quadtrap comparison 1

8/3/2019 QuadTrap Comparison 1

1/105


2/105

2

Comparison of Quads and Traps

Ion Traps Quadrupoles

Mass Separation in time Mass Separation in space

High sensitivity Full Scan Lower sensitivity Full Scan

Lower sensitivity SIM and SRM High sensitivity SIM and SRM

Offer multiple stages of MSn

Parent and neutral loss scans

Offers Only MS or MS/MS


3/105

3

LCQ Instrumentation

ClassicClassic

DecaDecaDuoDuo


4/105

4

Duo/Deca Comparisons

LCQ DUO LCQ DECA 500um capillary

1 square quadrupole & 1 octopole2 rotary pumps

400um capillary

2 octopolesOne rotary pump

Deca: approximately 10 x better signal than Duo


5/105

5

Current LCQ Generation


6/105

6

Advantage/XP Comparisons

450um Ion Transfer Tube Orthogonal Probes

550um Ion Transfer Tube Orthogonal Probes

XP: approximately 10 x better signal than Advantage


7/105


8/105

8

Mass Spectrometry Simplified

G

MS

D

enerate

oveelect

etect

Ion productionIon production

Ion opticsIon optics

Mass filterMass filter

Electron multiplierElectron multiplier


9/105

9

The Mass Spectrum

Red

Prism

Light,all colors Green

Blue

100

Ions,

various massesMass

Spectrometer200

300

T d d b i ll b d


10/105

10

Trace produced by summing all observedTrace produced by summing all observed

masses in each scanmasses in each scan

TotalTotal Ion Chromatogram or TICIon Chromatogram or TIC


11/105

11

Ionization vs. Fragmentation

Ionization HardSoft

No

Fragments

Fragments

API EICI


12/105

12

EI and CI Mass Spectra of Ephedrine

0

50

100

0 20 40 60 80 100 120 140 160 180

%

RelativeIntensity

m/z

EI

0

50

100

0 20 40 60 80 100 120 140 160 180

CI

58

148

166

MW = 165Th

(M+H)+


13/105

13

Ionization Techniques

ESI

GCPBI

TSPFAB

Molecular

W

eight

APCI

200,000

15,000

1,000

Non Polar Polar


14/105

14

What is API?

Atmospheric Pressure Ionization

Source Types:

1. Electrospray Ionisation (ES) Solution phase process (forthe most part).

2. APCI (Atmospheric Pressure Chemical Ionization) - Gas-phase process.

Source Purpose:1. Ionize the analyte (APCI) or transport ion in solution to the

gas phase.

2. Desolvate sample flow for introduction into mass

spectrometer.3. Baffle the first vacuum region of the MS from atmospheric

pressure in the source.

4. Pump away neutrals and opposite charged ions which would

otherwise interfere with the analysis of the desired polarity.


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15

LCQ Classic/Duo/Deca API Probes

Electrospray Ionization (ESI)

Peek insulator

Atmospheric Pressure

Chemical Interface (APCI)


16/105

16

LCQ Advantage/XP API Probes

Atmospheric Pressure

Chemical Interface (APCI)Electrospray Ionization (ESI)

Orthogonal ESI & APCI probes


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17

Chemistry Considerations

ESI:

Ions formed by solution chemistry

Good for Thermally labile analytes

Good for Polar analytes

Good for Large Molecules (Proteins / Peptides)

APCI:

Ions formed by gas phase chemistry

Good for Volatile / Thermally Stable

Good for Non-polar analytes

Good for Small Molecules (Steroids)


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18

ESI Versatility Advantage/XP

(Right: Picture represents

a low flow position,


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19

ElectrosprayBasic Layout

Heated Capillary

ESI Needle+/- 5 kV

Taylor Cone

Solvent evaporation and ion release

++

+++ ++

+

++

+++ +

++

+

+

+

+++

++

+

+

+

++

+++ +

++

++

+++ +

++

++

++

++

+

+

+

+

+

+

++

++

++

+

+

+

++

+

+ ++ +

+++

++

+

++

+

+++

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+


20/105

20

APCI Position on Advantage/XP

In/Out MovableCorona Discharge Pin

Total control for any

flow rate (200ul/min -

2000ul/min)


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21

LC Flow Rates

ESI:ESI:3 L/min - 1mL/minute

Optimal Flow Rate: 200 L/minGenerally, higher flow rates require higher heatedcapillary temperatures and higher gas flow rates.

