Mass Ionization 21 01 10

7/28/2019 Mass Ionization 21 01 10

1/67

Mass Spectrometry:

Ionization methods


2/67

Mass Spec Principles

Ionizer

Sample

+

_

Mass Analyzer Detector


3/67

How does a mass spectrometer work?

Ionization

method

MALDI

Electrospray

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


4/67

Ion sources for molecular mass

spectrometry


5/67

Soft Ionization Soft ionization techniques keep the molecule

of interest fully intact

Electro-spray ionization first conceived in1960s by Malcolm Dole but put into practice

in 1980s by John Fenn (Yale)

MALDI first introduced in 1985 by Franz

Hillenkamp and Michael Karas (Frankfurt)

Made it possible to analyze large moleculesvia inexpensive mass analyzers such as

quadrupole, ion trap and TOF


6/67

Soft Ionization Methods

337 nm UV laser

MALDI

cyano-hydroxy

cinnamic acidGold tip needle

Fluid (no salt)

ESI

+

_


7/67

Soft ion sources little excess energy in

molecule

reduced fragmentation


8/67

Hard ion sources leave excess energy in

molecule-extensive fragmentation


9/67

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


10/67

Sample introduction/ionization method

Ionization

method

Typical

Analytes

Sample

Introduction

Mass

Range

Method

Highlights

Electron Impact (EI)

Relatively

small

volatile

GC or

liquid/solid

probe

to

1,000

Daltons

Hard method

versatile

provides

structure info

Chemical Ionization (CI)

Relatively

smallvolatile

GC or

liquid/solidprobe

to

1,000Daltons

Soft method

molecular ionpeak [M+H]+

Electrospray (ESI)

Peptides

Proteins

nonvolatile

Liquid

Chromatography

or syringe

to

200,000

Daltons

Soft method

ions often

multiply

charged

Fast Atom Bombardment

(FAB)

Carbohydrates

Organometallics

Peptides

nonvolatile

Sample mixed

in viscous

matrix

to

6,000

Daltons

Soft method

but harder

than ESI or

MALDI

Matrix Assisted Laser

Desorption

(MALDI)

Peptides

Proteins

Nucleotides

Sample mixed

in solid

matrix

to

500,000

Daltons

Soft method

very high

mass


11/67

Electron Ionization

Also called as electron impact ionization

Oldest and best-characterized of all the ionization

methods.

A beam of electrons passes through the gas-phase

sample. An electron that collides with a neutral analyte

molecule can knock off another electron, resulting in a

positively charged ion.


12/67

Electron Ionization

The ionization process can either produce a molecular

ion which will have the same molecular weight andelemental composition of the starting analyte, or it can

produce a fragmention which corresponds to a smaller

piece of the analyte molecule.

Most mass spectrometers use electrons with anenergy of 70 electron volts (eV) for EI.

Decreasing the electron energy can reduce

fragmentation, but it also reduces the number of ionsformed.


13/67


14/67

EI Fragmentation of CH3OH

CH3OH CH3OH+

CH3OH CH2O=H+ + H

CH3OH+ CH3 + OH

CHO=H+ + HCH2O=H+

Why wouldnt Electron Impact be suitable

for analyzing proteins?


15/67

Why You Cant Use EI For

Analyzing Proteins

EI shatters chemical bonds

Any given protein contains 20 different

amino acids

EI would shatter the protein into not onlyinto amino acids but also amino acid sub-

fragments and even peptides of 2,3,4amino acids

Result is 10,000s of different signals from

a single protein -- too complex to analyze


16/67

Electron Ionization

Sample introduction

heated batch inlet

heated direct insertion probe

gas chromatography

liquid chromatography (particle-beaminterface)


17/67

Some typical reactions in EI

source


18/67

Fragmentation patterns


19/67

Fragmentation patterns


20/67

Electron Ionization

Benefits well-understood

can be applied to virtually all volatile

compounds reproducible mass spectra

fragmentation provides structural information

libraries of mass spectra can be searched forEI mass spectral "fingerprint"


21/67

Electron Ionization

Limitations

sample must be thermally volatile and

stable

the molecular ion may be weak orabsent for many compounds.

Mass range

Low Typically less than 1,000 Da.


