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Mass SpectroscopyLecture 3

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Prof. Nizam M. El-Ashgar

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Mass Spectrometer• Several Types. Only two will be described:• Quadrupole spectrometer and the time-of-flight spectrometer.• General description of instrument components:

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Inlet System:To introduce a very small amount of sample (µmol orless) into the mass spectrometer that converted togaseous ions. (a mean for volatilizing solid or liquidsamples is presents).Ion sources:Convert the components of a sample into ions.(generally the inlet system and the ion source arecombined into a single component). The output is astream of +ve or –ve ions are accelerated into the massanalyzer.Mass analyzerAnalogous to grating in an optical spectrometer.Dispersion is based upon the mass/charge ratios of theanalyte ions rather than upon the wavelength of photons.(Different categories of MS according to mass analyzer).

Detectors:Convert the beam of ions into an electricalsignal that can then be processed, stored inthe memory of a computer and displayed orrecorded in a variety ways.Vacuum System: To create low pressure(10-4 to 10-8 torr) in all the instrumentcomponents except the signal processorand readout. To prevent interaction ofcomponents with atmosphere so destroyed.

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Sample Inlet SystemFor permitting introduction of a representative sampleinto the ion source with minimal loss of vacuum. Varioustypes of inlets equipped to accommodate differentsamples: (Batch inlets, direct probe inlets,chromatographic inlets and capillary electrophoreticinlets).Batch Inlet Systems:Classical and simplest type.Sample is volatilized externally and then allowed to leakinto evacuated ionization region.The figure shown is for a one that applicable to gaseousand liquid samples having PB up to 5000C.

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Gaseous samples:• A small measured volume of a gas is trapped between

the two valves and then expanded into reservoir flask.For liquids:• A small quantity of sample is introduced into a reservoir

usually with a micrometer syringe.In either case vacuum system is used to achieve sampleP 10-4 – 10-5 torr.For samples with boiling points > 1500C:• T must be maintained at elevated T by oven and

heating tapes of maximum T of about 350 0C.• Sample is now in the gas phase is leaked into the

ionization area of the spectrometer via a metal or glassdiaphragm containing one or more pinholes.

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The Direct Probe Inlet.• Solids and non volatile liquids can be introduced

into the ionization region by means of a sampleholder or probe which is inserted through avacuum lock.

The lock system:vDesigned to limit V of air pumped after probe

insertion.vProbes also used for limited quantity samples

(few nanograms)(less wasted than batch system).vProbe: Sample is held on the surface of a glass or

Al capillary tube, a fine wire or a small cup..

v The probe is positioned within a few mm of the Ionizationsource and the slit leading to spectrometer.

v Vacuum used to maintain thermally unstable compoundsfor spectrum measurements before major decompositionoccurs. And to elevate nonvolatile conc. in the ionizationarea (carbohydrates, steroids, metal-organic species andlow molecular weight polymeric substances.

v Partial pressure attained is at least of 10-8 torr before onsetdecomposition.

Chromatographic and capillary electrophoretic inletSystem.vMS are often coupled with GC and HPLC or capillary

electrophoresis columns.v Separation and determination of the components of

complex mixture is obtained.v Specialized inlet systems is needed.٨

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MS – Direct Sample Introduction

External Sample Introduction System

Direct Sample Probe

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Mass AnalyzersSeveral devices are available for separating ions withdifferent m/z ratios.Mass analyzer should be:- Capable of distinguishing between minute mass

differences.- Allows passage of sufficient number of ions to yield

measurable currents.ResolutionResolution:: A measure of how well a mass spectrometerseparates ions of different mass.

•• LowLow resolutionresolution:: Refers to instruments capable ofseparating only ions that differ in nominal mass; that isions that differ by at least 1 or more atomic mass units.

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HHigh resolution:igh resolution: Refers to instruments capable of separating ions that differ in mass by as little as 0.0001 atomic mass unit.Resolution of mass spectrometer: RR = m/∆m = M1/ (M1 – M2) Where: m : mass of the first peak (or mean mass of the two peaks).∆m : mass difference between two adjacent peaks. ΔM = full width at half maximum (FWHM) Example:What R is needed to separate C2H4

+ and CH2N+, ions?∆m = 28.0313 - 28.0187 = 0.0126R =m/ ∆m = 28.025/0.0126 = 2.22x103

Where 28.025 is the mean mass for the two species

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Inte

nsi

ty (

%)

0

20

40

60

80

100

Mass [amu]111.95 112.00 112.05 112.10

Inte

nsi

ty (

%)

