Evolving Role of Mass
Spectrometry in
Bioanalytical Analysis
Daniel Pentek
February 1, 2007 -UCONN
Bioanalytical Chem 395
Topics to be covered:

Quick Review
MS Market & Technologies
 Basic ionization methods


Interfaces


Electrospray, APCI, APPI
Orifice, capillary
MS Analyzers


Performance Criteria
Strengths & weaknesses for bioanalytical analyses





Feb. 1, 2007
Quadrupole (single &triple quads)
Ion traps (3-D and 2-D)
Time of flight (linear, reflectron, MALDI)
Hybrids (Quad-TOF) and others…
FTMS (ICRs and orbitraps)
Bioanalytical Chem 395
What is a mass spectrometer?

An instrument that separates molecules or
atoms (e.g., ICP/MS) based on their
mass/charge ratio (m/z).
 In
order to control them, you need to put a
charge on them.
 In order to separate them, you need an
“analyzer” of some type…
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Bioanalytical Chem 395
LC/MS Historical Perspective

Industry was desperate for a decent, rugged
LC/MS interface.
 GC/MS required derivatization, etc.
 Not applicable to most biomolecules


API ionization ALONG WITH new interface
designs provided the solution.
Now, all interfaces are differentially pumped.
 Pumping of interfaces was critical
 Orifice – skimmer (nozzle) designs
 Heated

(MW, etc.)
(or cold) capillary designs
LC/MS is big business now!
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Analytical Instruments Technology Segments…
SDI data on analytical instrument technology shows mass spectrometry is a
$2B market in 2006, and predicts…
…that it will have fastest growth rate (8.3%) of any
analytical instrument technology through 2010
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*
Distribution of Mass Spectrometry Techniques*
Growth Opportunities
Avg. CGR >10%
$101M
$255M
$643M
$312M
$139M
$136M
$391M
CGR 2.3%
CGR 6.2%
CGR 9.5%
CGR 9.4%
CGR 17.0%
CGR 9.7%
CGR 4.9%
PKI has product offerings
*SDI Global 9th Ed. Sept. 2006
Feb. 1, 2007
$61M
$241M
CGR 3.8%
CGR 7.2%
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LC/MS(/MS) is a Marriage of Liquid
Chromatography and Mass Spectrometry




Marriage (like any other close relationship)
requires:
COMPROMISE!
LC Person: “MS is just another detector”
MS Person: “LC is just an inlet”
 What’s
good for LC may not be good for MS and vice
versa.
 LC was around a long time before they figured out
how to interface it to an MS.
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LC/MS Instrument Basics:
Single MS- Primarily used as a detector
LC
Ion Source
-ESI
-APCI
-APPI
Interface
Mass Analyzers
Detection
CEM
-Orifice – Sk
1- Quadrupole
-Capillary
2- Ion Trap (4 types) Discrete dynode
CCD
(Hot or cold) 3- Time of Flight
Photomultiplier
MS System – under vacuum
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Today’s LC/MS Ionization Methods
All Done at Atmospheric Pressure

On-line techniques:
 Electrospray (ESI) - Fenn @ Yale ~1984
 Shared Nobel Prize in 2002 for this work with K. Tanaka (MALDI)
and K. Wüthrich (NMR)
 Atmospheric Pressure Chemical Ionization (APCI)
 Irabarne & Thomson ~1979
 Atmospheric Pressure Photo Ionization (APPI)
 Emerging, not as widely used yet.

All of the above are done at atmospheric pressure
 Significant
change from traditional ionization methods
which were all done within the vacuum chamber.
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Ionization Techniques – Application range
Electrospray
APPI
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Electrospray Basics
(Spraying a charged “mist”)
Vacuum Interface
IonSpray inlet
charged droplets
High Voltage
–
+
+
Sample
+
+
+
+++
– ++
++ –
+
+
+
+
+
+
+
+
+
+
To Q0
-3
(8x10 Torr)
Ions
Nebulizer Gas
Ion Source
(atmosphere)
~10,000,000 ions on column
~4,000,000
- 40,000 ions
Operator Impact Area
(IonSpray is a AB-Sciex trademark name for nebulizer assisted electrospray.)
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~1000 ions
Electrospray – Based on Ion
Evaporation Theory
Rayleigh Limit = 10 cm2/V
-The key is to get rid of the solvent before the ion enters the MS.
-The higher the mobile phase flow rate, the more gas and heat that is required.
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Electrospray

Take home message:
 Electrospray
is concentration dependent
technique.
 Started out as very low flow technique, which
wasn’t very compatible with LC.
LC Person: “Use lower flows and narrower column”.
 MS Person: “Buy ESI probe that has higher gas flows
and desolvates better”.