22/67

Electron Ionization

Advantages of EI: high ion currents - sensitive

fragmentation aids identification

Disadvantages of EI:

weak or absent M+ peak inhibitsdetermination of MW

molecules must be vaporized (MW < 103 Da)

molecules must be thermally stable duringvaporization


23/67

Chemical Ionization (CI) Chemical ionization uses ion-molecule reactions to

produce ions from the analyte. The chemical ionization process begins when a

reagent gas such as methane, isobutane, or

ammonia is ionized by electron impact.

A high reagent gas pressure (or long reaction time)

results in ion-molecule reactions between the

reagent gas ions and reagent gas neutrals.

Some of the products of these ion-moleculereactions can react with the analyte molecules to

produce analyte ions.


24/67

Chemical Ionization (CI)Example (R = reagent, S = sample, e = electron, . =

radical electron , H = hydrogen):R + e ---> R+. + 2e

R+. + RH ---> RH+ + R.

RH+ + S ---> SH+ + R(of course, other reactions can occur)


25/67

Chemical Ionization (CI)Sample introduction

heated batch inlet heated direct insertion probe

gas chromatograph

liquid chromatograph (particle-beam interface)Benefits

often gives molecular weight information through

molecular-like ions such as [M+H]+, even when EIwould not produce a molecular ion.

simple mass spectra, fragmentation reducedcompared to EI


26/67

Chemical Ionization (CI)Limitations

sample must be thermally volatile and stable less fragmentation than EI, fragment pattern not

informative or reproducible enough for library

search results depend on reagent gas type, reagent gas

pressure or reaction time, and nature of sample.

Mass range

Low Typically less than 1,000 Da.


27/67

Field Desorption (FD)

The sample is deposited onto the emitter and the emitter is

biased to a high potential (several kilovolts) and a current is

passed through the emitter to heat up the filament.

Mass spectra are acquired as the emitter current is

gradually increased and the sample is evaporated from the

emitter into the gas phase.

The analyte molecules are ionized by electron tunneling atthe tip of the emitter 'whiskers'.

Characteristic positive ions produced are radical molecular

ions and cation attached species such as [M+Na]+ and [M-

Na]+. The latter are probably produced during desorption by the

attachment of trace alkali metal ions present in the analyte.


28/67


Sample introduction

Direct insertion probe. The sample is deposited onto the tip of the emitter

by

. dipping the emitter into an analyte solution

. depositing the dissolved or suspended sample

onto the emitter with a microsyringe


29/67


Benefits

simple mass spectra, typically one molecular ormolecular-like ionic species per compound.

little or no chemical background

works well for small organic molecules, manyorganometallics, low molecular weight polymersand some petrochemical fractions


30/67


Limitations

sensitive to alkali metal contamination and sampleoverloading

emitter is relatively fragile

relatively slow analysis as the emitter current is

increased the sample must be thermally volatile to some

extent to be desorbed

Mass range Low-moderate, depends on the sample. Typically

less than about 2,000 to 3,000 Da.

some examples have been recorded from ions withmasses beyond 10,000 Da.

Fi ld I i ti (FI)


31/67

Field Ionization (FI)

The sample is evaporated from a direct insertion probe,gas chromatograph, or gas inlet.

As the gas molecules pass near the emitter, they areionized by electron tunneling.

Sample introduction heated direct insertion probe

gas inlet

gas chromatograph

i i i


32/67


Benefits

simple mass spectra, typically one molecular ormolecular-like ionic species per compound.

little or no chemical background

works well for small organic molecules and somepetrochemical fractions

Fi ld I i i (FI)


33/67


Limitations

The sample must be thermally volatile.

Samples are introduced in the same way as forelectron ionization (EI).

Mass range

Low Typically less than 1000 Da.

F t At B b d t (FAB)


34/67

Fast Atom Bombardment (FAB)

The analyte is dissolved in a liquid matrix such as

glycerol, thioglycerol,m-nitrobenzyl alcohol,

ordiethanolamine and a small amount (about 1microliter) is placed on a target.

The target is bombarded with a fast atom beam

(for example, 6 keV xenon atoms) that desorbmolecular-like ions and fragments from theanalyte.

Cluster ions from the liquid matrix are alsodesorbed and produce a chemical background thatvaries with the matrix used.



35/67


Sample introduction

direct insertion probe LC/MS (frit FAB or continuous-flow FAB).

