How Mass Spectrometers Work

How Mass Spectrometers Work

•Principles

•New Technologies

•Application areas for specific technologies

Name of this game is technology, physics and engineering design.

J.J. Thompson

Discoverer of electron

1912, showed 2 neon isotopes deflected differently by magnet

His student Aston, (1918) designed more elaborate instruments.

Diverse Interfaces/Sources Let us Cope with a variety of Molecular Types

Need to get the molecule into the vapor phase before it can be ionized…. Unfortunately many more molecules are non-volatile than are volatile. The Interface problem

Then we have the problem that different structure types make ions with a variety of mechanisms and degrees of difficulty. The Source problem

We have a range of solutions to the Interface problem. These generally fall into:

Aerosolizing

Blasting off a surface

Heating

A Generic PictureAs liquid, in solution, flow from GC, eluent from LC, solid on probe or moving belt

Interface

Sample is volatilized and ionized either with electrons or charged gas molecules. Fragments break off the starting molecule

Different technologies to achieve this. E.g. Magnets, RF quadrupoles, “ion trap” (cyclotron) time-of-flight tube. All these, in different ways determine specific curves or paths through the separator based on molecular weight/charge. Some parameter e.g. Voltage is scanned, so that at some Vm only masses of m have proper curve and pass to detector

Source

To Vacuum

Typically this is a photomultiplier tube

Charged plate

collimator

Ion Separator

To Vacuum

Slit

Detector

To Vacuum

Sources

As liquid, in solution, flow from GC, eluent from LC, solid on probe or moving belt

Interface

Source

To Vacuum

Sources we will cover

Electron Impact

Chemical Ionization

Fast Atom Bombardment

Matrix Assisted Laser Desorption Ionization

Electrospray Ionization

Atmospheric Pressure Chemical Ionization

APPCI

Secondary Ionization (SIMS)

Different Classes of compounds may need different Ionization

techniquesMany compounds have adequate volatility on heated probe.

Often careful tracking of ion current vs. time will reveal mixtures of compounds selectively “boiling off” a heated probe.

Use by placing about a microgram in solution on probe tip, allow to dry, insert to source.

An important variation on this is Desorptive heating (very fast, like a “thermal shock”

Electron Impact Sources

Incandescent Rhenium filament, electron source, 70ev, can be lowered

To Vacuum, Maintains ca. 10-5 torr

Sample inlet, heated probe, etc. To mass

spectrometer

e-e-e-

Chemical Ionization (CI)-Why?

Variety of chemistry, tailor to the sample

One gas doesn’t work, try another

Dial in more fragmentation (hard, soft CI reagent gases)

Can be universal or compound selective

Positive or negative ion chemistry can be easily achieved

Most organics with mass < 800 Daltons have sufficient volatility for CI.

Chemical Ionization Sources

Inlet, choice of gasses (RG), ca. 1torr.(maintained by valve, pumping system)

Incandescent Rhenium filament, electron source

To Vacuum

Sample inlet, heated probe, etc.

RG+

RG+ RG+ RG+

RG+

RG+

RG+RG+RG+

RG

RG+

To mass spectrometer

RGRG

RG

Ion Chemistry and Chemical Ionization

Proton transfer (proton addition or abstraction)

Charge transfer

Electron capture

Addition of Reagent ion

Higher adduct

Cluster formation

Governed by the heat of formation of the various products vs the reactants

Example for CIOverdose case, gastric contents examined for drugs.

Here, the soft ionization potential of Isobutane as CI reagent gas could be counted on to provide molecular ions of all the compounds.

Milne, et al. Anal. Chem. 1970 (42) 1815-1820.

Different Reagent Gases give somewhat different Mass Specs

We can see why CI data don’t make it into “fingerprint” databases

R.G.Cooks, et al. Org Mass Spec. 1976, 11, 975-983

Better Molecular Ions for fragile compounds

The Ion-molecule chemistry can be diagnostic for stereoisomers

You might say we rationalize the proclivities based on ease of H atom abstraction from para positionHarrison and Lin, Org. Mass Spec. 1984, 19, 67-71,

See also, Keogh, et.al. Anal Chem 1984, 56, 1849-1852.

