 800 900 1000

%

0

100

S1494 9 (0.317) 1: TOF MS ES+ 4.79e3241.0

141.0

Instrument Resolution : 13,500

Theoretical Mass (C9H11N4S2+H) : 240.0498

Measured Mass : 240.0499

Error : 0.42 ppm

Ionisation mode and

polarity Electrospray ionisation (+) mode

Type of mass analyser

S1494

m/z222 224 226 228 230 232 234 236 238 240 242 244 246 248 250 252 254 256 258 260 262 264

%

0

100

%

0

100

S1494 (0.036) 1: TOF MS ES+ 8.04e12241.1

242.1 243.1

S1494 10 (0.345) 1: TOF MS ES+ 9.42e3241.0

242.0 243.0

theoretical

experimental

Ionisation mode and

polarity

Type of mass analyser

Ion counts