0

20

40

60

80

100

Mass [amu]111.95 112.00 112.05 112.10

Inte

nsi

ty (

%)

0

20

40

60

80

100

Mass [amu]111.95 112.00 112.05 112.10

•RP= 3,000 •RP= 5,000 •RP= 7,000

•C6H5OF•C6H5Cl

Resolving Power ExampleResolving Power Example

• High resolution data reports include ppm estimate– ppm = parts per million (1 ppm = 0.0001%)• 5 ppm @ m/z 300 = 300 * (5/106) = ±0.0015 Da• 5 ppm @ m/z 3,000 = 3,000 * (5/106) = ±0.015 Da• A molecule with mass of 44 could be C3H8, C2H4O,

CO2, or CN2H4.• If a more exact mass is 44.029, pick the correct

structure from the table:

C3H8 C2H4O CO2 CN2H4

44.06260 44.02620 43.98983 44.03740

High Resolution MSHigh Resolution MS

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– C3H6O and C3H8O have nominal masses of 58 and 60, andcan be distinguished by low-resolution MS.

– C3H8O and C2H4O2 both have nominal masses of 60.– Distinguish between them by high-resolution MS.

C2 H4 O2

C3 H8 O

60.0211260.05754

60

60

MolecularFormula

Nominal Mass

PreciseMass

ResolutionResolution

High resolution MS can replace elemental analysis for chemical formula confirmation

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R needed in MS depends upon its application.Example 1: same nominal mass ions: C2H4

+, CH2N+,N2

+ and CO+

(All ions of nominal mass 28 Da).Exact masses: 28.0313, 28.0187, 28.0061 and

27.9949 Da respectively.These requires an instrument with a resolution of

several thousands.Example 2: Low MWt ions with a unit mass difference

or more: NH3+ (m = 17) and CH4

+ (m = 16) Rinstrument of 50 or less is sufficient.

Commercial MS are available with R range of 500 -500,000.

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Types of Mass AnalyzersMagnetic Sector Analyzers• Employ a permanent magnet or an

electromagnet.• Cause beam from the ion source to travel in a

circular path most commonly of (180, 90 or 60deg)

• Schematic of a 90-deg magnetic sectorspectrometer

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Magnetic Sector: Single Focusing: A magnetic field is used tofocus ions based on their momentum as they are ejected froman ion source at high energy. Resolution <2000

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Operation:• Ions formed by Electron impact are accelerated through slit

B into the metal analyzer tube internal P ≈ 10-7 torr.• Ions of different mass can be scanned across the exit slit by

varying the field strength of the magnet or the acceleratingpotential between slits A and B.

• Ions passing through the exit slit fall on a collector electrode,ion current resulted that amplified and recorded.

• Translation or KE of an ion of mass m bearing z exiting slit Bis given by:

KE = ZeV = ½ mv2

Where V is voltage between A and Bv is velocity of the ion after acceleratione is the electronic charge = 1.60x10-19 C

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v Note: all ions having the same number of charges areassumed to have the same KE after acceleratingregardless of their mass. (approximately true).

v All ions leaving the slit have approximately same KE, theheavier ions must travel at lower velocities.

v The path in sector by ions of a given m/z represents abalance between two forces acting upon them.

The magnetic force FM:

FM =BzeVWhere B is MF strengthThe balancing centripetal force:

Fc = mv2/rWhere r is the radius of curvature of the magnetic sector.For ion to traverse the circular path to the collector, FM and FC must be equal

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FM =BzeV = Fc = mv2/rv = Bzer/m

Substituting the previous equation in ZeV = ½ mv2

m/z = B2r2e/2VMass Spectra:vVarying one of three variables (B, V or r) while

holding the other two constant.vModern MS ions are sorted by holding V and r

constant while varying the current in the magnetand thus B.vIn sector MS (using photographic recording) B and

V are constants, r is the variable.

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What accelerating potential will be required to direct a singlycharged water molecule through the exit slit of a magneticmass spectrometer if B = 0.240 T and r of curvature of the ionthrough the magnetic field is 12.7 cm?SI units:Ez = 1.60x10-19 C x 1 r = 0.127 m

B = 0.240 T = 0.240 W/m2

V = B2r2ez/2m= [0.240 W/m2]2[0.127m]2[1.60x10-19C]

2x2.99x10-26kg

= 2.49 x10 W2C/m2kg or volts

gkgx

molOHxmolOHg

m 3

223

2 10/1002.6

/02.18 −+

+

=

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Double focusing spectrometersPrevious are single-focusing spectrometers.Limitation: Low precision1- directional distribution of ions2- energy distribution of ionsv Same m/z ratio but with small diverging directional

distribution are focused so limiting the resolution ofmagnetic sector instruments (R ≤ 2000).

vThis because of the translational E distribution of ionsleaving a source (Boltzmann dist.) arises fromenergies of original molecules and source fieldinhomogeneities.

vSpread of KE causes a broadening of the beamreaching the transducer and a loss of resolution.

