intro to ms nrao

8/22/2019 Intro to MS Nrao

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Introduction

toQuadrupole Mass

Spectrometry


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Components of a Mass

Spectrometer

SampleIntroductionMethod to vaporize sample

Inlet

Ionization/ desorption

Analyzer

Mass Sorting

Ion Detection

Detection

Data Analysis

Source

+

1330 1340 1350

100

75

50

25

0

Solid

Liquid

Vapor

Detect

ions

Form ions(charged

molecules)Sort Ions by Weight


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LC-MS Components

Sample introduction

e.g., FIA, HPLC

Sample ionization Nebulizer System

Sample transfere to high vacuum regionAPI Interface

Ion mass-to-charge filtering Mass Analyzer

Ion detection Detector

Data acquisition and analysis

Data System


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Ionization

Techniques

ElectronImpact

(EI)

ChemicalIonization

(CI)

APIMALDI

Hard, Fragments

Fast AtomBombardment

(FAB)

Soft, Intact


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LC-MS Sample Inlets

Themost common sample inlets for LC-MS are

Atmospheric Pressure Ionization (API)

Sources

Two distinct types of API sources:

Heated Nebulizer Source

ElectroSpray Source

IonSpray

TurboIonSpray


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Heated Nebulizer (APCI)

Heated Nebulizer:

Atmospheric Pressure Chemical Ionisation (APCI)

corona discharge needle

polar to unpolar and thermally stable compounds


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Heated Nebulizer:





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Heated Nebulizer:





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Heated Nebulizer:





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Heated Nebulizer:





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Heated Nebulizer

1st picture inside of APCI/HN, with corona needle offset between orifice andquartz tube

2nd picture back of APCI/HN unit with articulations for the HN on back and

the corona needle on top.


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Heated Nebulizer (APCI) Inlet

Suitable for polar, thermally stablecompounds

Usually, Molecular Weight < 1000 amu

Probe is heated to facilitate vaporization

Requires nebulizer and auxiliary gas

Requires corona discharge needle to produce

ionization (APCI)


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ElectroSpray Inlet

(Ion Evaporation)

++++

+ +++ +

+++

++

Liquid

5 kv

++ +++++++++++

+

+

+

+++

++

++

Droplet

Formation

Droplet

Evaporation

Coulomb

Explosion

Ion Evaporation

Liquid Phase Ionization

MolecularIon

H+

++ +++

++++++

++++++

+++

+++

+++

+++

+++

+++


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TurboIonSpray Inlet


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TurboIonspray Source


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TurboIonSpray Inlet

Pros:

Hot drying gas improves evaporation of charged

droplets

no thermal degradationbetter sensitivity at higher flow rates

tolerates higher aqueous solvent compositions

Improved performance over a wide range of LC flow

rates without splitting (5L/min to 1mL/min)

Detection limits are very good


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Photo Ionization Source


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Operating Principle

Samples are sprayed with the aid of a nebulizing gasinto a heated probe, as in APCI.

A dopantcompound is vaporized in the auxiliary gaswithin the heated nebulizer probe.

In the ionization region, dopant molecules arepredominantly ionized by UV radiationand formphotoions.

The photo ionsinitiate a cascade of ion-moleculereactions leading to the formation of the ionizedanalytes in the form [MH]+ (by proton transfer) or [M]+(by charge transfer).


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The role of the Dopant

The dopant acts as an intermediate in the

ionization process

Opens up an alternative reaction channel

Increases analyte ionization efficiency

Two dopants were extensively tested

Toluene

Acetone


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PhotoIonization lamp

Krypton lamp

10 eV

CompoundIonizationPotential(eV)

Nitrogen15.58Water12.62

Acetonitrile12.20Oxygen12.07Methanol10.84

MethylPentanoate10.40Hexane10.13Heptane9.93Acetone9.70Pyridine9.26Benzene9.24

Amphetamine8.99Toluene8.83

Naphtalene8.14Reserpine7.88

Triethylamine7.53


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Ion Source Schematics


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PhotoSprayTMSource Block


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PhotoSpray Source

Pros: Entire mobile phase and sample vaporized into the gas phase (with

heat), then ionized

Uses Dopant (Toluene/Acetone) to promote ionization

Accommodates high LC flow from (0.2 - 2 mL/min)

Uses heat (400500 C) & nebulizer gas to vaporizeHPLC eluent and steam distill sample into the gasphase for photoionization

