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Training

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Luiz Fernando A. Santos

luiz.santos@bruker.com.br

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Ion Generation: Atmospheric

Pressure Ionization (API)

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ESI is works very well for polar analytes. APCI and APPI can be used

as complementary techniques for less- or non-polar compounds.

Molecular W

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Analyte Polarity

very polarnonpolar

100,000

10,000

1,000 APCI

Electrospray

APPI

Relative Applicability of LC/MS Techniques

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Generation of Ions – ESI Source

The heated drying gas creates a

warm environment in the source.

As the heated gas flows round

the front of the capillary and out

of the spray shield, it acts as a

counter-current gas to the ion

stream, therefore reducing the

flow of neutrals and unwanted

ions into the ion optics.

The glass capillary itself is not

heated.

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Formation of charged droplets at the

needle tip within the spray chamber

Capillary (4

kV)

Nebulizer

GasSample

Solution

Dry Gas

Spray Needle

grounded

Dry Gas

Capillary Cap (4 kV)

Generation of Ions – ESI Source

Nebulization

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Generation of Ions - Desolvation

When the force of the Coulomb repulsion exceeds the surface tension of

the droplet, the droplet explodes, producing charged daughter droplets that

are subject to further evaporation.

EvaporationRayleigh

Limit

Reached

Coulomb

Explosion

Droplet

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Generation of Ions – Ion Evaporation

Evaporation (Multiply Charged)

Ion

Droplet

As solvent evaporates from the droplet, the surface becomes highly

charged. When the field created by the ions at the surface of the droplets

exceeds the surface tension, ions are emitted directly into the gas phase.

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Fundamentals of

Mass Spectrometry:

Mass Resolution

Isotope Patterns

Monoisotopic Mass

Mass Accuracy

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It is a measure of a mass spectrometer’s ability to distinguish two

compounds of nearly equal mass.

600 800 1000 1200 1400 1600 1800 2000 2200 2400 m/z

100

200

300

400

500

600

700

800

900

1000

1100

a.i.

1618 1623 1628 m/z

<-301->

<-273->

“easy” “not so easy”Resolution

What is meant by ‘Resolution’?

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The narrower the FWHM, the higher the resolution.

With the same FWHM, higher masses will have higher

resolution than lower masses.

Resolution = (m/z) / FWHM

FWHM Full Width at Half

Maximum

How do we measure mass resolution?

1521.9743

1522.9772

1521 1522 1523 1524

m/z

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Isotopic Distribution Patterns

1312

100

90

8070

60

50

40

3020

10

0

C1

122121120

100

90

8070

60

50

40

3020

10

0

C10

1,2061,2041,2021,200

100

90

8070

60

50

40

3020

10

0

C100

12,03012,02012,01012,000

100

90

8070

60

50

40

3020

10

0

C1000

Isotopes are atoms with the same number of protons in the nuclei, but with different numbers of neutrons. Only 21 elements have only one stable isotope. All

other elements are mixtures of at least 2 stable isotopes, and the proportions of these isotopes can vary greatly depending on the element. Carbon has 2 stable

isotopes, C-12 and C-13, with natural abundance of 98.892% and 1.108% respectively. As the number of carbons increase in a molecule, the isotopic

distribution pattern will reflect the mass contribution of the isotopes with their extra neutrons.

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pQLYENKPRRPYIL

MW 1672.9

Res. 1’000

1,6811,6791,6771,6751,6731,671

100

90

8070

60

50

40

3020

10

1673.9Average Mass

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1,6821,6801,6781,6761,6741,672

100

908070

60

50

3020

10

0

Res. 10’000[M+H]+

[M+H+1]+

[M+H+2]+

[M+H+3]+

[M+H+4]+

1672.9 Monoisotopic Mass

Neurotensin

Resolution = (m/z) / FWHM

Average and monoisotopic masses

Lower resolution results in an “average”

-inaccurate determination of peak center

-calculated average mass is inaccurate

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Defining the TOF mass measurement

Mass accuracy can be expressed as a percentage or ppm:

e.g. % mass accuracy =Measurement error

True massx 100%

Very small percentage errors (<0.01%) are expressed as

parts per million or ppm. 0.01% = 100ppm

e.g. ppm =Measurement error

True

mass

x 106

Measured mass: 1296.970

True mass: 1296.685 0.02% error

ppm = 0.001 per thousand

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Precision and mass accuracy

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Calibration for ESI-TOF

Good instrument calibration is mandatory for any accurate mass measurement !

