LC/MS WORKSHOP
IOWA 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
Micromass GCT
LC/MS
Agilent QTOF 6540
m/z
Inlet
Direct Probe
Flow Injection
GC, LC
Ion Source
EI, CI
APCI, ESI
Analyzer
Q, QQQ
EB,
Ion Trap
TOF, QTOF
FT, Orbitrap
Detector
EM, PM
Data System
Applications of Mass Spectrometry
•Pharmaceutical analysis
•Biomolecule characterization
Proteins and peptides
Oligonucleotides
•Environmental analysis
Pesticides on foods
Soil and groundwater contamination
•Forensic analysis/clinical
TOF EQUATION
TIME OF FLIGHT SCAN
QTOF 6540 CAPABILITIES
MS and MS/MS
Mass Accuracy of better than 3ppm
Resolution (FWHM) 45,000 for mass 1000
Rapid 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
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•
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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 together
Resolution = 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
SelenoMethinine MW196
MethylSelenoCysteine MW 182
MW 163
QUALITATIVE DATA PROCESSING
MASS 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
SELECTED IONS FROM SEMIX
ESI MASS SPECTRA OF SEMIX
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