APCI:APCI:200 L/min - 2mL/minute.Optimal Flow Rate: 500 L/minGenerally, higher flow rates require more sheath andauxiliary gas, but do not require higher heatedcapillary temperatures.


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22

Theoretical Increase in Response

DConc max =2

Column 1D

2Column 2

Col. Diameter mm 1.0

0.05

21

0.5

2.3

3.04.6

0.2

5

2.0 Capillary

Flow Rate ml/min 1 < 10 l / min1Theoretical

Increase

LC Additi


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23

LC Additives

Acids

Do not use inorganic acids (may cause source corrosion)

Formic and acetic acid are recommended

Bases

Do not use alkali metal bases (may cause source corrosion)

Ammonium hydroxide is recommended

Surfactants (surface active agents)

Detergents and other surface active agents may suppressionization

Trifluoroacetic Acid (TFA)

May enhance chromatographic resolution, but causes ion

suppression in both negative and positive ion mode

Isopropyl Alcohol

May Enhance Negative Ion Formation

B ff ( H)


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24

Buffers (pH)

Avoid using non-volatile HPLC additives such as:

Alkali Metal Phosphates

Borates

Citrates

Keep Buffer concentrations below 20 mM using volatilesalts such as ammonium acetate.

When using buffers, more frequent cleaning of the heatedcapillary and API stack will be necessary

LC/MS Additi d B ff S


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25

LC/MS Additives and Buffers Summary

Acetic Acid

Formic Acid

Ammonium Hydroxide

Ammonia Solutions

Trichloroacetic Acid (< 0.1% v/v)

Trifluoroacetic Acid (< 0.1% v/v)

Isopropyl Alcohol

(10% of organic phase)

Ammonium Acetate

Ammonium Formate

Proton Donors

Proton Acceptors

Chromatographic

Separation

Negative ion

formation

Buffers

C LC/MS S l t


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26

Common LC/MS Solvents

Methanol

AcetonitrileWater

IsopropanolDichloromethane

Chloroform

Hexane

Effects of Solvents and Additives on ESI


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27

Effects of Solvents and Additives on ESI

50/50 MeOH/H2O

50/50 ACN/H2O

100 H2O

100 MeOH

100 ACN

50/50 MeOH/H2O 1% Acetic

50/50 MeOH/H2O 0.1% Formic

50/50 ACN/H2O 1% Acetic

50/50 ACN/H2O 0.1% Formic

50/50 MeOH/H2O 5mM NH4OAc

50/50 MeOH/H2O 10mM NH4OAc

50/50 MeOH/H2O 0.1% TFA

50/50 MeOH/H2O 0.05% TFA

50/50 MeOH/H2O 0.02% TFA50/50 ACN/H2O 0.1% TFA

50/50 ACN/H2O 0.05% TFA

50/50 ACN/H2O 0.02% TFA

50/50 MeOH/H2O 0.1% NH4OH

50/50 ACN/H2O 0.1% NH4OH

Solve

ntSystem

Counts (protonated ion species)