 Ease
of use and higher LC flow compatibility
drove source development.
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Example: High Flow Electrospray Source
AB-Sciex TurboIonSpray Source
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Example: High Flow Electrospray Source
AB-Sciex TurboIonSpray Source
Electrospray Probe
Heater Gas Probe
High Voltage
Connector
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Temperature and
Source ID Connector
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Newer Ion Sources are have Orthogonal
Design: 90º to Ion Entrance (Orifice) of MS.
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Electrospray- Tips
 Modifiers
Organic acids (e.g. formic, acetic) promote
ionization of basic compounds (sp3 N- containing)
 Neutral compounds containing nucleophilic lone
pairs (sp2 N, sp3 O) can be desorbed by
cationization with alkali metal or ammonium ions.
 Ammonium formate or acetate are recommended
as buffers ( 2-10 mM optimum, can see
suppression effects over 20 mM)

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Electrospray- Tips
 Modifiers (cont.)
Salts can interfere with ionization and can cluster
to complicate spectrum (but also aid in
identification)
 Strong bases or quaternary amines can interfere
with positive mode analytes
 Sulfonic acids interfere with negative mode
analytes
 DO NOT USE PHOSPHATE BUFFERS

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Atmospheric Pressure Chemical Ionization
(APCI)
(“Steam distill” LC eluent past a HV needle)
To the MS…
To MS

Liquid flows up to 2 mL/min are handled by using 2
additional gas flows and heat.
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Heated Nebulizer – APCI Probe
Designed to Deliver Mist to Needle
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Atmospheric Pressure Chemical
Ionization (APCI) Basics
(“Steam distill” LC eluent past a HV needle)
 APCI
utilizes corona discharge
 APCI is a “three” step process:
1) Needle at high voltage ionizes nebulizing gas
(air or nitrogen) forming primary ions
 2) Primary ions react immediately with solvent
molecules forming reagent ions
 3) Reagent ions react (by proton transfer) with
analyte molecules forming (M+H)+ in positive ion
mode or (M-H)- in negative ion mode

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Atmospheric Pressure Chemical
Ionization (APCI)

Corona discharge example - positive ion
EI on atmosphere cause e- removal from
N2, O2 forming N2+•,O2+•
 2) In a complex series of reactions N2+•,O2+•
react with H2O, CH3OH forming H3O+ and
CH3OH2+ as reagent ions for CI.
 3) H3O+, CH3OH2+ donate protons to analyte
forming [M+H]+
 1)
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APCI- Tips
 Buffers:
Buffers/modifiers not required for ionization
 Volatile buffers tolerated up to 50 mM
 Very polar modifiers may reduce sensitivity to less
polar analyte

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APCI- Heated Nebulizer Summary
HN is a high flow (0.5-2.0 mL/min.) inlet
 Suitable for polar, thermally stable cmpds
 Usually, MW < 1000 amu
 Probe is heated to facilitate vaporization
 Requires nebulizing and auxiliary gas
 Requires corona discharge needle to
produce ionization (APCI)

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ESI or APCI? - Which is better?



For some applications, the choice
is obvious…
For analytes <1000 Da, you had to
try both and see which one yielded
the best sensitivity.
Now, vendors are starting to offer
“dual mode” sources to speed up
method development…
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Dual Mode Source…
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Atmospheric Pressure Photo
Ionization (APPI)

Emerging technique (about 5 years old)
 Uses
typical 10 eV UV lamp (similar to Photoionization lamps for GC).
 Similar to APCI, but applicable to broader
range of compounds.
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LC/MS Instrument Basics:
Interfaces- 2 basic designs…
LC
Ion Source
-ESI
-APCI
-APPI
Interface
Mass Analyzers
-Orifice – Sk
-Capillary
(Hot or cold)
Quadrupole
Ion Trap
Time of Flight
Magnet
Detection
CEM
Discrete dynode
CCD
Photomultiplier
MS System – under vacuum
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Example: Orifice-Skimmer Interface
(AB-Sciex) Differentially pumped
+
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Vacuum Interface: Curtain Gas &
Differentially Pumped Interfaces
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Vacuum Interface: Curtain Gas
&Differentially Pumped Interfaces
 UHP
nitrogen curtain gas (CG) keeps nonionized species out of the orifice and analyzer
region
 CG aids in ion declustering (with CID
potentials)
 Two stage transition from atmosphere to low
pressure region of analyzer (1 x 10-5 torr)
 Curtain gas and ions are drawn in due to;
Pressure differential (both ions & CG)
 Electric field gradients (ions only)