Detection limits are very good

Improvements over ESI and APCI compound dependant

Wider application range

Cons: Thermally labile analytes may degrade when vaporized

Low mol weights only (


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PhotoSpraysource vs APCI vs

TurboIonspray

Sensitivity

Polar compounds: TIS > APCI > PhotoSpray

Non Polar compounds: PhotoSpray > APCI > TIS

Chemical Background

TIS > APCI > PhotoSpray

Suceptibility to the MP compositionPhotoSpray > TIS > APCI


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Ion Sources Sensitivity

SampleCharacteristics

ESI APCI PhotoSpray

Ionic

Ionizable

Polar Non-ionic

Nonpolar


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Ion Sources Characteristics Molecular

Weight Consideration

ChemicalProperties

ESI APCI PhotoSpray

Ionic Ionizable Polar Non-ionic Nonpolar - - molecular weight >100000

>2000 >1000 >500


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PhotoSprayTMPotential Application

Areas

Pharmaceutical Companies/CROs

Steroids/Hormones/Apolar Vitamins

Relative Apolar drugs

Alternative Markets

Petroleum/Oil Industry

Polymer Analysis

Food Analysis

Sugars

Carotenoids

Essential Oils

Environmental Analysis

PAHs; PCBs; Dioxines


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Areas

Clinical

Vitamins

Food Analysis

Sugars, Carotenoids. Essential Oils, Flavonoids

Environmental Analysis

PAHs; PCBs; Dioxines, Pesticides

Petroleum/Oil Industry

Polymer Analysis


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Areas

Prohibited classes of substances

Stimulants

Narcotics

Anabolic Agents

Anabolic androgenic steroids

Beta-2-agonists

Diuretics

Peptide hormones, mimetics and analogues

Masking agents

Beta-Blockers


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API Inlets

Heated Nebulizer, TurboIonSpray and Photo

Ionization Inlets are soft ionization sources

They produce abundant molecular or protonated ions

with little or no fragmentation

Ideally suited for high sensitivity (quantitative)

analyses

Qualitative analyses can be enhanced with the

presence of characteristic fragment ions

I T f f At h t


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Ion Transfer from Atmosphere to

a High Vacuum Environment

2 Tor Declust. RegionProblem of all conventionalinterface designs:

Matrix, e.g., salt praticles, may

cause clogging of the orifice

and/or form deposition inside the

interface or further downstream

How to overcome this problem:

Off-axis spraying

Z spray

Orhtogonal sprayingStill not good enough

Clustered

I ons

Declustering

on

Off -axis spray

direction

Th API P t t d C t i G


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The API Patented Curtain Gas

Interface

2 Tor Declust. RegionAll AB Sciex LC/MS use thepatented Curtain Gas Interface

Combination of simple off-axisspraying with active gas protection

Simple design

Easy to optimize

Curtain gas protects MS fromcontamination

dirt forms deposition off-axis oncurtain gas plate

Matrix particles (neutrals)can not pass N2pressure barrier

reduces matrix induced drift to aminimum

very infrequent cleaning

CurtainGas

Clustered

I ons

Declustering

on

Off -axis spraydirection


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1. Curtain Gas assists in droplet evaporation

ATMOSPHERIC PRESSURE VACUUMCUR DP SK

FP

N2

+ ++

+

+

+

How the curtain gas works


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FP

N2

+

+ +

+

++



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2. Curtain Gas declusters ions


FP

N2

+



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FP

N2

+



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FP

N2

+



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State-Of-The-Art API Interface

N2gas curtain and orificevoltage assist in

declustering ions

Increasing orifice voltage can

cause analyte fragmentation(compound dependant)

advantageous with Single

Quad MS for achieving

structural information

A Vi B hi d th API I t f


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A View Behind the API InterfaceThe RF0 Ion Guide


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Ion Sampling Region

Expansion of gas and ions into vacuum

random

motion

of

ions andgas

760 torr 1 torr

250 micronorifice

atmosphere vacuum

barrel shock

silent zone

-mach disk-

gas velocity breaks

sound barrier

ion motion

alligned and

confined to

gas

4.5 mm

Different Ion Sampling


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Different Ion Sampling

Approaches

ions

gas

opticsRf onlyquadrupole

ions

highly charged

clusters

ions

opticsRf only quad

gas

skimmer

ring

electrode

ions

highly charged

clusters

ions

gas

ions

highly charged

clusters

focusing

ring

A. On Axis Sampling

B. Off Axis Sampling

C. L (dog leg) Sampling


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Patented RF Ion Guide (Q0)

Ion beam expands behind Skimmerdifferential pressure difference

space charge RF Ion Guide prevents loss of ions and focuses them into and

through Ion Guide

8 mTorr Ion Guide region promotes collisional focussing

+

+

SkimmerOrifice

Patented Collisional Focussing


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Patented Collisional Focussing

vs. Conventional Ion Guides

Collisional Focussing

dampens beam

amplitudeFocussed ion beam

improved transmision

Conventional Ion

Guides

poorer ion transmisson(ions are lost)

Sensitivity of Ion Transmission vs


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Sensitivity of Ion Transmission vs.