0 100000 200000 300000 400000 500000

Tyr-Gly-Gly-Phe-Leu

Leucine Enkephalin

API Stack


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28

API Stack

LCQClassic, LCQDUO,

LCQDECALCQ DECAXP,LCQAdvantage

Ion Transfer Tube and Removal Tool


29/105

29

Ion Transfer Tube and Removal Tool

Removal

tool

Ion transfer tube

Heated capillary

Vent Prevent Mechanism


30/105

30

Vent Prevent Mechanism

Heated Tube

in-situ

Heated Tube

removed

Tungsten Vent

Prevent

Ion Optics


31/105

31

Ion Optics

First

multipole

Lens

Intermultipole

Lens

Second

multipoleLens

Octapole

Mount

Vacuum

Baffle

Analyzer

Mount

IONS

IN

IONS

OUT

Multipole Potential Wells


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32

Multipole Potential Wells

Octapole

Mass RangeTransEfficiency

Round

Quadrupole

Square

Quadrupole

Mass Analyzer (Ion Trap)


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33

Mass Analyzer (Ion Trap)

Vacuum System


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34

Vacuum System

Every mass analyzer must operate under vacuum inEvery mass analyzer must operate under vacuum inorder to minimize both ion/molecule andorder to minimize both ion/molecule and

molecule/ molecule collisions.molecule/ molecule collisions.

At atmospheric pressure, the mean free path of a typicalAt atmospheric pressure, the mean free path of a typicalion is only ca. 52 nm and at 1ion is only ca. 52 nm and at 1 mTorrmTorr, it is 40 m., it is 40 m.

Without vacuum, the ions produced in the source wontWithout vacuum, the ions produced in the source wontmake it to the detector.make it to the detector.

The LCQ vacuum is maintained by a both rotary andThe LCQ vacuum is maintained by a both rotary andturbomolecularturbomolecularpumpspumps

Ion Optics (Operating Pressures)


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Ion Optics (Operating Pressures)

1.3 torr760 torr 1.7x10-3 torr 2.0 x10-5 torr (1.0x10-5 torr He)

3.5x10-3 torr He

220 L/sec60 m

3

/hr 100 L/sec

Steps to Ion Trap Scan Functions


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36

Steps to Ion Trap Scan Functions

Trapping- all scans

Isolation- SIM and MSn

Excitation- MSn

Ejection- all scans

Helium as a Damping Gas


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37


+

+

+

+Without HeliumWithout Helium

He

collision HeHe

He

He

+

+

+

+

With HeliumWith Helium

Discovery of the Effects of Helium


38/105

38

y



39/105

39

p g

Ion Trap ResolutionEffect of Damping Gas


40/105

40

p p g

Traps injected ions by removing kinetic energy

Damps ion motion to center of trap

Result...Increase in resolution and sensitivity

Helium Effect


41/105

41

S#:1 RT:0.00 AV:1 SM:7G NL:2.50E7T:+ p Full ms

514 516 518 520 522 524 526 528

m/z

0

20

40

60

80

100

RelativeAb

undance

524.3

525.3

S#:23-32 RT:0.71-1.00AV:10 SM:7G NL:5.61E7T:+ p Full ms

500 1000 1500 2000

m/z

0

20

40

60

80

100

RelativeAb

undance

1522.04

1621.971322.06

1721.89

1222.141821.95524.26

1122.21 1921.88195.15

1022.09


514 516 518 520 522 524 526 528m/z

0

20

40

60

80

100

RelativeAbun

dance

522.6523.0

521.8

521.2 523.9

520.7


500 1000 1500 2000m/z

0

20

40

60

80

100

RelativeAbun

dance

1620.791520.26

1720.441320.95

1220.75523.01 1919.96

1120.90

192.17

Helium shut off and not flowing into trapHelium shut off and not flowing into trap


514 516 518 520 522 524 526 528

m/z

0

20

40

60

80

100

RelativeAb

undance

524.3

525.3


500 1000 1500 2000

m/z

0

20

40

60

80

100

RelativeAb

undance

1522.04

1621.971322.06

1721.89

1222.141821.95524.26

1122.21 1921.88195.15

1022.09


514 516 518 520 522 524 526 528m/z

0

20

40

60

80

100

RelativeAbun

dance

522.6523.0

521.8

521.2 523.9

520.7


500 1000 1500 2000m/z

0

20

40

60

80

100

RelativeAbun

dance

1620.791520.26

1720.441320.95

1220.75523.01 1919.96

1120.90

192.17

Helium shut off and not flowing into trapHelium shut off and not flowing into trap