Benefits rapid

simple

relatively tolerant of variations in sampling

good for a large variety of compounds

strong ion currents -- good for high-resolutionmeasurements



36/67


Limitations

high chemical background defines detection limits

may be difficult to distinguish low-molecular-weightcompounds from chemical background

analyte must be soluble in the liquid matrix

no good for multiply charged compounds with morethan 2 charges

Mass range

Moderate Typically ~300 Da to about 6000 Da.

Secondar Ion Mass Spectrometr (SIMS)


37/67

Secondary Ion Mass Spectrometry (SIMS)

Dynamic SIMS is nearly identical to FAB except

that the primary particle beam is an ion beam(usually cesium ions) rather than a neutral beam.

The ions can be focused and accelerated to higher

kinetic energies than are possible for neutralbeams, and sensitivity is improved for highermasses.

The use of SIMS for moderate-size (3000-13,000Da) proteins and peptides has largely beensupplanted by electrospray ionization.

S d I M S t t (SIMS)


38/67


Sample introduction

Same as for FAB

Benefits

Same as for FAB, except sensitivity is improved forhigher masses (3000 to 13,000 Da).

S d I M S t t (SIMS)


39/67


Limitations

Same as for FAB except

target can get hotter than in FAB due to moreenergetic primary beam

high-voltage arcs more common than FAB

ion source usually requires more maintenance thanFAB

Mass range Moderate Typically 300 to 13,000 Da.

Electrospray Ionization (ESI)


40/67


Sample dissolved in polar, volatile buffer (no salts)

and pumped through a stainless steel capillary (70

- 150 mm) at a rate of 10-100 mL/min

Strong voltage (3-4 kV) applied at tip along with

flow of nebulizing gas causes the sample tonebulize or aerosolize

Aerosol is directed through regions of highervacuum until droplets evaporate to near atomic

size (still carrying charges)


41/67


El t I i ti (ESI)


42/67


Can be modified to nanospray system

with flow < 1mL/min

Very sensitive technique, requires lessthan a picomole of material

Strongly affected by salts & detergents

Positive ion mode measures (M + H)+ (addformic acid to solvent)

Negative ion mode measures (M - H)- (add

ammonia to solvent)


43/67

Positive or Negative Ion Mode?

If the sample has functional groups thatreadily accept H+ (such as amide andamino groups found in peptides andproteins) then positive ion detection isused-PROTEINS

If a sample has functional groups that

readily lose a proton (such as carboxylicacids and hydroxyls as found in nucleicacids and sugars) then negative iondetection is used-DNA



44/67


Sample introduction

flow injection LC/MS

typical flow rates are less than 1 microliter perminute up to about a milliliter per minute



45/67


Benefits

good for charged, polar or basic compounds permits the detection of high-mass compounds at

mass-to-charge ratios that are easily determined

by most mass spectrometers (m/z typically less

than 2000 to 3000). best method for analyzing multiply charged

compounds

very low chemical background leads to excellentdetection limits

can control presence or absence of fragmentation

by controlling the interface lens potentials

compatible with MS/MS methods



46/67


Limitations

multiply charged species require interpretation andmathematical transformation (can sometimes bedifficult)

complementary to APCI. No good for uncharged,

non-basic, low-polarity compounds (e.g.steroids) very sensitive to contaminants such as alkali

metals or basic compounds

relatively low ion currents relatively complex hardware compared to other ion

sources

Mass range

Low-hi h T icall less than 200 000 Da.

Atmospheric Pressure Chemical Ionization


47/67


(APCI)

Similar interface to that used for ESI.

In APCI, a corona discharge is used to ionize theanalyte in the atmospheric pressure region.

The gas-phase ionization in APCI is more effective

than ESI for analyzing less-polar species.

ESI and APCI are complementary methods.



48/67


(APCI)

Sample introduction

same as for electrospray ionization

Benefits

good for less-polar compounds

excellent LC/MS interface

compatible with MS/MS methods



49/67


(APCI)

Limitations

complementary to ESI

Mass range

Low-moderate Typically less than 2000 Da.

Matrix-Assisted Laser Desorption


50/67


Ionization (MALDI)

The analyte is dissolved in a solution containing anexcess of a matrix such as sinapinic acid ordihydroxybenzoic acid that has a chromophorethat absorbs at the laser wavelength.

