Mass spectrometry

ByKommineni.vidyachowdhary M.pharm

Vaagdevi pharmacy collegewarangal

1

MASS MASS SPECTROMETRYSPECTROMETRY

Mass spectrometry is the most accurate method for determining the molecular mass of the compound and its elemental composition.

It is also called as positive ion spectra or line spectra.

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Introduction• Mass spectrometry (Mass Spec or MS) uses high energy electrons to

break a molecule into fragments.

• It does not involve the absorption or emission of light.• A beam of high-energy electrons breaks the molecule apart.

• The masses of the fragments and their relative abundance reveal information about the structure of the molecule.

• Separation and analysis of the fragments provides information about:

– Molecular weight

– Structure

Mass Spec Principles

IonizerIonizer

Sample

+_

Mass AnalyzerMass Analyzer DetectorDetector

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Schematic of Mass Spectrometry

Ionizer

↓Mass-to-charge ratio Analyzer

↓

Detector

How does a mass spectrometer work?

• Ionization method– MALDI– Electrospray(Proteins must be charged

and dry)

• Mass analyzer– MALDI-TOF

• MW – Triple Quadrapole

• AA seq– MALDI-QqTOF

• AA seq and MW– QqTOF

• AA seq and protein modif.

Create ions Separate ions Detect ions

• Mass spectrum

• Database analysis

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What is a Mass Spectrometer?

A Mass Spectrometer is a machine that weighs molecules ! (by measuring the mass to charge ratio of ions)

SourceEICIESIAPCIAPPIMALDI

DispersionTOFFT-ICRSectorQuadTrap

DetectorFaraday CupChanneltronMCP

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Mass Spectrometry Categorization• Based on ionization:

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometryElectrospray ionization (ESI) mass spectrometryInduction-coupled plasma mass spectrometry (ICP-MS)Atmospheric Ionization Mass SpectrometrySecondary Ionization Mass Spectrometry (SIMS)

• Based on M/Z Separation:Quadrupole Mass spectrometryIon trap mass spectrometrymagnetic sector mass spectrometrytime-of-flight mass spectrometryFourier Transform Ion Cyclotron Resonance Mass SpectrometryIon Mobility Mass Spectrometry

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Mass Spectrometry Categorization(continued)

• Based on Applications:Environmental Mass SpectrometryBiological Mass SpectrometryCell Mass SpectrometryPortable Mass Spectrometry

• Based on Configuration:Tandem Mass Spectrometry

• Based on Sample Introduction:GCMS; LCMS, Electrophoresis Mass Spectrometry

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MS Operation• Nearly all mass spectrometers need to operate under

high vacuum condition with the pressure less than 10 -5 Torr with the only exceptions of an ion trap mass spectrometer (milli-Torr) and an ion mobility mass spectrometer (Torr).

• Never turn on a mass spectrometer without knowing the chamber pressure.

• A tour to major mass spectrometry facilities in Genomic Research Center, Sinica will be arranged.

Mass spectrometersMass spectrometersLinear Tim e Of Flight tube

Reflector Tim e Of Flight tube

detector

reflector

ion source

ion source

detector

tim e of flight

tim e of flight

• Time of flight (TOF) (MALDI)Time of flight (TOF) (MALDI)– Measures the time required for ions to fly down the length

of a chamber. – Often combined with MALDI (MALDI-TOF) Detections from

multiple laser bursts are averaged. Multiple laser

• Tandem MS- MS/MSTandem MS- MS/MS-separation and identification of compounds in complex mixtures- induce fragmentation and mass analyze the fragment ions. - Uses two or more mass analyzers/filters separated by a

collision cell filled with Argon or Xenon

• Different MS-MS configurations– Quadrupole-quadrupole (low energy)– Magnetic sector-quadrupole (high)– Quadrupole-time-of-flight (low energy)– Time-of-flight-time-of-flight (low energy)

Typical Mass Spectrometer

LC/LC-MS/MS-Tandem LC, Tandem MS

Typical Mass Spectrum• Characterized by sharp, narrow peaks

• X-axis position indicates the m/z ratio of a given ion (for singly charged ions this corresponds to the mass of the ion)

• Height of peak indicates the relative abundance of a given ion (not reliable for quantitation)

• Peak intensity indicates the ion’s ability to desorb or “fly” (some fly better than others)

m/z ratio:

Molecular weight divided by the Charge on this protein

All proteins are sorted based on a

mass to charge ratio (m/z)