CI in C6F6

Chemical Ionization-Diversity of Chemistry possible

Different reagent gases

Take advantage of chemical affinities to tailor the ionization

“Hard Ionization” gasses (big-∆H) produce high energy MH+, leading to more fragmentation. Example, Hydrogen

Energy scale in reagent gasses

H2>>CH4>iC4H10>NH3>CH3-ONO>NF3>N2O(last three are proton abstraction reagent gases for negative ion MS. N2O is also pos. CI gas)

Compare proton affinities (PA) of conjugate acid of reagent gas to that of substrate. e.g., C4H9

+ is a strong enough acid to give a H+ to any nitrogenous base.

Contrary example, NH4+is not acidic enough to protonate ethers

Other Reagent Gases

Ar

CS2

N2

Electrospray Ionization (ESI)

Liquid Mobile phase from LC or direct injection of solution

Coulombic explosion after desolvation in vacuum

(M+nH)n+

(M+nH)n+

(M+nH)n+

(M+nH)n+

(M+nH)n+

(M+nH)n+

Lower vacuum

MM

M

H+

H+

H+ H+H+

H+

H+

skimmers

To

mas

s sp

ec

Capilliary Potential at 3-8 kVolts

Limitations on E-Spray

Compound must be polar enough to spray in a substantially aqueous mobile phase

Relies on electrostatic charging of aerosol droplets

Non-volatile salts in mobile phase can foul the ion optics. (Buffers)

Ion optics in a source after using phosphate buffer. Can work for a while but salts on highly charged surfaces can arc etc.

Atmospheric Pressure Ionization(API) Mass Spec

Source works in near to atmospheric pressure

Uses heated nebulizer

Corona discharge uses solvent CI

Useful in Normal Phase HPLC

Logical choice for very non-polar molecules.

Atmospheric Pressure Ionization

Heater

High-velocity nebulizer gas

Liquid sample (LC)

High voltage needle “corona” discharge, ionizes, breaks down, the gas close to it

Vacuum, to mass spec

SolventH+

Sufficient concentration to act as Chemical Ionization Reagent gas

SH+

M

SH+SH+

SH+

SH+

M

M M

M

MH+

MH+

MH+

MH+

And fragments!

Atmospheric Pressure Photoionization Chemical

Ionization (APPCI)

Cutting edge source technology

Uses UV photon flux to transit chromophores to excited states, able to ionize other molecules.

When there is no chromophore, a dopant that has a chromophore, like acetone is used.

Atmospheric Pressure Photoionizaton (APPI)

Atmospheric Pressure Ionization

Heater

High-velocity nebulizer gas

Liquid sample (LC)

MH+

MH+

MH+

MH+ Vacuum, to mass spec

SH+SH+

SH+ SH+

SH+

M

M MM

M

And fragments!

UV light source

h

hh

h

hh

Ion Separation

Ion Separator

To Vacuum

Various Ion Separation Technologies

•Different for different applications

•Big variable in cost

•Different in resolving power

•Differ in the accessible mass range

•We will cover:

Magnetic sector

Quadrupole

Time-of-flight

Ion Trap cyclotron

Combinations, triple quad, Q-TOF, double sector

Magnetic Sector Ion Separation

magnet

Ions from source

Curvature of pathway varies as function of mass/charge

As the magnetic field is scanned, only one mass at a time has the right curvature to make it through the slit

Detector

€

zV =mv2

2

HzV =mv2

R

R =mvHz

mz

=H 2 R 2

2V

Quadrupole mass analysis

Mass range 10–4000 daltons (amu)

Resolution, typically 1000

Scan rate 5000 daltons/min

Accuracy .1–.2 daltons

Not for high resolution mass spec

Great for most organic chemistry applications

How do Quadrupoles separate ions of different m/z?