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LC/MS Instrument Basics:
Mass analyzers- where most of the differences occur…
LC
Ion Source
-ESI
-APCI
-APPI
Interface
Mass Analyzers
-Orifice – Sk
-Capillary
(Hot or cold)
Quadrupole
Ion Trap
Time of Flight
Magnet
Detection
CEM
Discrete dynode
CCD
Photomultiplier
MS System – under vacuum
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Analyzer (and System) Criteria

Analyzer Considerations:
 Resolution
 Mass accuracy
 Scan speed
 Dynamic range

System Considerations:
 “Sensitivity”
 Sample thru-put
– Fast LC?
 Primary application: quantitation, qualitative or…
 Software- application based
 Ease of use!!!
 Price…
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Single MS and MS/MS Systems:
 Single
MS systems (quadrupole or TOF)
Have the added dimension of mass vs. UV or
diode array detectors.
 However, chemical noise is the limiting factor for
sensitivity (S/N) and dynamic range.

 “MS/MS

Systems”
Many variations now…
2 quadrupole MS’s, separated by a collision cell
 Quadrupole front end, collision cell, TOF back end
 TOF-TOF, separated by collision cell
 “MSn” analyzers- ion traps, FTMS’s and others…


Feb. 1, 2007
Purposes essentially the same (regardless of
analyzer type), select one ion from all the others,
fragment it, andBioanalytical
study Chem
the395
fragment ions
2003 SDI- Price vs. Resolution
SDI MAP October 2003
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2003 SDI- Price vs. Mass Range
Q-TOF
LC-TOF
Gen Purpose
IT Nth
TQ
Mag Sector
QTrap
SDI MAP October 2003
Feb. 1, 2007
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2003 SDI- Mass Range vs. Resolution
SDI MAP October 2003
Feb. 1, 2007
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Resolution- What is it?


Ability to separate (resolve) adjacent ions
Typically defined as: M/∆M
 M:

Mass
∆M: Full Width at Half Max.
Quadrupoles: scan at constant peak width
 30/1=30, 300/1=300, 3000/1=3000…
 Resolution increases as you go to higher mass

TOFs: scan at constant resolution
 10k res: m/z 10.001, 100.01, 1000.1, 10,001
 Peak width increases as you go to higher mass
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Analyzer Types: Quadrupoles
(and hexapoles, octopoles, etc.)
Fundamental parts of virtually all LC/MS
systems
 Serve one of two purposes:

 Ion

Capture and transmit ions from one place to
another…
 Ion

Feb. 1, 2007
transmission devices (quad, hex, oct…)
filtering devices (quadrupoles only…)
Act as a mass filter (analogous to a magnet)
Bioanalytical Chem 395
Quadrupole Theory

Quad. as a mass filter
 Separates
ions based
on m/z ratio

Quad. made of 4 rods
 “A pole”
- vertical rods;
“B pole” - horiz. rods (by
convention)

y
L
DC, RF volt. imposed:
 U=(DC)A-(DC)B (FDC)
 V:
+
+
ro
r
x
z
RF volt. peak-peak
(RFp-p)
 V = 7.22 * M * r02 * f 2 ;
i.e., V ~ M
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Feb. 1, 2007
Quadrupole Theory (cont.)
Resolution (pk width) of quad. defined by:
 M/ M =0.126/[0.168-U/V]

 M:Mass
M:Full Width at Half Height
 As slope approaches 0.168, resolution
approaches infinity (no signal)

Resolution at any mass depends on U/V
ratio (∆DC / ∆RFp-p)
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Typical “Single quad” MS Ion Path:
Basic, “single MS” analyzer
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API-3000 “Triple Quad” Ion Path
OR
IQ1
CEM
IQ2
RNG
Q0
ST
RO1
ST3
RO3
DF
RO2 (LINAC)
1 Torr
S25B Pump
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6 mTorr
Varian 550
Leybold 361
Backed by D10E
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API-3000 Mass Filter RailCollision Cell
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All Triple Quads- Collision Cells
must Overcome “Crosstalk”
•
Crosstalk: can occur when measuring common
fragment from 2 different precursor ions
•
•
ABI-Sciex uses “Linac” (linear accelerator)
•
•
•
•
It takes time for ions to exit a collision cell
Eliminates cross-talk and allows faster MS/MS
scanning without sensitivity losses
Q2 rods are tilted and separate DC potentials are
applied to each pair of rods to create an axial electric
field
Waters (Micromass) uses “T-Wave”
Agilent uses something (they have to…)
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API-3000 Collision Cell - Linac
•
•
Q2 Linac (linear accelerator) eliminates
cross-talk and allows faster MS/MS
scanning without sensitivity losses
Q2 rods are tilted and separate DC
potentials are applied to each pair of
rods to create an axial electric field
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AB-Sciex’s LINAC Technology
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Triple Quad Scanning Modes:
MS/MS - Product Ion Scan