Typical Mass Spectrum

aspirin

Relative Relative AbundanceAbundance

120 m/z-for singlysingly charged ion this is the mass

Resolution & Resolving PowerResolution & Resolving Power• Width of peak indicates the resolution of the MS

instrument

• The better the resolution or resolving power, the better the instrument and the better the mass accuracy

• Resolving power is defined as:M is the mass number of the observed mass (M) is the

difference between two masses that can be separated

Resolution in MSResolution in MS

Resolution in MSResolution in MS

QTOF

783.455

784.465

785.475

783.6

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Introduction to Mass Spectrometry

Sample introduction

IonizationMinimize collisions, interferences

Separatemasses

Count ionsCollect results

Nier-type mass spec

The sample cone isolates thetorch from the interior.

The torch box of an Agilent 7500 ICPMS

spray chambertorchAr feed

RF coil

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N

SSource magnets

Filament

Collector

Sample Inlet+

+ ++

+

+++

+

++

+

+

+

+

+

Extraction lenses

Electron ionization

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In this type anode and cathode are arranged with a very

fine gap (0.5 to 2mm) which may serve as a slit.

The gaseous sample introduced at the anode points where

the electric field is concentrated.

The ionisation of the sample takes place by extraction of

electrons from the sample by microtips of the anode.

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N

SSource magnets

Filament

Collector

Sample Inlet+

+ ++

+

+++

+

++

+

+

+

+

+

Extraction lenses

+

+++

++

++

+ +

+

+

+

+

Chemical ionization

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These are the devices used to separate the ions produced in the ion source into their individual m/z ratios and focus them on the detector.

A number of different mass analysers exist in mass spectrometry.

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Magnetic sector – single focussing - double focussing

Quadrupole analysers

Ion Trap (Quistor) devices

Time-of-Flight (TOF) analysers

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Magnetic sectors – single focussing•Generally bulky and expensive.•Earliest type of analyser and still popular.•High accelerating potential especially in comparison with other methods (usually 4-10kV).•Magnets wedge shaped and must provide homogeneous fields.•Ion beam enters and exits at exactly 90˚.•Focuses ions according to their momentum.

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Magnetic sectors – double focussing

•Adds a second electric sector to provide energy focussing of ions independent of mass.•Double focussing means that both the energy and momentum focus is designed to coincide at the collector slit.•Very high mass resolution can be achieved by this arrangement.•Very bulky and expensive but high performance.

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Quadrupole Mass Analyzer

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+

+

++

The quadrupole consists of four parallel rods. The opposing rodshave the same polarity whilst adjacent rods have opposite polarity.

Each rod is applied with a DC and an RF voltage.Ions are scanned by varying the DC/Rf quadrupole voltages.

Only ions with the selected mass tocharge ratio will have the correct oscillatory pathway in the Rf field.

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• Consists of ring electrode and two end caps

• Principle very similar to quadrupole

• Ions stored by RF & DC fields• Scanning field can eject ions

of specific m/z• Advantages - MS/MS/MS…..

- High sensitivity full scan MS/MS

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• In this type of analyser the sorting of the ions is done in absence of magnetic field.

• It operates on the principle that, if the ions produced are supplied with equal energy and allowed to travel predetermined distance then they will acquire different velocities depending of their masses.

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• The detector records the charge induced when an ion passes by or hits a surface

• Electron Multipliers (EM)*– Most common detector– -Can Detect positive and negative ions

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• Faraday Cup– Least expensive detector– Captured ions transfer charge to cup– used to calibrate other MS detectors

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• Photographic detection: This detector system is most

sensitive than any other detector because the photoplate integrates the ion signal over a period of time.

The photoplates are processed by the usual photographic techniques and read with the aid of densitometer.

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Different Ionization Methods• Electron Impact (EI - Hard method)

– small molecules, 1-1000 Daltons, structure

• Fast Atom Bombardment (FAB – Semi-hard)– peptides, sugars, up to 6000 Daltons

• Electrospray Ionization (ESI - Soft)– peptides, proteins, up to 200,000 Daltons

• Matrix Assisted Laser Desorption (MALDI-Soft)– peptides, proteins, DNA, up to 500 kD

Electron Impact IonizationElectron Impact Ionization

• Sample introduced into instrument by heating it until it evaporates

• Gas phase sample is bombarded with electrons coming from rhenium or tungsten filament (energy = 70 eV)

• Molecule is “shattered” into fragments (70 eV >> 5 eV bonds)

• Fragments sent to mass analyzer

EI Fragmentation of CHEI Fragmentation of CH33OHOH

CH3OH CH3OH+

CH3OH CH2O=H+ + H

CH3OH + CH3 + OH

CHO=H+ + HCH2O=H+

Why wouldn’t Electron Impact be suitable for analyzing proteins?