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Double Focusing Spectrometers:v Correction for both the directional and E distribution of ions.v Both directional and E aberrations of a population of ions are

simultaneously minimized by use of carefully selectedcombinations of electrostatic and magnetic fields.

Magnetic sector double focusing: Anelectrostatic analyzer is used serially with amagnet to select monoenergetic ions. Highresolution >10,000 mass up to 100,000Da

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• The ion beam is first passed through an electrostaticanalyzer (ESA).

• Consists of two smooth curved metallic plates acrosswhich a dc potential is applied which limiting the KE of theions reaching the magnetic sector to a closely definedrange.

• Ions with E greater than average strike the upper side ofESA slit and lost to the ground.

• Ions with E less than average strike the lower side of theESA slit and are thus removed.

• Directional focusing occurs along the focal plane (d).• Energy focusing takes place along the plane e.• Only ions of one m/z are double focused at the

intersection of d and e for any given V and B.• The collector slit is located at this locus of double focus.

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Focus EFocus mom.

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Quadrupole Mass SpectrometerComparing with magnetic sector instrument it is:• Less expensive and more rugged.• More compact• Found in commercial benchtop ms• Low scan times (<100 ms) which is useful for

chromatography.• Most common mass analyzers in use today.Quadrupole mass analyzer is responsible for filtering thesamples ions.

v Consists of four parallel metal rods.

v Each rod pair is connected together electrically.

v A radio frequency voltage is applied between one pair ofrods then the other.

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Operation:v Ions travel down the quadrupole in between the rodsv Electric field separates ionsv Ions are subjected to complex forcesvOnly Ions of a particular m/z reaches the detectorAdvantages• Inexpensive• Easily Interfaced to Many Ionization MethodsDisadvantages• Low Resolution (<4000)• Low Accuracy (>100ppm)• MS/MS requires multiple analyzers• Low Mass Range (<4000)• Slow Scanning

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Time-of-Flight MS (TOF)

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TOF:v+ve ions are produced periodically by bombardment of

the sample with brief pulses of electrons, secondaryions or laser generated photons.

vThe ions are then accelerated into a field-free drifttube by an electric field pulse of 103 – 104 V.

vSeparation of ions on the basis of mass occurs duringthe transit of the ions to the detector located at theend of the tube.

vAll ions have same KE but their velocities areinversely proportional to their masses.

vLighter particles arrived earlier.vTypical flight times are 1 to 30 µs.

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Time-of flight analyzer & time of flight reflectron

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Reflectron TOF-MS Improved mass resolution in MALDI TOF-MS hasbeen obtained by the utilization of a single-stage or a dual-stagereflectron (RETOF-MS). The reflectron, located at the end of the flighttube, is used to compensate for the difference in flight times of thesame m/z ions of slightly different kinetic energies by means of anion reflector. This results in focusing the ion packets in space andtime at the detector

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Advantages• Simplicity and ruggedness.• Ease of accessibility of the ion source.• Extremely High Mass Range (>1 MDa)• Fast ScanningDisadvantages• Low Resolution (4000)• Low sensitivity• Low Accuracy (>200ppm)• MS/MS not possible

Linear Time-of-Flight (TOF)

Advantages• High Resolution (>20,000

in some models)• High Accuracy (<5ppm)• 10,000 Mass Range• Fast Scanning

Disadvantages• Low Resolution for

MS/MS (PSD)

Reflectron Time-of-Flight (TOF)

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Ion Trap AnalyzersvGaseous anions or

cations can be formedand confined for extendedperiods by electric and/ormagnetic fields.

vSeveral types, A simplertype of ion trap that usedfor GC/MS

vNow used to obtain massspectra of a variety ofanalytes.