Helium flowing into trap

Ion Trap Stability Diagram


42/105

42

y

The region shaded blue indi-cates a (DC) and q (RF)

values which provide stable

trajectories in the r-directionThe region shaded yellow

indicates the z-stable a and q

combinations

The green area where the r-

and z-stable regions overlap

indicates the a and q combi-nations under which ions will

be stable in the trap

Stability Diagram for Commercial Traps


43/105

43

q kV

m ez =

( / )

LCQ Scan-Out (Ejection) Rates


44/105

44

Normal Scan (5500 amu/sec)

Common full, SIM, or MSn (SRM and CRM)scanning

Resolution (FWHM) = 0.50, Mass Accuracy = 0.05

Zoom Scan (280 amu/sec)

Increases resolution and mass accuracy across anarrow range (allows charge state determination)

Resolution (FWHM) = 0.15, Mass Accuracy = 0.02

Turbo Scan (55,000 amu/sec)

Decreases total scan time of a full scan, thus

increasing number of scans across achromatographic peak

Resolution (FWHM) = 3.0, Mass Accuracy = 0.5Used for better quantitation due to an increase

of scans across a chromatographic peak

What is AGC and Why Is it Important?


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45

Camera AE

Too much light degrades the image

stored on film, causing a loss of

color and image resolution.

Too little light results in dark

picture with no fine details visible.

Cameras with high quality light

meters and AE controls produce

high quality pictures over a wide

dynamic range of lighting

conditions.

LCQ Series AGC

Controls amount of ions (light)

entering the ion trap (film)

Too many ions degrade the

spectral quality in the trap,

causing loss in mass resolution

and mass assignment. Too fewions result in poor sensitivity to

low level or minor components.

AGC ensures excellent quality

MS, SIM and MS/MS spectra,

as well as excellent sensitivity

over a wide dynamic range.

Automatic Gain Control (AGC)


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46

Prescan before the analytical scan

- Measures the # of ions in the trap for a

pre-defined time (10 ms)

-Allows software to determine optimum

ion injection time

No AGC Spectrum of Ultramark 1621, Caffeine,MRFA Calibration Mixture space charging


47/105

47

MRFA Calibration Mixture space charging

Spectrum of Ultramark 1621, Caffeine, MRFACalibration Mixture with AGC


48/105

48

Calibration Mixture with AGC

AGC (Ion Population Control)


49/105

49

~ 300 Ions ~ 1500 Ions ~ 3000 Ions ~ 6000 Ions

530m/z

0

20

40

60

80

100

Relative

Abundance

524.3

525.3

526.3

530m/z

0

20

40

60

80

100

Relative

Abundance

524.4

525.4

526.3

527.5

530m/z

0

20

40

60

80

100

Relative

Abundance

524.5

525.5

526.5

527.5

530m/z

0

20

40

60

80

100

Relative

Abundance

524.8

525.7

526.7

522 522 522 522

Poor ResolutionGood Resolution

Calculation of Ion Time


50/105

50

Constant During Prescan

AGC Prescan Signal =

Multiplier Gain x Prescan TimeNumber of Ions x

(3x105 counts) (10 ms)

Calculated Ion Time = Target Value

AGC Prescan Signal(how long the gate lens is open)

Triplicate Injection of 5 nmol of MRFA (AGC ON)


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51

Scan Number

UnscaledTIC(counts

)

0.0E+00

2.0E+074.0E+07

6.0E+07

8.0E+071.0E+08

1.2E+08

1.4E+08

1.6E+081.8E+08

2.0E+08

0 100 200 300 400 500 600

Unscaled TIC

0

10

20

30

40

50

60

0 100 200 300 400 500 600Scan Number

InjectionTime(ms)

Injection Time

Scaled TIC

0.0E+00

1.0E+08

2.0E+08

3.0E+08

4.0E+08

5.0E+08

ScaledTIC(counts)

8.0E+08

7.0E+08

6.0E+08

0 100 200 400 500 600300

Scan Number

Isolation of Ions


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Ion we wish to isolateIon we wish to isolate

7

0.908qz

0.0

Ions at different qz values oscillate at

different frequencies (

o)

22

qzo

Isolation Waveforms


53/105

53

~ m/z 200

q axis .908

500 Hz

16 msec

q axis.908

Why MS/MS or MSn ?