Product ion scan- common MS/MS mode
 After
identification, the precursor ion is sent
into the collision cell and fragmented
 Q1 is fixed, Q3 sweeps a given mass range
 Used for structural information gathering and
identification of product ions
 First step to developing quantitative method
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MS/MS - Product Ion Scan (cont.)
Product Ion Scan
m1+ set
Product ion spectrum of a particular compound
m1+
m2+
m2+
m2+
m2+ scan
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Triple Quad Scanning Modes:
MS/MS - Precursor Ion Scan

Precursor ion scan
 Q1
sweeps a given mass range, Q3 is fixed
 Used to determine the “origin” of particular
product ion(s) created in the collision cell
 Frequently used for drug metabolite
identification (common product ion observed
in the metabolites)
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MS/MS - Precursor Ion Scan (cont.)
Precursor Ion Scan
+
m1 scan
A set of compounds with a common product ion
m2+
m1+
m1+
m2+ set
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m1+
Triple Quad Scanning Modes:
MS/MS Constant Neutral Loss

Neutral loss scan
 Q1
& Q3 both scan a given mass range but
with a constant difference between ranges
scanned
 Spectrum indicates which ions lose a neutral
species equal to Q1 - Q3 difference
 Complement to Precursor Ion Scan
 Neutral “gain” indicates a multiply charged
precursor ion was fragmented
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MS/MS Constant Neutral Loss (cont.)
Constant Neutral Loss Scan
+
m1 scan
A set of compounds with a common neutral fragment
m1+
m2+
-m
m
m2+
m1+
-m
m2+ scan
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Triple Quad Scanning Modes:
Multiple reaction Monitoring (MRM)
If Q1 and Q3 width=0, then MRM
 Many precursor to product ion pairs can
be monitored (A-B, A’-B’, A”-B”, etc.)
 MRM analysis is the best way to maximize
signal intensity of product ions
 MRM used primarily for quantitation
studies

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MS/MS - Multiple Reaction Monitoring (MRM)
Precursor ion
set
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Fragmentation
(CAD)
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Product ion
set
Triple Quad MS/MS Example…
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MS Analyzer Comparison- Mass Accuracy
Quadrupoles
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Analyzer Types: Ion Traps -MS/MSn
Systems
3D Ion Trap
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4 “Types” of Ion Traps…
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3D Ion Trap- MS/MS Operation
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Strengths/Weaknesses of 3D Traps
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Triple Quads vs. Ion Traps
Complementary MS/MS Approaches:
Tandem in Space: Triple Quads
Poor scanning sensitivity
Great for quant (MRM)
Very selective scans
Tandem-in-Time: Ion Traps
Very sensitive scanning
Only product ion scans
Only scanning
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Ion Traps -2D (2002)

“Ion bottles” for optical
spectroscopy.
 Minimize
fringing fields
to maximize
performance.

Linear Traps
3-D Traps
Feb. 1, 2007

Ion accumulation for
enhanced ms
sensitivity.
High quality mass
spectrometer:
 RCM,
Bioanalytical Chem 395
526.
2002, 16, 512-
AB-Sciex Q TRAP™ System
Ion Path
Dipolar Aux AC
N2 CAD Gas
Skimmer
Q0
Orifice
Q1
IQ1
Q2
IQ3
IQ2
LINAC
Feb. 1, 2007
Q3
Bioanalytical Chem 395
Exit
linear ion trap
3-4x10-5 Torr
Trapping Forces in a Linear Ion
Trap
Radial Trapping RF Voltage
Axial
Trapping
DC
Voltage
Axial
Trapping
Exit Lens
Radial Trapping RF Voltage
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Resonance Excitation
Run Thermo 2D Ion Trap
Simulation…
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Linear vs. 3-D Ion Traps:

Linear Trap
 Trapping



Efficiency
3-D Trap
 Trapping
No quadrupole field on
center line.
Longer flight path.