A small amount of this solution is placed on thelaser target.

The matrix absorbs the energy from the laserpulse and produces a plasma that results invaporization and ionization of the analyte.


51/67

MALDI Ionization

++

+

+

-

--

++

+

+

-

---++

Analyte

Matrix

Laser

+

++

Absorption of UV radiationby chromophoric matrix andionization of matrix

Dissociation of matrix,phase change to super-compressed gas, chargetransfer to analyte molecule

Expansion of matrix atsupersonic velocity, analytetrapped in expanding matrixplume (explosion/popping)

+

+

+

MALDI


52/67

MALDI

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 mL of 1 pmol/mL sample

P i i l f MALDI TOF MASS


53/67

Principal for MALDI-TOF MASS

+

+

+++

+

+

+

+

+

pulsedUV or IR laser(3-4 ns)

detector

vacuum

strong

electricfield

Time Of Flight tube

peptide mixture

embedded inlight absorbingchemicals (matrix)

cloud ofprotonatedpeptide moleculesacc

V

P i i l f MALDI TOF MASS


54/67

Principal for MALDI-TOF MASS

Linear Time Of Flight tube

Reflector Time Of Flight tube

detector

reflector

ion source

ion source

detector

time of flight

time of flight



55/67

Matrix Assisted Laser Desorption

Ionization (MALDI)

Sample introduction

direct insertion probe

continuous-flow introduction

Benefits

rapid and convenient molecular weightdetermination



56/67

Matrix Assisted Laser Desorption

Ionization (MALDI)

Limitations

MS/MS difficult

requires a mass analyzer that is compatible withpulsed ionization techniques

not easily compatible with LC/MS

Mass range

Very high Typically less than 500,000 Da.


57/67


58/67


59/67


60/67


61/67

How is the sample introduced into the


62/67

Mass Spectrometer?

For EI and CI there are four main options Heated reservoir septum inlet for pure gases or

volatile liquids. Basically this is a heated reservoir

(~200oC) with a small restriction bleed into the ion

source. Sample is injected into the reservoir

through a septum.

Direct insertion probe for volatile solids. Sample isloaded into a quartz tube at the end of a probe and

inserted directly into the ion source. The end of the

probe can then be heated, if required, up to

temperatures in excess 400oC.


63/67



64/67

Mass Spectrometer?

For EI and CI there are four main options Particle Beam interface for semi-volatile compounds that

are amenable to EI and CI.

Samples are dissolved in a suitable solvent and the solution

is introduced into the mass spectrometer using a suitable(e.g. HPLC) pump.

The liquid is nebulised with helium gas to form an aerosol of

solvent droplets. The stream of liquid droplets passes

through a desolvation chamber and then a series of nozzles

and skimmers that remove the solvent and helium, allowing

a stream of solid particles (the sample) to enter the EI/CI ion

source.



65/67

Mass Spectrometer?

For FAB / LSIMS Sample can be dissolved directly into the matrix on thetarget or applied dissolved in a miscible solvent. The probeis then inserted in to the mass spectrometer. A dynamicversion of FAB exists, often referred to as continuous flowFAB. Here, the sample, in matrix, is introduced from outsidethe instrument and is then pumped to the target via a lengthof fused silica tubing. Interfacing chromatographictechniques such as HLPC and CZE has been successfully

achieved with this option, although practical difficulties areoften encountered. Dynamic FAB has largely beensuperseded by ESI, which is a more robust technique.



66/67

Mass Spectrometer?

FOR MALDI:The sample is dissolved in matrix, spotted onto the

target, allowed to crystallise and then inserted intothe instrument



67/67

Mass Spectrometer?

For ESI For pure samples dissolved in the mobile phase, a directinjection loop can be used. The technique really comes intoits own however, when interfaced to chromatographicprocedures such as CZE, CEC and in particular HPLC.Historically, mass spectrometry and HPLC have beenunhappy bedfellows. ESI has changed all that and the twoare now routinely and reliably interfaced together. Also, withsmaller columns and ever decreasing flow rates being used,

the amount of sample required for successful analyses isalso reduced, thus effectively increasing the sensitivity of thetechnique dramatically. With CEC being coupled to ananospray (e.g. 50nl/min) ESI interface, attomole detection

Documents

Mass Ionization 21 01 10