54/105

54

Signal to Noise Improvement

0 1 2 3 4 5 6

Stages of Analysis

Intensity Signal

Noise

S/N

MS/MS Parameters


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Precursor ion m/z

Excitation voltage

Excitation qz

1.0 2.0 3.0

Excitation Voltage (V)

Intensity

Precursor ion

(Product ions)

0.0 4.0

77001-1285

970219

Resonant Excitation qz Value


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Fragmentation

Energy

0.908

qz

0.00.2250.225

0.908

qz

0.0

0.908

qz

0.0

0.300.30

0.450.45

Fragment ions

not trapped

Product Ion

m/z Range

1/41/4

1/31/3

1/21/2

Ion Trap Scan Functions


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1. Collect

2. Isolate

3. Fragment

4. Eject

Summary (Ion Trap Functions)


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1)1)CollectionCollection

2)2)IsolationIsolation

3)3)ExcitationExcitation

4)4)Ejection

For Scans: All

By: Ring Electrode

Method: Alternating RF

frequency (760 kHz) at a set

amplitude along with Hedampening gas traps and

cools the ions to the center of

the trap.Ejection



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1)1)TrappingTrapping



4)4)Ejection

For Scans: SIM, MSn

By: Endcap Electrodes

Method: a) Tailored waveform

applied to all ions in the trap

except ion of interest

b) Thus, only ions of interest

remain in the trap.

Ejection



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4)4)Ejection

For Scans: MSn

By: Endcap Electrodes

Method: a) Cool ion of

interest back to set q value

(default = 0.25). b) Apply

custom RF waveform inresonance with the set q

value, activation time

(default = 30 msec), andoptimized activation

amplitude.

Ejection



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4)4)Ejection

For Scans: All

By: Ring Electrode

Method: Ramp ring RFpower to increase the q values

of all ions in desired scan

range, low mass to high mass.(i.e. Mass Selective Scanning)

Also, ramp the RF amplitude

on the endcap electrodes toconsolidate the ions to a

group (Resonance Ejection)

Ejection

Tune Page

convection gauge


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62

Ion gauge

Pressure


63/105

63

Caffeine stock: 1 mg/ml in methanol

MFRA stock: Dissolve 3.0 mg MRFA in 1 ml 50:50 methanol:water

Ultramark stock: Measure 10 l of Ultramark 1621, and dissolve it in10 ml acetonitrile

ESI calibration solutionESI calibration solution

Into a clean vial pipette 100 l of caffeine stock, 5 l of MRFA stock and2.5 ml of Ultramark stock. Add 50 l of glacial acetic acid and 2.34 ml50:50 methanol:water

Ion Trap Animation


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The Eighth Generation Triple Quad


65/105

65

TSQ 15,

TSQ 45,

TSQ 46,TSQ 70,

TSQ 700,TSQ 7000,

TSQ,TSQ Quantum

Smaller because


66/105

66

25 cm quad

25 cm quad

25 cm quad

25 cm quad

8degrees

90 degrees

TSQ 7000

Quantum

90 Degree

Square

quad

collisioncell

HyperQuadsTMHyperbolic Quadrupoles


67/105

67

Forms Pure Quadrupolar Fields

Reduces Fringing Effects

Significantly Improves Resolution Improves Transmission

Improves Peak Shapes

TSQ 7000

1993 to 2000

r0 = 4 mmL = 250 mm

TSQ Quantum

2001 to

r0 = 6 mm

L = 250 mm

Quadrupole Mass Analyzer


68/105

68

+

+

++

The ion is transmitted along the quadrupole in a

stable trajectory Rf field. The ion does not have astable trajectory and is ejected from the quadrupole.