Efficiency
Quadrupole field gives
amplitude and phase
dependent injection eff’s.
~1 cm to lose injection
energy.
Linear trap is ~10X better
 Extraction Efficiency
 Extraction Efficiency

18-20% (measured)

< 50% (Probably ~30%)
3-D trap is ~2X better
 Ion Capacity:
 Ion Capacity:
5-inch linear trap:
45X greater capacity
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FTMS- Ion Cyclotrons (ICR)
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Thermo FTMS (ICR) with 2D Ion
Trap Front End…
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FTMS (ICR)…
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FTMS Data Example…
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New Analyzer: FTMS Ion Trap- Orbitrap

Finnigan LTQ Orbitrap FTMS
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Finnigan LTQ Orbitrap FTMS
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Orbitrap (Brochure) Data
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2003 SDI- Price vs. Resolution
Orbitrap
SDI MAP October 2003
Feb. 1, 2007
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‘Time-of-Flight’ Mass Spectrometry
TOF Analyzer
 Linear
Mode
 Reflectron Mode
Common
Ionization Methods for TOF MS
 MALDI
 ESI
Sample Application
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Simplified Schematic- TOF-MS
analyzer
Time-of-Flight (TOF)
drift tube
The analyser, detector and ionisation
source are under high vacuum to allow
unhindered movement of ions
 Operation is under complete data
system control

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TOF Mass Analyzer – The ‘Drift Tube’
+
+
+
-
+
+
+
+
+
+
time
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LINEAR MALDI TOF MS
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REFLECTRON MALDI TOF MS
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Ionization Methods —MALDI
The Mechanism of MALDI
(Matrix-Assisted Laser Desorption/Ionization):
Ion Desorption
 The Formation of a ‘Solid Solution’
 Matrix Excitation
 Analyte Ionization
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MALDI Mass Spectrometry
Mass Spec
MALDI = Matrix-Assisted Laser
Desorption / Ionization
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MALDI TOF Mass Spectrometry
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SELDI MS (Surface Enhanced Laser
Desorption/Ionization)
1. Add sample
2. Washing
3. Add matrix (‘EAM’)
4. Detection by TOF-MS
EAM = Energy absorbing molecule
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gifsChem
from395
http://www.bmskorea.co.kr/new01_21-1.htm
Bioanalytical
Orthogonal MALDI TOF MS
PerkinElmer pro-TOF
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MALDI-TOF Ex. Data
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Sample Applications (TOF MS)
Biomarker Discovery
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Quadrupole- TOF MS/MS Systems (QTOF)
 Introduced
commercially by Micromass
around 1995.
Brilliant innovation, first commercial hybrid MS/MS.
 They charged “what the market would bear”

$500-600k
 No competition!
 Great qualitative analyzer


TOF analyzer provided:
Incredibly fast scan rates
 Accurate mass capability (MW confirmation)
 Higher resolution (8k initially, now 10-12k w/ 1 reflectron)

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QSTAR® XL System—Schematics
770 L/s
250 L/s
4-anode detector
DC Quad
Accelerator
column
Sample
Ions
Q1
Q0
Q2
10 mTorr
2.5 Torr
Curtain
Gas
LINAC
liner
770 L/s
10-2 Torr
• eliminate cross-talk
• fast switching MS  MS2
Effective Flight
Path = 2.5 m
Field Free
Drift region
7x10-7 Torr
Ion Mirror
(reflector)
• broad dynamic range
• saturation correction
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Conducting
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Micromass QTOF Premier
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What can a QTOF do for you…?
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TOF/TOF System (2001)
TOF 1
CID
Beam XY
Deflector 1
Gas in
TOF 2
Detectors
Source 2
TIS
U
U
U
U
Lens 1
Source 1
Source 1
Grid
Feb. 1, 2007
Source 2
Collision Grid
Cell
Beam XY
Deflector 2
Mirror
Bioanalytical Chem 395
ABI 4700 TOF/TOF
Automation Sample Loader
Collision 200 hz
Cell
Laser
Fast
Sample
Stage
Integration –
Oracle Database
Feb. 1, 2007
Bioanalytical Chem 395
Patented
TOF-TOF™
Analyzer
New Model: ABI-4800 TOF-TOF
Feb. 1, 2007
Bioanalytical Chem 395
Ion Trap- TOF New Hybrid Design
Feb. 1, 2007
Bioanalytical Chem 395
Summary
 Role
and importance of MS in bioanalytical analysis
continues to grow and evolve
MS is typically no longer the bottleneck (sample handling
and data acquisition/processing slow thru-put)
 Power of new MS technology providing new dimension of
information on biomolecules

 There
are 3 fundamental MS analyzer technologies,
each with it’s advantages and disadvantages.

‘Hybrids’ can take advantage of best of 2 technologies
 LC/MS

continues to evolve at a rapid rate
Better, faster, cheaper…
Feb. 1, 2007
Bioanalytical Chem 395