Accelerated beam of all the ions

pos

Neg

DC voltage

And AC (Radio Frequency)

Ions with wrong spiral, crash into sides and don’t get through

Ions with proper spiral make it to the detector

The Quadrupole Orbits

Where RF and DC are the dc and ac voltages m is the mass and e is the charge, is the radiofrequency and r is the space between the quad rods

€

Q =4eRF

mr02ω 2

A =8eDC

mr02ω 2

Q

AConstant A/Q ratio: steeper slope=lower resolution

Regions of orbit stability Lifetimes are 50-

100 sec

A “Mathieu” diagram

Ion TrapsUse RF fields to bring the ions into orbits (like Quadrupole, but made into a ring.

Scanned RF can sequentially make different masses have stable orbits

Ion chamber is swept clean thousands of times per second, then the RF voltage changed for a new orbit.

Ideal for MS-MS. In this technique we can keep a selected mass in orbit for a long time, then introduce a collision gas for secondary Chemical Ionization.

Either get new fragment, or use to track the origin of smaller fragments (Did that fragment come from the molecular ion, or from fragmentation of a product ion?)

Ion Traps

Ring Electrode

End Cap Electrodes

DC voltage

Electron Multiplier

RF, frequency can be swept.

Ion Source

Ions held in orbits

Ion Trap Equation of motion

€

qz=

4zVmr

0

2ωRF

2

From the stability digram, qz

gives instability at 0.98

V is the peak voltage between ring and endcap electrodes.

Is the RF frequency, ca. MHz for a 1 cm gap(radius)

Resolution and Mass Spec

C20H9+

C19H7N+

C13H19O2N3+

??? All same nominal mass

249

Done with TOF, FT mass spec or double sector magnetic instrument

Exact mass calibration compounds used.

5ppm precision is sufficient

C19H7N+` C20H9+ C13H19O2N3

+

249.058 249.070 249.1479

Exact Mass MS

Curves back, focusses

4-sector instruments;2 electric field, 2 magnet sectors

Highly refocussed, accuracy is is ca 10ppm

detector

Three sector Quad Mass SpecsMS-MS

A second mass separation sector

Can pass single ion m/z for selective Ion Monitoring.

We can exploit this as way to eliminate chemical noise from a dirty matrix

Can have collision cell for further CI on a specific, selected ion or fragment ion.

Collision cell, hexapole acceleration region without selectivity accd to mass

A normal quadrupole for mass separation

Ions from source

Time-of-Flight Mass Spectrometry

Totally different concept. Have a fixed electric field down a “flight tube”

A fixed distance from start to detector.

Accelerate ions to the field, start timing. The time to the detector is inversely proportional to mass (smaller mass gets there first)

Time therefore is calibrated to mass

Great precision, accuracy--can do exact Mass determination

Time-of-Flight mass spectrometry

Simple principle: Drift time over a fixed distance is related to momentum. “time” of transiting over this distance is proportional to

€

mz

Best technique for macromolecules. (have done over 500kDa). Has very high mass resolution, fortunately.

Since biomolecules like this suffer from bad volatility issues, TOF is happily paired with MALDI ionization.

How Flight Tubes work

Electric field “reflectron” gives longer path for resolution

Ion path

Accelerate ions (“pusher”) From source

Detector

MatrixAssistedLaserDesorptiveIonization

Or… What to Do, when your sample has no volatility whatsoever

How do you get a protein to oblige you by vaporizing nicely and going down the mass spec?

laser

Dilute biomolecule in e.g. cinnamic acid for proteins, picolinic acids for nucleotides

Molecular ions

Exquisite sensitivity

Great marriage with TOF for high mass range, resolution

MALDI-TOF

1048.9

1537.6

1296.7

1060.6

A mixture of peptides ca. 10 amino acid in length

Injected to mass spectrometer 20 femtomoles (10-15)each, of angiotensin I, angiotensin II, bradykinin, and fibrinopeptideA

Mass Spec Detectors

Ions+ Voltage to Detector

Conversion Dynode

+ +

+++

+ +

+

Phosphor plate

h

hh

h

hhe-

e-e-

e-

e-e-

Ph

otoMu

ltiplier

Tu

be

Specific Detectors

Cascade of Faraday Cups

The important point is the 106-fold amplification by these stages (e- per ion)