How does the Quadrupole work ?


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The quadrupole consists of four parallel rods. The opposing rodshave the same polarity while adjacent rods have opposite polarity.

-ve

+ve

Each rod is applied with a DC and

an RF voltage. Ions are scanned byvarying the DC/Rf quadrupole

voltages.

Only ions with the selected massto charge ratio will have the

correct oscillatory pathway in the

Rf field.

Effect of Peak Width On Transmission


70/105

70

0

20

40

60

80

100

%

T

r

a

n

s

m

i

s

s

i

o

n

2 1.5 0.7 0.5 0.2 0.1

Peak Width FWHM

HYPERQUAD ROUND RODS

Effect of Peak Width on Resolution


71/105

71

0

2000

4000

6000

8000

10000

12000

0 200 400 600 800 1000 1200

m/z

R

esolution

Quad 0.7 FWHM

Quad 0.1 FWHM

Sector

Quantum, API 4000, Ultima

at 0.7 FWHM at m/z 1000

R = 1428

R is relatively flat across m/z

Quantum operating at

0.1 FWHM at m/z 1000

R = 10,000

The Power of Resolution


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Separation of ions with samenominal m/z value

Unequivocal determination ofcharge state (ESI)

High resolution precursor ionselection for MS/MS

High resolution product ion for

charge state determination

Effect of changing resolution on peak shape


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1.0 FWHM 0.7 FWHM 0.5 FWHM

0.3 FWHM0.2 FWHM 0.1 FWHM

Resolution vs. Intensity


74/105

74


2.5e6 2.1e61.9e6

Resolution vs. Intensity


75/105

75


0.8 e61.5e6 1.4e6



76/105

76

If one MS scan between m/z 100 and 500is completed in one second, then eachm/z will be allowed to pass for 2.5 ms.

1000 ms== 2.5 ms/amu

400 amu

+

++

+

+

To

detector

+



77/105

77

And, for the same peak, for example, thequadrupole performs 5 complete scansfrom 100 500 Da each taking 1 sec.

5 sec

100 500 Da

100 500 Da

100 500 Da

100 500 Da 100 500 Da

Ion Trap Pre-Scan


78/105

78

The length of time that the trap staysopen to collect ions is determined by a

pre-scan which measures total ion current(prevents space charging, so no ghostpeaks)

5 sec

Pre-Scan

Ion Trap Pre-Scan Contd


79/105

79 5 sec

Across a peak 5 sec. wide the trap mightfill and empty 5 times. So, a group of ionsare collected ca. every 1 sec each group

is then ejected to the detector, smallerions first. 1 sec

1 sec

1 sec

1 sec

1 sec



80/105

80

But for a trap, each m/z (andall m/z at the same time) is/are

collected for ca. 750 ms (taking

pre-scan and interscan timesinto account) and then scanned

to the detector.

For the quadrupole,

each m/z is scanned

(sequentially)

to the detector for 2.5

ms.

+

+

++



81/105

81

A trap will fill similar tofilling a glass with water. All

ions entering the trap will be

collected until the trap fillswith a pre-defined amount of

ions (AGC target value).

At any one particularinstant, a quad will only

scan/look for only one

m/z. All other m/z willbe ignored. In this

example, each m/z is

scanned for 2.5 ms.

+

+

++



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82

So, for full scan MS, a trap will givebetter sensitivity because there aremore ions representing each m/zarriving at the detector for each scan.

+

++

+

How Much More???


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83

Accounting for pre-scan/interscantiming, the trap produces ca. 300 times(750 ms/2.5 ms) for the collection ofeach m/z compared to a quadrupole (i.e

2 orders of magnitude).