The „ideal“ calibrant must have:

• known elemental composition

• no interferences (ion suppression, isobaric interferences) with the analytes

The „ideal“ calibrant nice to have:

• should cover the desired mass range

• ionizable both in positive and negative mode

• well soluble in common HPLC solvents and stable in solution

• not „sticky“ to surfaces (memory effects)

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External calibration: Calibrant is injected separately from sample.

+ no interference between analyte and calibration standard masses.

- Higher demands on control of laboratory environment (e.g. temperature) compared to

internal calibration mode.

Internal calibration: Calibrant is introduced simultaneously with the sample.

+ Better mass accuracy due to identical instrumental conditions.

- Interference between sample and internal standards possible

A: ion suppression

B: isobaric interferences

Calibration for ESI-TOF

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A. Injection of standard via 6-port-valve at the beginning/end of the LC-MS run

+ no suppression effects or isobaric interference possible

- slight time offset between calibrant and analyte spectra

B. Introduction of calibrant with second ESI sprayer (continuous or discontinuous)

+ calibration standard present in every spectrum

- suppression effects possible, isobaric interference

C. Add calibration standard via T-piece post-column

+ calibration standard present in every spectrum

- suppression effects possible, isobaric interference

D. calibration based on compounds with known elemental composition in the

analysis

Instrumental solutions

Calibration – micro and standard LC

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Valve position “Waste”

LC flow directly enters the micrOTOF

The valve can be switched via time segments in the acquisition method.

Injection of calibrant via 6-port valve

Valve position “Source”

Calibrant is introduced into the micrOTOF

autosamplercolumn

ESI-TOF

waste

syringe pump

Cal.Std. Flow 2-10 µl/min

pump

autosamplercolumn

ESI-TOF

waste

syringe pump

Cal.Std. Flow 2-10 µl/min

pump

Configuration for External Calibration

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Determining Multiple Charge States

Example: m1 = 1000, isotope m2 = 1001

Charge (z) = 1 1000/1, 1001/1 m/z = 1000, 1001

Charge (z) = 2 1000/2, 1001/2 m/z = 500, 500.5

Charge (z) = 3 1000/3, 1001/3 m/z = 333.33, 333.66

The m/z difference is incremental to the charge.

Charge 2 = 0.5

Charge 3 = 0.333

Charge 4 = 0.25

2+3+

1+

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Ion Generation:

Interfaces

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Positive Ion Mode Negative Ion Mode

Formation of protonated molecular ions Formation of deprotonated species

M + HA � [M+H]+ + A- M + B � [M-H]- + BH +

Example: Example:

�

R

NRR

+H

R

NR RHA A-+++ �

O

COR -

O

COR H

B: + B-H+

Analytes with a basic character, like for instance compounds carrying amino groups, are

generally ionized in positive mode. The sample molecule picks up a proton from the more

acidic solvent.

Analytes that are more acidic might to be ionized in negative ion mode (e.f. carboxylic or

sulfonic acids). The molecule looses a proton to a base in solution and becomes negatively

charged.

A mean for the basic or acidic character of a sample is its pKa value.

Electrospray Chemistry

Sample Chemistry

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Cluster Formation

In addition to the formation of adducts clustering of ions and solvent molecules can be observed,

e.g. [M+Na+CH3CN]+, [M+H+MeOH]+, …

For higher concentrated samples very often clusters of the analyte molecules themselves are

formed:

[2M+H]+, [2M+Na]+ , [2M-H]-, [2M+Na-2H]- , …

Common observation:

the dimer often is formed predominatly as [2M+Na]+ even if the monomer prefers formation of

[M+H]+

Electrospray Chemistry

Adducts and Clusters

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Electrospray Solvents and Buffers

Electrospray requires polar solvents. Commonly used are water, methanol, acetonitrile and iso-

propanol

The pH of the solvent has a major effect on analytes which are ionic in solution:

Acidic pH (<7.0; 5 preferred) for positive ions

Basic pH (>7.0; 9 preferred) for negative ions

Acidic additives for positive ion mode:

●Formic acid, 0.1-1.0%

●Acetic acid, 0.1-1.0%

●Trifluoroacetic Acid ≤ 0.05%

The TFA anion forms ion pairs with positive analyte ions and thus leads to signal

suppression! But TFA concentrations of ≤ 0.05% are acceptable.

Basic additives favour the negative ion mode:

● Ammonium hydroxyde (pH 10-11)

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Advantages ☺☺☺☺

• Soft ionization method, providing molecular ions, e.g. M+H +, M+Na+

• Suited for a wide range of moderate to high polarity compounds

• Extended mass range for multiply charged analytes, e.g. proteins,

oligonucleotides

• Very sensitive interface for LC-MS coupling.