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v It consists of a central doughnut-shaped ring electrode and apair of endcap electrodes.

v A variable RF voltage is applied to the ring electrode whilethe two end-cap electrodes are grounded.

v Ions of appropriate m/z value circulate in a stable orbit withinthe cavity surrounded the ring.

v By increasing RF voltage the orbit of heavier ions becomedestabilized , while lighter become destabilized causing themto collide with the wall of the ring electrode.

v By RF voltage scanning the trapped ions destabilized andleave the ring electrode cavity via openings in the lower endcap the emitted to a transducer.

v Rugged compact and less costly than sector or quadrupoleinstruments.

v Capable of resolving ions that differ in mass by unit in themass range of 500-1000 Da

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Operation of an Ion Trap MS

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Tandem Mass Spectrometry : MS/MS or MSn

Mass spectrometry is a very powerful method to analysethe structure of organic compounds, but suffers from 3major limitations:1. Compounds cannot be characterised without clean

samples2. This technique has not the ability to provide sensitive

and selective analysis of complex mixtures3. For big molecules like peptides spectra are very

complex and very difficult to interpret.Tandem:

The technique has been used for:1. Structure elucidation of unknown 2. Analysis of complex mixture with minimum sample

clean up.

nfCID

p mmm + → ++

What is MSMS?MS/MS means using two mass analyzers (combined in oneinstrument) to select an analyte (ion) from a mixture, thengenerate fragments from it to give structural information.

Ion source MS-2MS-1

Mixture of ions Single ion Fragments

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What is MS/MS?

MS/MS+

+

+ +

+

1 peptide selected for

MS/MS

The masses of all the pieces give an MS/MS spectrum

Peptidemixture

Have only masses to start

Interpretation of an MSMS spectrum to derive structural information is analogous to solving a puzzle

+

+

+ +

+

Use the fragment ion masses as specific pieces of thepuzzle to help piece the intact molecule back together

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-HN--CH--CO--NH--CH--CO--NH-

Ri CH-R’

ci

zn-i

R”

di+1

vn-i wn-i

low energy

high energy

Cleavages Observed in MS/MS of Peptides

ai

xn-i

bi

yn-i

Peptide Fragmentation

E G S F F G E E N P N V A R

175.10246.14345.21459.25556.30670.35799.39928.43985.45

1132.521279.591366.621423.641552.69

=>

=

=

=

E=GluG=GlyS=SerF=PheN=AsnP=ProV=ValA=AlaR=Arg

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MS – MS (TANDEM MS) INSTRUMENTS

- Employs two or more stages of mass analyzers- Example is two quadrupoles coupled in series- First analyzer selects ion (precursor ion) and second

analyzer selects the fragments of the precursor ion- Used to obtain more information about the structure of

fragment ions.- Fragment ions may be dissociated into lighter fragment

ions or converted into heavier ions by reaction withneutral molecule

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Tandem Mass Spectrometry (MS/MS)• Mass spectrometers are commonly combined with separation

devices such as gas chromatographs (GC) and liquidchromatographs (LC).

• The GC or LC separates the components in a mixture, and thecomponents are introduced, one by one, into the massspectrometer.

• MS/MS is an analogous technique where the first stageseparation device is another mass spectrometer.

• .•

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• Suppose that we analyze a mixture of components by a"soft" ionization method (such as chemical ionization, fastatom bombardment, or electrospr ay ionization). Eachcomponent produces chaacteristic ionic species such as[M+H]+.

• To keep the discussion simple, let's assume that eachcomponent of the mixture has a unique molecular weight.

• The mass spectrum of the mixture contains peaks foreach compound present in the mixture.

• Now, suppose that we would like to identify one of themixture components.

• All the mass spectrum tells us is the molecular weight,but we would really like to see fragment ions that providestructural information for the component of interest.٤٩

• The simplest form of tandem mass spectrometry combines twomass spectrometers.

• The first mass spectrometer is used to select a single(precursor) mass that is characteristic of a given analyte in amixture.

• The mass-selected ions pass through a region where they areactivated in some way that causes them to fall apart to producefragment (product) ions.

• This is usually done by colliding the ions with a neutral gas in aprocess called collisional activation (CA) or collision-induceddissociation(CID).

• The second mass spectrometer is used to separate thefragment ions according to mass.

• The resulting "MS/MS" spectrum consists only of product ionsfrom the selected precursor.

• Chemical background and other mixture components areabsent.

•

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Tandem mass spectrometry (MS/MS)

v Enhance selectivity by combining complimentary techniques

v Number of Stages =1. GC2. GC/MS3. GC/MS/MS4. GC/MS/MS/MS

1 2 3 4

Number of S tages of Analysis

•Signal

•Chemical• Noise

•S/N

Tandem Mass Spectrometry : MS/MS or MSn

Tandem Mass Spectrometry : MS/MS or MSn

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•MS2•MS1

• The ion selected by the MS1 is the parent ion and can bea molecular ion resulting from the primary fragmentation.