+

+++

But


84/105

84

What if I wanted to pass (filter) only onem/z ion to the detector (i.e. SIM or SRM) then I could spend more time on that ion

+

+ ++



85/105

85

Yes, on a quadrupole,

interscan times are

relatively short and sothe quadrupole remains

fixed on that one ion a

duty cycle of close to100%

But a trap will still only

collect ions in batches-

and prescan/interscantimes afford a duty cycle of

about 75%

+

+++

What is a Duty Cycle?


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86

Scan 1 ISD Scan 2

Definition:-

Time taken to acquire 1 scan and be ready to acquire

the next one

Interscan delay (ISD) is the time taken to return all

system voltages to the start values and reach a stable

stateThis is dead time and should be minimized

Duty Cycle

In Addition

Whil h b i i i l


87/105

87

While the beam instrument is continuouslydetecting one particular m/z a trap builds acurve from an average over each collection

time and the points are least frequent atthe most important region for quantitation

(the take off).

For Example

SRM f 5 Al l ith LCQ D


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88

SRM of 5 pg Alprazolam with LCQ Decagives a %RSD of 6.11 while

SRM of 750 fg Alprazolam with TSQ 7000gives a %RSD of 1.87.

Signal to noise ratio (S/N) are similar

(20:1 and 28:1 respectively).

100

RT: 4.26

SN: 20

%RSD 6 11%RSD 6 11 SRM 5SRM 5 Al lAl l

90

80

an

70ce


89/105

89

0

10

20

30

40

50

Relative

%RSD 6.11%RSD 6.11withwithLCQLCQDecaDeca

SRM 5pgSRM 5pg AlprazolamAlprazolam60A

bundan

RT: 4.41

SRM 750fgSRM 750fg AlprazolamAlprazolam with TSQwith TSQ

SN: 28

%RSD 1.87%RSD 1.87

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5Time (min)

0

10

20

30

40

50

60

70

80

90

100

The Effect of Ion Trap Scan Speeds onQuantitative Performance


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90

In general, faster scanning produces more points

across a chromatographic peak, hence better

precision and lower LOQs.

In fact, for an ion-trap, scan speed refers only to

the time taken to scan ions from the trap duringmass analysis.

Scan speed does not refer to the total analyticalcycle. In an ion trap device, an MSn analytical

scan comprises at least four events:



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91

1- AGC Pre-scan

2- Ion Injection (usually the rate-determining step)

3- Isolation and activation of the parent ion within

the trap

4- Scanning the ions out of the trap (mass analysis)

MS Scan FunctionMass

Analysis

Ion


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92

IonActivation

Ion

isolation

Ion

Injection

Analytical ScanAGC Prescan

Scan TerminologyScan Terminology


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93

Prescan Mass Analysis Prescan Mass Analysis Prescan Mass Analysis

1st Microscan 2nd Microscan 3rd Microscan Save Data

Complete Scan Cycle



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94

The injection time constitutes the majority of the

total scan time.

For good quantitative reproducibility, it is

necessary to take enough points to precisely

determine the chromatographic peak particularly

at the take-off point.

Increasing the scan speed in the trap does not

significantly increase the number of data pointstaken across the peak.

Quantitation m/z 500 scanning 135 510

Pre-scan 0 msec

Isol /Activ / Download 80

Pre-scan 0 msec


Scan speeds in ion traps


95/105

95

Pre-scan 60 msec

Isol/Activ/ Download 80

375 amu @ 5500 amu/sec 70

Injection time 500

Total scan time 710

Scans / 10 sec wide peak 14

Pre-scan 60 msec

Isol/Activ/ Download 80

375 amu @ 5500 amu/sec 70

Injection time 500

Total scan time 710


Quantitation m/z 500 scanning 135-510Pre-scan 60 msec

Isol /Activ / Download 80375 amu @ 13,000 amu/sec 30

Injection time 500

Total scan time 670


Pre-scan 60 msec


375 amu @ 13,000 amu/sec 30

Injection time 500

Total scan time 670



375 amu @ 13,000 amu/sec 30

Injection time 500

Total scan time 610



375 amu @ 13,000 amu/sec 30

Injection time 500Total scan time 610




96/105

96

The only significant way to increase the sampling

rate across the peak is to reduce the injectiontime which can be achieved in two ways:

1- Set a lower max injection time which reduces the

number of ions in the trap, hence sensitivity

2- Increase the efficiency of the source and lenses

to improve the transmission of ions; I.e filling thetrap to the same level in a shorter period of time.