Q0 Pressure

M A l


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Mass Analyzers

All mass spectrometers sort ions based on their mass-to-

charge ratios (m/z) in a vacuum

Common analyzer types:

Quadrupole

single quadrupole

triple quadrupole

Time of flight

Ion trap

Magnetic and electric sector

Fourier transform ion cyclotron resonance

Quadrupole Fundamentals


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Mass

Voltage

-DC RF amplitude

Voltage

Mass

+DC RF amplitude

r0

+

+

A quadrupole is a tunable band-pass filter

RF/DC ratio is constant

filter is tuned by raising

voltage amplitude


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Selecting Quadrupole Resolution

DC

RF


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+

+

+

1. Ion enters the quadrupole system


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+

+

+

2. Electrical repulsion and attraction,

respectively, between quadrupole rods and ion


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+

+

+

3. Movement of the ion into direction of the

nearest quadrupole rod with the opposite charge


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+

+

+

4. RF-voltage changes polarity and electrical repulsion

and attraction, respectively, between quadrupole rods

and ion


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+

+

+

5. Movement of the ion into direction of the nearest

quadrupole rod with the opposite charge


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+

+

+



and ion


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+

+

+




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+

+

+



and ion


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+

+

+




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Start of the animation:How is the oscillating RF voltage working?


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+

+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+


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+

+



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Stable & Unstable Ion Trajectories

At any RF/DC potential setting only ions of one particular

mass/charge ratio have a stable trajectory

Scanning Using a Quadrupole


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Mass Filter

3 7 4 . 0

4 8 2 .5

263.3

347.3

459.4

518.5300 360 420 480 540

m/z, amu

10

20

30

40

5060

70

80

90

%I

ntensity

2 29 1 .2 7 .24

Pi t f Q d l


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Picture of a Quadrupole

++ +

-

-

Single Quadrupole MS


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(generic API 150 schematics)

- Patents numbers 4,963,736 and 5,179,278

RF-only Ion Guide Filt. QuadStubbies

8 mT Ion-Guide Region 2x10-5 Tor MS Region2 Tor Declust. Region

Curtain Gas

Clustered

Ions

on

Declustering MS-filtrationFocusing Extraction

Patented Curtain Gas interface

Patented collisional focussing quadrupole ion guide

Schematics of a Triple


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Quadrupole MS

RF Ion Guide: Focusses ion beam into Q1

Q1: Analyzer Quadrupole 1can be scanned or set to pass one specific m/z

Q2: Collision Cell

can be pressurized with N2for fragmentation of ionscan be at low pressure for bandpassing all ions

Q3: Analyzer Quadrupole 3

can be scanned or set to pass one specific m/z

N2 Inlet

RF

Ion Guide

Q1 Q2

Coll is ion Cel l

Q3 Detector

Fragmentation in a Tripple


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Quadrupole MS

precursor ion

molecular weight

information

possible fragment ions

structural information

Cross Talk in a Conventional High

P C lli i C ll


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Pressure Collision Cell

Collision cell transit time: 50-100 msec

(conventional design)

Cross-talk between consecutivelymeasured MRMs with same Q3mass

Q1 mass Q3 mass Time

( msec )

136.1 43.1 45.0

136.1 51.1 45.0

189.2 56.1 45.0232.2 56.1 45.0

204.1 56.1 45.0

246.1 56.1 45.0

218.1 56.1 45.0

152.1 59.1 45.0

Cross Talk in a Conventional High

P C lli i C ll


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Pressure Collision Cell

The Problem:potential systematic errors

fragment ions having identical mass but differentprecursors can not be differentiated

sensitivity loss when using very short dwell times forMRM

Scan speed and number of analytes/sample(throughput) must be reduced

Solutions:

Software or firmware (clean pulse)work-arounds

LINAC (AB Sciex Patent)

API Linac Collision Cell


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(Linear Accelerator, Patented)

y

x

y

x

X < y X > y

+5.75 v +4.25 v+1.5v field gradient

entrance exit

Q2 Linac (linear accelerator) eliminates cross-talk

allows faster MS/MS scanning withoutsensitivity loss

API Linac Collision Cell


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(Linear Accelerator, Patented)