• DISSOCIATION occurs in the fragmentation region.

• The daughter ions are analysed in the SecondSpectrometer (MS2).

• In fact, the MS1 can be viewed as an ion source for MS2.

Triple Quadrupole Mass Spectrometer

electrospray sourceelectrospray source

QQ11 QQ22 QQ33

collision cellcollision cell detectordetector

••microcapillarymicrocapillary

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Ion Trap Mass Spectrometer

electrospray sourceelectrospray source

microcapillarymicrocapillary

end capend capring electrodering electrode

detectordetector

end capend cap

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Fourier Transform (FT) instruments

Mass analysis performed by detecting cyclotronfrequencies of ions (depend on m/z) in uniform B, in timedomain. Then F.T. to frequency domain (mass spectrum).FTMS provide:• improved signal to noise ratios.• Greater speed.• Higher sensitivity.• Higher resolution.The heart of FTMS is an ion trap within which ions can

circulate in a well-defined orbits for extended periods.

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The Ion Cyclotron Resonance (ICR)• Gaseous ion drifts into or formed in a strong MF.

• The motion becomes circular in a plane that is ⊥ to the directionof the field.

• The angular frequency of this motion is called cyclotronfrequency ωc. ωc= v/r = zeB/m

• In fixe field the ωc depends only upon the inverse of the m/zvalue.

• Increases the velocity of an ion will be accompanied by increaseof rotation of the ion.

• This circulated trapped ion in the MF is capable of absorbing Efrom an ac EF.

• So the EF frequency matches the ωc.• The absorbed E increases v of the ion and r of travel without

disturbing ωc

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MS – Fourier Transform Analyzer

Ion Cyclotron Resonance

Magnetic Field

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• Inner solid line represents the original path to the ion.• Dashed line shows spiral path when switch is moved briefly to

position 1.• Outer solid line is new circular path when switch again opened.• For ensemble ions of the same m/z ratio between the two

plates; when ac signal is applied the cyclotron resonancefrequency sets all the particles into coherent motion that is inphase with the field.

• Ions of different m/z ratios (different ωc) are unaffected by theac field.

Measurement of the ICR signal:v Coherent circular motion of resonant ions creates image current

observed current after termination of the frequency sweepsignal (from position 1 to 2).

v The current decreases exponentially with time.v It is a capacitor current induced by circular movement of a

packet of ions with the same m/z ratios.

vExample:v +ve ions approaches the upper plate electrons attracted

from circuit common to this plate causing a momentarycurrent.

v This current reversed at the other plate as ions reach theother plate.

vAn ac current produced depends on number of ions in thepacket

v Frequency of ac current is characteristic of m/z value ofthe ions in the packet.

v This current measures conc. of ions.v The induced image current decays with time (few tenth of

s to several s) by losing energy with collisions betweenions (ions reach the thermal equilibrium) (time domainsignal).٦٠

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Diagram of the cell used in pulsed ICR & in FT-MS

Pulsed ICR uses a single-frequency rf excitation, whereas a scanned frequency is used in FT-ms. A voltage 1-5 V is used to trap +ions. The grid is used for pulsing the ion beam.

B┴ to front and back plates of cell

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vGenerally equipped with a trapped ion analyzer cell.vGaseous sample molecules are ionized in the center of the

cell by electrons that are accelerated from the filamentthrough the cell to a collector plate.

v A pulsed voltage applied at the grid serves as a gate toswitch the electron beam on and off periodically.

v The ions are held in the cell by a 1 to 5 V potential applied tothe trap plate.

v The ions are accelerated by a radio-frequency signal appliedto a transmitter plate.

v The receiver plate is connected to a preamplifier to amplifythe image current.

v Ions could be stored for several minutes.v The dimensions of the cell are not critical (usually a few cm)

on a side.

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Ø AdvantagesØ No slits or ion optic lenses to

adjust.Ø All ions detected simultaneously

for a single ionizing pulse.Ø Detection sensitivity

independent of m.Ø Extremely High Mass Resolution

(>500,000)highØ e.g. m/rm ~ 220000 for m/z =

84 at B = 1.2TØ Very Good Accuracy (<1 ppm)Ø MS/MS in one analyzer

Disadvantages• Expensive• Requires

Superconducting Magnet

• Slow MS/MS

FT-ICR-MS

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Ion image current generation

few ms; v varied linearly

what is B/z range?

FT-ICR

Rapid scan ICR

Ions subjected to slower scan. When v matches cyclotronresonance frequency of ion it is excited and detected.

wide-range ms