1 of 26

Mass Spectrometry

by

Dr. Avinash Upadhyay

2 of 26

What does a mass spectrometer do?

1. It measures mass better than any other technique.

2. It can give information about chemical structures.

What are mass measurements good for?

To identify, verify, and quantitate: metabolites, recombinant proteins, proteins isolated from natural sources, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers

3 of 26

Pharmaceutical analysis

Bioavailability studies

Drug metabolism studies, pharmacokinetics

Characterization of potential drugs

Drug degradation product analysis

Screening of drug candidates

Identifying drug targets

Biomolecule characterization

Proteins and peptides

Oligonucleotides

Environmental analysis

Pesticides on foods

Soil and groundwater contamination

Forensic analysis/clinical

Applications of Mass Spectrometry

4 of 26

Inlet

Ionization

Mass Analyzer

Mass Sorting (filtering)

Ion

Detector

Detection

Ion Source

• Solid

• Liquid

• Vapor

Detect ions

Form ions

(charged molecules)

Sort Ions by Mass (m/z)

1330

1340

1350

100

75

50

25

0

Mass Spectrum

Summary: acquiring a mass spectrum

5 of 26

6 of 26

Inlet

Ion

source

Mass

Analyzer

Detector

Data

System

High Vacuum System

Mass Spectrometer Block Diagram

7 of 26

Inlet

Ion

source

Mass

Analyzer

Detector

Data

System

High Vacuum System

Mass Spectrometer Block Diagram

Turbo molecular pumps

8 of 26

Inlet

Ion

Source

Mass

Analyzer

Detector

Data

System

High Vacuum System

HPLC

Flow injection

Sample plate

Sample Introduction

9 of 26

Inlet

Ion

Source

Mass

Analyzer

Detector

Data

System

High Vacuum System

MALDI

ESI

FAB

LSIMS

EI

CI

Ion Source

10 of 26

Methods for Ionization�

Method of Ionization is selected on the basis of sample

Volatile
Non-volatile

Ionization of volatile compound:

Two methods are commonly used for volatile compounds:

Electron Impact Ionization (IE)
Chemical Ionization (CI)

11 of 26

a) Electron ionization (EI)

Sample taken on the probe tip placed near to a heated filament which provides electron and is heated in the ionization chamber causing the sample to form vapour.

Electrons are accelerated from the hot filament to an anode through a potential difference of 70eV

70eV is enough for ionization and causes excessive fragmentation

The molecules are bombarded with high energy electron by removal of an electron. The product is a cation radical.

ABC + e 🡪 [ABC]^+* + 2e

12 of 26

Chemical ionization�

Reagent gas is used in this method such as methane, isobutane or ammonia .
Reagent gas is passed through ion chamber at low pressure
Gas is ionized by electron impact, it undergoes following reaction

A soft ionization method

Reagent gas reagent ion (Z+) M [MZ]+

Examples:

CH₄ + e CH₄ ⁺ + 2e

CH₄ ⁺+ e CH₅⁺+ CH₃

CH₅⁺ + M CH₄ + MH⁺ ( molecular ion, (M + 1)

Little fragmentation; find M+ which is unstable under EI conditions

13 of 26

High voltage applied

to metal sheath (~4 kV)

Sample Inlet Nozzle

(Lower Voltage)

Charged droplets

+

MH⁺

MH₃⁺

MH₂⁺

Pressure = 1 atm�Inner tube diam. = 100 um

Sample in solution

N₂

N₂gas

Partial�vacuum

Electrospray ionization:

Ion Sources make ions from sample molecules�(Ions are easier to detect than neutral molecules.)

14 of 26

15 of 26

�Electrospray ionization �

16 of 26

hν

Laser

1. Sample is mixed with matrix (X) and dried on plate.

2. Laser flash ionizes matrix molecules.

3. Sample molecules (M) are ionized by proton transfer: �XH⁺ + M 🡪 MH⁺ + X.

MH⁺

MALDI: Matrix Assisted Laser Desorption Ionization

+/- 20 kV

Grid (0 V)

Sample plate

17 of 26

MatrixMatrix--assisted laser desorption/ionization assisted ionization

18 of 26

Matrix assisted laser desorption/ionization Matrix-

19 of 26

Inlet

Ion

source

Mass

Analyzer

Detector

Data

System

High Vacuum System

Time of flight (TOF)

Quadrupole

Ion Trap

Magnetic Sector

FTMS

Mass Analyzer

20 of 26

Operate under high vacuum
measure mass-to-charge ratio of ions (m/z)

Resolution:

Resolution is the ability of a mass spectrometer to distinguish between ions of different mass-to-charge ratios. Therefore, greater resolution corresponds directly to the increased ability to differentiate ions.

Quadrupole
time-of-flight
ion traps

Mass analyzers separate ions based on their

mass-to-charge ratio (m/z)

21 of 26

Quadrupole Mass Analyzer

Uses a combination of RF and DC voltages to operate as a mass filter.

Has four parallel metal rods.

Lets one mass pass through at a time.�
Can scan through all masses or sit at one fixed mass.

22 of 26

Quadruple Mass Analyzer

It is more compact, less expensive, High scan rate, get entire spectrum in .1 sec!
A quadrupole analyser consists of 4 rods of hyperbolic or circular cross-section arranged symmetrically.
A voltage consisting of a DC component U and an RF (Radio Frequency) component V₀ is applied between adjacent rods.
Opposite rods are connected. Ions entering the rod assembly at one end with an energy of 10eV-20eV oscillate in the x and y directions.
The resolution is selected by changing the value of U . High values of U give high resolution. This reduces the region of stable oscillation, and thus the sensitivity.
Quadruple instruments have a constant value of DM throughout the mass range, so might have a resolution of 300 at mass 300, and 1000 at mass 1000.

23 of 26

mass scanning mode

m1

m3

m4

m2

m3

m1

m4

m2

single mass transmission mode

m2

m3

m1

m4

m2

Quadrupoles have variable ion transmission modes

24 of 26

TOF analyser is usually coupled with a MALDI source .

If an ion is accelerated with a voltage, its resulting velocity is a function of its mass/charge ratio.

In a TOF analyser, separation is by velocity, and the mass of an ion may be determined by the time taken for it to travel a fixed distance.

Ions are accelerated by electric field of 1000-10000V for a pulse of 25 seconds.�
Ions then drift through an evacuated ‘drift tube’�
All ions enter with same KE because same acceleration V

Same KE, larger masses move slower therefore larger ions take longer Flight time and small molecules get to the far end first ; takes shorter flight time.

Time-of-flight (TOF) Mass Analyzer

25 of 26

Time-of-flight (TOF) Mass Analyzer

+

Source

Drift region (flight tube)

detector

V

Ions are formed in pulses.
The drift region is field free.
Measures the time for ions to reach the detector.
Small ions reach the detector before large ones.

26 of 26

Ion Trap analyser:

In the ion trap, a ring of hyperbolic internal cross-section is enclosed by two end caps.

The end caps are at earth potential and an RF voltage is applied to the ring.

For a particular RF amplitude, ions above a threshold mass are “trapped” in

stable orbits within the assembly.

Scanning is accomplished by trapping all ions above a low threshold, and turning off the ion source.

The RF amplitude is then increased and ions escape the trap into the detector in order of increasing mass.