Data Dependant Acquisition of MSn Spectra

Data Dependant Acquisition: Intelligent decision-making software that

selects precursor ion for MSn experiments based on user define criteria


97/105

97

selects precursor ion for MSn experiments based on user-define criteria.

Critical for metabolite screening experiments.

Scan event 1

Full-Scan MSScan event 2

Full-Scan MS2

Software selects most intenseion from scan event 1 as

precursor ion for ms2

experiment in scan event 2,

provide that its intensity is

above a user selected threshold

m/zm/z

Dynamic Exclusion-- MS and MS/MS of Co-Eluters

MS

MS/MS


98/105

98

407

407MS

Time

452

452

365

206

377255

MS/MSm/z

377

231

Threshold

MS MS/MS

MS/MS

MS

MS/MS

MS

MS MS

MS

m/z

m/z

m/z


Major Strengths of Triple Quads


99/105

99

Major Strengths of Triple Quads

SRM Sensitivity

Neutral Loss Scan Mode Parent (Precursor) Scan Mode

Major Strengths of the LCQ Deca XP Plus

MSn Scan Mode

Full Scan MS/MS Sensitivity

Consecutive Reaction Monitoring (CRM)

What are neutral loss scans ?

Both Q1 and Q3 are scanned together


100/105

100

Both Q1 and Q3 are scanned together

Q3 is offset by the neutral loss under

investigation The precursor ions collide with Argon gas inQ2 to create fragment ions

Only those compounds which give a fragmenthaving that specific loss are detected

Since both Q1 and Q3 are scanning, neutralloss scan mode is slower than any other mode

Neutral loss scans

Neutral loss scans are used for screening experiments where agroup of compounds all give the same loss


101/105

101

g pgroup of compounds all give the same loss

NN2H

N2H

N

N

N2H

N2H - m/z 84

N

N

N2H

N 2H

N

N 2H

N2H

- m/z 84

NN

N - m/z 84

N

NOH

N

OH

- m/z 84

What are precursor ion scans ?


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102

Precursor ion scans also known as parent ion scans

Q1 is scanned

Q3 is set to allow only a fragment ion of one m/z topass; (Q3 fixed)

ions collide with Argon gas in Q2 to create fragment or

product ions

Only those compounds which give that specificfragment ion are detected

Precursor ion scans

Precursor ion scans are used for screening experiments where agroup of compounds all give the same fragment ion


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103

g p p g g

N

N

N

N

NOH

N

N

N

N

N2H

N 2H

N

N

N2H

N2Hm/z

84

m/z 162m/z 192

m/z 192

m/z 238

m/z 268

Comparison of MSn in a Triple Quad versesan Ion Trap Instrument.


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104

Triple Quad(nonresonant excitation): Acceleration voltage applied

equally to all masses. Get a mix of ms2

, ms3

msn

products.

Ion Trap(resonant excitation): Excitation energy is in resonance with

only one mass at a time. Fragments, once formed, can not be furtherexcited unless they are purposely selected for next stage of MS.

Allows one to take apart a molecule in a controlled, step-wise fashion.


Ion Traps Quadrupoles


105/105

105

Mass Separation in time Mass Separation in space

High sensitivity Full Scan Lower sensitivity Full Scan

Lower sensitivity SIM and SRM High sensitivity SIM and SRM

Offer multiple stages of MSn

Parent and neutral loss scans

Offers Only MS or MS/MS

quadtrap comparison 1

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