Why You Can’t Use EI For Analyzing Why You Can’t Use EI For Analyzing ProteinsProteins

• EI shatters chemical bonds

• Any given protein contains 20 different amino acids

• EI would shatter the protein into not only into amino acids but also amino acid sub-fragments and even peptides of 2,3,4… amino acids

• Result is 10,000’s of different signals from a single protein -- too complex to analyze

Soft Ionization MethodsSoft Ionization Methods

337 nm UV laser

MALDI

cyano-hydroxycinnamic acid

Gold tip needle

Fluid (no salt)

ESI

+_

Soft IonizationSoft Ionization • Soft ionization techniques keep the molecule of interest

fully intact

• Electro-spray ionization first conceived in 1960’s by Malcolm Dole but put into practice in 1980’s by John Fenn (Yale)

• MALDI first introduced in 1985 by Franz Hillenkamp and Michael Karas (Frankfurt)

• Made it possible to analyze large molecules via inexpensive mass analyzers such as quadrupole, ion trap and TOF

Ionization methodsIonization methods• Electrospray mass spectrometry (ESI-MS)

– Liquid containing analyte is forced through a steel capillary at high voltage to electrostatically disperse analyte. Charge imparted from rapidly evaporating liquid.

• Matrix-assisted laser desorption ionization (MALDI)– Analyte (protein) is mixed with large excess of matrix (small organic molecule)– Irradiated with short pulse of laser light. Wavelength of laser is the same as absorbance max

of matrix.

Electrospray IonizationElectrospray Ionization• Sample dissolved in polar, volatile buffer (no salts)

and pumped through a stainless steel capillary (70 - 150 m) at a rate of 10-100 L/min

• Strong voltage (3-4 kV) applied at tip along with flow of nebulizing gas causes the sample to “nebulize” or aerosolize

• Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size (still carrying charges)

Electrospray (Detail)Electrospray (Detail)

Electrospray IonizationElectrospray Ionization• Can be modified to “nanospray” system with flow < 1

L/min

• Very sensitive technique, requires less than a picomole of material

• Strongly affected by salts & detergents

• Positive ion mode measures (M + H)+ (add formic acid to solvent)

• Negative ion mode measures (M - H)- (add ammonia to solvent)

Positive or Negative Ion Mode?Positive or Negative Ion Mode?

• If the sample has functional groups that readily accept H+ (such as amide and amino groups found in peptides and proteins) then positive ion detection is used-PROTEINS

• If a sample has functional groups that readily lose a proton (such as carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative ion detection is used-DNA

Matrix-Assisted Laser Desorption Matrix-Assisted Laser Desorption IonizationIonization

337 nm UV laser

MALDI

cyano-hydroxycinnamic acid

MALDIMALDI

• Sample is ionized by bombarding sample with laser light

• Sample is mixed with a UV absorbant matrix (sinapinic acid for proteins, 4-hydroxycinnaminic acid for peptides)

• Light wavelength matches that of absorbance maximum of matrix so that the matrix transfers some of its energy to the analyte (leads to ion sputtering)

HT Spotting on a MALDI Plate

MALDI IonizationMALDI Ionization

++

+

+

-

--

++

+

+

-

---++

Analyte

Matrix

Laser

+

++

• Absorption of UV radiation by chromophoric matrix and ionization of matrix

• Dissociation of matrix, phase change to super-compressed gas, charge transfer to analyte molecule

• Expansion of matrix at supersonic velocity, analyte trapped in expanding matrix plume (explosion/”popping”)

+

+

+

MALDIMALDI• Unlike ESI, MALDI generates spectra that have just a singly

charged ion

• Positive mode generates ions of M + H

• Negative mode generates ions of M - H

• Generally more robust that ESI (tolerates salts and nonvolatile components)