• Robust and low maintenance

• Interface for routine and automated use

Disadvantages ����

• Solution chemistry influences ionization process

Ion suppression/Matrix effect:

Quantification is challenge for co-elution; need appropriate internal

standards. Stable-isotopic labeled internal standards are optimal.

• Adduct ions (other than M+H) possible with some analytes, no unambiguous

ionization for unknown compounds

• For higher concentrations saturation effects limit the linear range

ESI – Advantages and Disadvantages

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Basics and Theory

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Dry Gas Heater Dual Ion Funnel

Analytical

Quadrupole

Collision

Cell

Orthogonal Accelerator Detector

Reflectron

Flight Tube

Glass

Capillary

Collision

Gas Supply

API Spray Chamber

Sprayer

Hexapole

Functional Overview

ESI-Qq-oTOF

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Driving forces from source to detector: Pressure gradient

Potential gradient

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micrOTOF-Q: MS

No isolation in Q :

RF only

Low collision

energy: no

CID

Dry Gas Heater

Dual Ion Funnel

AnalyticalQuadrupole

Collision Cell

Orthogonal Accelerator Detector

Reflectron

Flight Tube

Glass Capillary

Collision Gas Supply

API Spray Chamber

Sprayer

Hexapole

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isolation in Q

high collision

energy: CID

Dry Gas Heater

Dual Ion Funnel

AnalyticalQuadrupole

Collision Cell

Orthogonal Accelerator Detector

Reflectron

Flight Tube

Glass Capillary

Collision Gas Supply

API Spray Chamber

Sprayer

Hexapole

micrOTOF-Q: MS/MS

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micrOTOF-Q ISCID

High voltage step between

funnels: CID in medium

pressure region

Dry Gas Heater

Dual Ion Funnel

AnalyticalQuadrupole

Collision Cell

Orthogonal Accelerator Detector

Reflectron

Flight Tube

Glass Capillary

Collision Gas Supply

API Spray Chamber

Sprayer

Hexapole

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338.340

609.280

0.0

0.5

1.0

1.5

5x10

Intens.

200 400 600 m/z

195.065

397.212

609.281

0.00

0.25

0.50

0.75

1.00

4x10

Intens.

200 400 600 m/z

397.213

0

2000

4000

6000

8000

Intens.

100 200 300 400 500 600 m/z

174.092

227.119

365.187

397.211

0

250

500

750

1000

1250

Intens.

50 100 150 200 250 300 350 400 450 m/z

ISCID

Isolation

Fragmentation

Reserpine

MS3 by In Source CID and CID (q)

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Quadrupole

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Mass Filter !

• RF and DC

• RF: Responsible for ion transmission (ALL ions)

• DC: select specific m/z

+

+

- -

+

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Think of the TOF operation as a drag race between vehicles of different sizes, but all

having identical engines:

• “Start line” = orthogonal accelerator; “Finish line” = TOF detector

• Just as all vehicles have the same engine (i.e., horsepower), all ions are pulsed up

the flight tube with the same kinetic energy.

• Since m = 2E/v2 (E = ½ m·v2), the smaller vehicles/ions will reach the finish

line/detector before the larger ones.

START FINISH

Principle of the TOF Mass AnalyzerTOF: The Great Race

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Separation of Ions by m/z – TOF Assembly

Orthogonal Accelerator

The ion ‘packages’ enter the field free time-of-

flight tube.

In the flight tube they are separated because

of their different velocities resulting in

different flight times.

As all ions have the same kinetic energy they

travel with a velocity that is inversely

proportional to their m/z.

Mass-to-charge ratios are determined by

measuring the time that ions take to move

from the orthogonal acceleration to the

detector.

Field

-free drift reg

ion

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The aim of an electrostatic reflector, also called

reflectron is to improve mass resolution. It creates a

retarding field that acts as an ion mirror by deflecting

the ions and sending them back through the flight

tube.

The reflector corrects the energy dispersion of ions

with the same m/z ratio. Indeed, ions with more

kinetic energy will penetrate the reflectron more

deeply and will spend more time in the reflectron.

Thus they reach the detector at the same time as

slower ions of the same m/z.

Ions receive kinetic energy from electric field.

E = 1/2mv2

Resolution of Ions – TOF Assembly

m1 = m2, but E1 < E2Orthogonal Detector

accelerator

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