Q2 Linac (linear accelerator) eliminates cross-talk

allows faster MS/MS scanning withoutsensitivity loss

Q2 rods are tilted and separate DC potentials are appliedto each pair of rods to create an axial electric field

gradient

Ions exit collision cell much faster

potentialgradie

nt

ionvelocity collisions with N2

reduce ion ion velocity

Cross-Talk without Linac


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Cross Talk without Linac

3.67e4 cpsXIC of +MRM (4 pairs): for 136.1 / 91.1 amu from Seleg 2500pg No-LINAC

Dwell 100msec

0.5 1.0 1.5 2.0Time, min

20

40

60

80

%I

ntensity

1.04e6 cpsXIC of +MRM (4 pairs): for 188.2 / 91.1 amu from Seleg 2500pg No-LINAC

0.5 1.0 1.5 2.0Time, min

20

40

60

80

%I

ntensity

25 ng Selegiline

Cross-talkfor amphetamine

100

100

Injection of selegiline to determine the level of cross-talk in

the amphetamine MRM transition

Different precursor masses, same fragment mass

No Cross-Talk with Linac


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No Cross Talk with Linac

Injection of selegiline to determine the level of cross-talk in the

amphetamine MRM transition with a Linac Q2

8.66e3 cpsXIC of +MRM (4 pairs): for 136.1 / 91.1 amu from Seleg 2500pg w LINAC

0.5 1.0 1.5 2.0Time, min

2000

4000

6000

8000

Intensity,cps

3.76e6 cpsXIC of +MRM (4 pairs): for 188.2 / 91.1 amu from Seleg 2500pg w LINAC

0.5 1.0 1.5 2.0Time, min

1e6

2e6

3e6

Intensity,cp

s

25 ng Selegiline

Cross-talk for amphetamine

No Cross-Talk with Linac


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No Cross Talk with Linac

0.5 1.0 1.5 2.0 2.5 3.0Time, min

10

20

30

40

50

60

70

80

90

%I

ntensity

10025

msec

50mse

c

100msec

250msec

Selegiline

Selegiline response using different MRM dwell times


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MSMS scanning in an

Quadrupole LCMSinstrument

Q1/Q3 Scanning Modes


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Q1/Q3 Scanning Modes

Q1 Full Scan(StartStop):Q1 always used as single MS analyzer

Used primarily for identification of precursor ion

Q3 operates in RF-only mode

Q3 transmits all ions to detectorQ3 acts as ion guide for product ions

In all MS/MSmodes, Q3 is a mass filter

Ions are separated based on mass/charge ratio

Q2 is source of product ions entering Q3

N2 Inlet

RFIon Guide

Q1 Q2Coll ision Cell

Q3 Detector

Single MS Operation


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Selected Ion Monitoring (SIM) (Width = 0):Used to optimizeanalyzer for specific ions

SIM used for quantitative analyses

Q1 SIM used to optimize precursor ion

Maximize signal in preparation for MS/MS

MS/MS - Product Ion Scan


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After identification, the precursor ion is sent into the

collision cell and fragmented

Q1 is fixed, Q3 scans a defined mass range

Provides structural information and identification of product

ions

Product Ion Scan


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Select

Precursor Ion

Scan ProductsCAD

m1

+

m2+

m2+

m2+

Product ion spectrum

of a particular compoundm1+ set

m2+ scan

Quadrupole 1 Coll is io n Cell (Q2) Quadrupole 3

MS/MS - Product Ion Scan


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After identification, the precursor ion is sent into the

collision cell and fragmentedQ1 is fixed, Q3 scans a defined mass range

Provides structural information and identification of

product ions

MS/MS - Precursor Ion Scan


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Precursor ion scan

Q1 scans a defined mass range, Q3 is fixed

Used to determine the origin of particular

product ion(s) created in the collision cellFrequently used for drug metabolite identification

(common product ion observed in the

metabolites)

MS/MS - Precursor Ion Scan (cont.)


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MS/MS Precursor Ion Scan (cont.)

MS/MS Constant Neutral Loss


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Neutral loss scan:

Q1 & Q3 both scan a given mass range but with a

constant difference between the 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

MS/MS Constant Neutral Loss (cont.)


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MS/MS Constant Neutral Loss (cont.)

MS/MS - Multiple reaction Monitoring (MRM)


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MS/MS Multiple reaction Monitoring (MRM)

If Q1 and Q3 width=0, then MRM

Many precursor to product ion pairs can be

monitored

MRM analysis is the best way to maximizesignal/noise ratio of compounds

MRM used primarily for quantitation studies

Multiple Reaction Monitoring (MRM)


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p g ( )

Precursor

ion set

Product

ion setFragmentation

(CAD)

Ion Detection


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Detectors Channel Electron Multiplier (CEM)

Discrete Dynode Detector

Data Acquisition

& Post Analysis Evaluation


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& Post Analysis Evaluation

Two computer and software platforms are offered

Windows NT and MAC

Automatic Resolution

Optimization


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Optimization

Ability to pre-define Unit,High and Low Resolution

Auto Resolution

optimization and Auto-Calibration


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intro to ms nrao

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