• Easier to use and maintain, capable of higher throughput

• Requires 10 L of 1 pmol/L sample

Principal for MALDI-TOF MASS Principal for MALDI-TOF MASS

++

+++

++

+ ++

p u lse dU V o r IR la se r(3 -4 n s )

d e te c to r

vacuum

strong electric field

Time Of Flight tube

p e p tid e m ixtu ree m b e d de d in ligh t a b so rb in g ch em ica ls (m a trix )

c lo u d o fp ro ton a te dp e p tid e m o lecu le sa ccV

Principal for MALDI-TOF MASSPrincipal for MALDI-TOF MASS

Linear Time Of Flight tube

Reflector Time Of Flight tube

detector

reflector

ion source

ion source

detector

tim e of flight

tim e of flight

MALDI MALDI == SELDISELDI

337 nm UV laser

MALDI

cyano-hydroxycinnaminic acid

MALDI/SELDI SpectraMALDI/SELDI Spectra

Normal

Tumor

Background of fragmentation

• The impact of a stream of high energy electrons causes the molecule to lose an electron forming a radical cation.– A species with a positive charge and one unpaired

electron

+ e-C H

H

HH H

H

H

HC + 2 e-

Molecular ion (M+)

m/z = 16

Background

• The impact of the stream of high energy electrons can also break the molecule or the radical cation into fragments.

(not detected by MS)

m/ z = 29

molecular ion (M+) m/ z = 30

+ C

H

H

H

+ H

HH C

H

H

C

H

H

H C

H

H

C

H

H

H C

H

H

+ e-H C

H

H

C

H

H

H

m/z = 15

Background

• Molecular ion (parent ion):– The radical cation corresponding to the mass of the

original molecule

• The molecular ion is usually the highest mass in the spectrum– Some exceptions w/specific isotopes– Some molecular ion peaks are absent.

HH

H

HC H C

H

H

C

H

H

H

Background

• Mass spectrum of ethanol (MW = 46)M+

Background

• The cations that are formed are separated by magnetic deflection.

Background

• Only cations are detected.– Radicals are “invisible” in MS.

• The amount of deflection observed depends on the mass to charge ratio (m/z).– Most cations formed have a charge of +1 so the

amount of deflection observed is usually dependent on the mass of the ion.

Background

• The resulting mass spectrum is a graph of the mass of each cation vs. its relative abundance.

• The peaks are assigned an abundance as a percentage of the base peak. – the most intense peak in the spectrum

• The base peak is not necessarily the same as the parent ion peak.

Background

M+base peak

The mass spectrum of ethanol

Background

• Most elements occur naturally as a mixture of isotopes.– The presence of significant amounts of heavier

isotopes leads to small peaks that have masses that are higher than the parent ion peak.

• M+1 = a peak that is one mass unit higher than M+

• M+2 = a peak that is two mass units higher than M+

Easily Recognized Elements in MS

• Nitrogen:– Odd number of N = odd MW

CH3CNM+ = 41

SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/2/09)

Easily Recognized Elements in MS Bromine:

M+ ~ M+2 (50.5% 79Br/49.5% 81Br)

2-bromopropane

M+ ~ M+2


Easily Recognized Elements in MS• Chlorine:

– M+2 is ~ 1/3 as large as M+

Cl


M+2

M+

• Sulfur:– M+2 larger than usual (4% of M+)

Easily Recognized Elements in MS

M+

Unusually large M+2

S


Easily Recognized Elements in MS• Iodine

– I+ at 127– Large gap

Large gap

I+

M+


I CH2CN

Fragmentation Patterns

• The impact of the stream of high energy electrons often breaks the molecule into fragments, commonly a cation and a radical.– Bonds break to give the most stable cation.– Stability of the radical is less important.


• Alkanes– Fragmentation often splits off simple alkyl groups:

• Loss of methyl M+ - 15• Loss of ethyl M+ - 29• Loss of propyl M+ - 43• Loss of butyl M+ - 57

– Branched alkanes tend to fragment forming the most stable carbocations.

Fragmentation Patterns• Mass spectrum of 2-methylpentane

Fragmentation Patterns• Alkenes:

– Fragmentation typically forms resonance stabilized allylic carbocations

Fragmentation Patterns• Aromatics:

– Fragment at the benzylic carbon, forming a resonance stabilized benzylic carbocation (which rearranges to the tropylium ion)

M+

CH

H

CH Br

HC

H

H

or

Fragmentation Patterns• Alcohols

– Fragment easily resulting in very small or missing parent ion peak

– May lose hydroxyl radical or water• M+ - 17 or M+ - 18

– Commonly lose an alkyl group attached to the carbinol carbon forming an oxonium ion.• 1o alcohol usually has prominent peak at m/z =

