Karteikarten
Mass Spectrometry Hyphenated methods
Karten in diesem Stapel (36)
- 1overview• What is mass spectrometry
– Principles of ionisation and mass analysers
• Interpretation
– The mass spectrum
– Interpretation – peaks and isotopes
• Hyphenated methods GC-MS and LC-MS
– Analysis in the pharmaceutical industry / forensics - 2Principles of MS
- 3Examples mass spectra
- 4Interpretation: Mass, m/z, isotopes What is mass?• SI unit for mass is kg
• