LC/MS WORKSHOP IOWA STATE UNIVERSITY Kamel Harrata Instrument Description Data Acquisition Data...

LC/MS WORKSHOPIOWA STATE UNIVERSITY

Kamel Harrata

Instrument Description

Data Acquisition

Data Processing

WHAT IS MASS SPECTROMETRY?

Mass spectrometry is an analytical technique that involves the study in the gas phase of ionized molecules with the aim of one or more of the following:

• Structural characterization • Gas phase reactivity study • Qualitative and quantitative analysis of

components in a mixture. • Molecular weight determination

LC/MS and GC/MS Mass Spectrometers

GC/MS

LC/MS

InletIon

SourceAnalyzer

Detector

m/z

Data SystemDirect Probe

Flow InjectionGC, LC

EI, CIAPCI, ESI

Q, QQQEB,Ion TrapTOF, QTOFFT, Orbitrap

EM, PM

Micromass GCT Agilent QTOF 6540

•Pharmaceutical analysis•Biomolecule characterization

Proteins and peptidesOligonucleotides

•Environmental analysisPesticides on foodsSoil and groundwater contamination

•Forensic analysis/clinical

Applications of Mass Spectrometry

TOF EQUATION

TIME OF FLIGHT SCAN

QTOF 6540 CAPABILITIES

MS and MS/MSMass Accuracy of better than 3ppmResolution (FWHM) 45,000 for mass

1000Rapid switching between positive

and negative ion modes

LC SYSTEM

4 reservoir bottles A1, A2, B1, B2

Binary Pump can handle up to 8000 psi

UV detector

LC Column housing

Autosampler

ELECTROSPRAY IONIZATION

Desolvation + Coulomb Explosions

AGILENT ELECTROSPRAY ION SOURCE (POSITIVE MODE)

ELECTROSPRAY SPECTRUM OF MYOGLOBIN

RELATIVE APPLICABILITY OF LC/MS TECHNIQUES

ADVANTAGES OF ELECTROSPRAY

• Soft ionization

• LC/MS interface with best sensitivity

• Extends mass range for multiply charged analytes

• Works with a wide range of moderate to high polarity compounds

• Low maintenance

ELECTROSPRAY SOLUTION CHEMISTRY

- Mobile phase pH has a major effect for analyte that are ions in solution

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

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

- Manipulation of pH can enhance performance for analytes that are not normally ionized in solution

QTOF 6540 ION PATH ELEMENTS

MASS SPECTROMETER TYPE: MASS ACCURACY

• Quadrupole 200-300 ppm• Linear Time of Flight 200 ppm• Reflectron Time of Flight 25-50

ppm• OR-Time of Flight 1-3 ppm• Fourier Transform MS 0.1-1

ppm

MASS ACCURACY-DEVIATION IN PPM

EXACT MASS CALCULATION AND DEVIATION

HOW MUCH MASS ACCURACY IS NEEDED?

Elemental composition implication

TOF RESOLUTION

Resolution is the ability to distinguish between two mass peaks that are extremely close togetherResolution = M / Peak Width

(FWHM: full width at half maximum)

LC/MS OF SEMIX (10 UG/ML) 10 PPM

LC/ESI pos of : L-Selenocysteine (Peak#1); MethSeCy (#2); SeMethionine(#3); Etionine(#4)

Agilent XDB C18, 1.8 um, 4.6mm ID, 150 mm L

L-Selenocysteine MW 168 MethylSelenoCysteine MW 182

SelenoMethinine MW196 MW 163

QUALITATIVE DATA PROCESSINGMASS HUNTER QUAL SOFTWARE

Open File

Use default layout: Chromatogram, Mass Specta

Process and print data

Display mass spectrum

Average few scans

Background substraction

Peak integration

POSITIVE ION MODE LC/ESI OF SEMIX

6x10

0.52

0.54

0.56

0.58

0.6

0.62

0.64

0.66

0.68

0.7

0.72

0.74

0.76

0.78

0.8

0.82

0.84

0.86

0.88

0.9

0.92

0.94

0.96

0.98

1

1.02

1.04

1.06

1.08

1.1

1.12

1.14

1.16

1.18+ESI TIC Scan Frag=175.0V QTOF1100112.d

1

Counts vs. Acquisition Time (min)0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8

SELECTED IONS FROM SEMIX

1x10

0

1

2

3

+ESI TIC Scan Frag=175.0V QTOF1100112.d

2.05720.86140.6812

1.3037

0.5993 1.8606 2.3520

2x10

0

0.25

0.5

0.75

1+ESI EIC(336.9181) Scan Frag=175.0V QTOF1100112.d

0.6976

0.77951.0907 1.9425

2x10

0

0.25

0.5

0.75


0.8614

2x10

0

0.25

0.5

0.75


1.3037

0.6812 1.5166

2x10

0

0.25

0.5

0.75


2.0572

Counts (%) vs. Acquisition Time (min)0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

ESI MASS SPECTRA OF SEMIX

3x10

0

0.5

1

1.5

2

2.5

3

3.5

4+ESI Scan (0.6321-0.7304 min, 7 scans) Frag=175.0V QTOF1100112.d Subtract

247.8689

336.9161

167.9531 288.9178

4x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

+ESI Scan (0.8287-0.9269 min, 7 scans) Frag=175.0V QTOF1100112.d Subtract

166.9578

227.9477

4x10

0

0.2

0.4

0.6

0.8

1

1.2


180.9732

134.9684

4x10

0

0.5

1

1.5

2

2.5

3

3.5

4


147.0449

208.0347

Counts vs. Mass-to-Charge (m/z)120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580

LC/ESI OF SURFACTINE MIXTURES

14 DALTON DIFFERENCE BETWEEN COMPOUNDS

POSITIVE ION MODE APCI OF CAROTENOIDS

m/z

Positive ion mode LC/APCI is the method of choice for analyzing and quantifying Carotenoids

LC/MS WORKSHOP IOWA STATE UNIVERSITY Kamel Harrata Instrument Description Data Acquisition Data...

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