31 corresponding to H2C=OH+


• MS for 1-propanol

M+M+-18

CH3CH2CH2OH

H2C OH



• Amines– Odd M+ (assuming an odd number of nitrogens

are present)– -cleavage dominates forming an iminium ion

CH3CH2 CH2 N

H

CH2 CH2CH2CH3 CH3CH2CH2N CH2

H

m/ z =72

iminium ion

Fragmentation Patterns86

CH3CH2 CH2 N

H

CH2 CH2CH2CH3

72


• Ethers– -cleavage forming oxonium ion

– Loss of alkyl group forming oxonium ion

– Loss of alkyl group forming a carbocation


H O CHCH3

MS of diethylether (CH3CH2OCH2CH3)

CH3CH2O CH2H O CH2


• The impact of the stream of high energy electrons often breaks the molecule into fragments, commonly a cation and a radical.– Bonds break to give the most stable cation.– Stability of the radical is less important.


• Alkanes– Fragmentation often splits off simple alkyl groups:

• Loss of methyl M+ - 15• Loss of ethyl M+ - 29• Loss of propyl M+ - 43• Loss of butyl M+ - 57

– Branched alkanes tend to fragment forming the most stable carbocations.

Fragmentation Patterns• Mass spectrum of 2-methylpentane

Fragmentation Patterns• Alkenes:

– Fragmentation typically forms resonance stabilized allylic carbocations

Fragmentation Patterns• Aromatics:

– Fragment at the benzylic carbon, forming a resonance stabilized benzylic carbocation (which rearranges to the tropylium ion)

M+

CH

H

CH Br

HC

H

H

or

Fragmentation PatternsAromatics may also have a peak at m/z = 77 for the benzene ring.

NO2

77M+ = 123

77

Fragmentation Patterns• Alcohols

– Fragment easily resulting in very small or missing parent ion peak

– May lose hydroxyl radical or water• M+ - 17 or M+ - 18

– Commonly lose an alkyl group attached to the carbinol carbon forming an oxonium ion.• 1o alcohol usually has prominent peak at m/z =

31 corresponding to H2C=OH+


• MS for 1-propanol

M+M+-18

CH3CH2CH2OH

H2C OH



• Amines– Odd M+ (assuming an odd number of nitrogens

are present)– -cleavage dominates forming an iminium ion

CH3CH2 CH2 N

H

CH2 CH2CH2CH3 CH3CH2CH2N CH2

H

m/ z =72

iminium ion

Fragmentation Patterns86

CH3CH2 CH2 N

H

CH2 CH2CH2CH3

72


• Ethers– -cleavage forming oxonium ion

– Loss of alkyl group forming oxonium ion

– Loss of alkyl group forming a carbocation


• Aldehydes (RCHO)– Fragmentation may form acylium ion

– Common fragments:

• M+ - 1 for • M+ - 29 for

RC O

R (i.e. RCHO - CHO)

RC O

Fragmentation Patterns• MS for hydrocinnamaldehyde

M+ = 134C C C H

H

H

H

H

O

133

105

91105

91



• Ketones– Fragmentation leads to formation of acylium ion:

• Loss of R forming

• Loss of R’ forming

RC O

R'C O

RCR'

O

Fragmentation Patterns• MS for 2-pentanone

CH3CCH2CH2CH3

O

M+

CH3CH2CH2C O

CH3C O



• Esters (RCO2R’)– Common fragmentation patterns include:

• Loss of OR’ – peak at M+ - OR’

• Loss of R’ – peak at M+ - R’

Frgamentation Patterns

M+ = 136

C

O

O CH3

105

77 105

77


Rule of Thirteen• The “Rule of Thirteen” can be used to identify

possible molecular formulas for an unknown hydrocarbon, CnHm.

– Step 1: n = M+/13 (integer only, use remainder in step 2)

– Step 2: m = n + remainder from step 1

Rule of Thirteen• Example: The formula for a hydrocarbon with

M+ =106 can be found:

– Step 1: n = 106/13 = 8 (R = 2)

– Step 2: m = 8 + 2 = 10

– Formula: C8H10

Rule of Thirteen

• If a heteroatom is present, – Subtract the mass of each heteroatom from the

MW– Calculate the formula for the corresponding

hydrocarbon– Add the heteroatoms to the formula

Rule of Thirteen

Example: A compound with a molecular ion peak at m/z = 102 has a strong peak at 1739 cm-

1 in its IR spectrum. Determine its molecular formula.

References

• Chatwal GR, Anand SK. Instrumental method of chemical analysis, Himalaya publishing house.

• Sharma YR. Elementary organic spectroscopy.• Willard,merritt,dean. Instumental methods of

analysis. • www.google.com/images

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Mass spectrometry

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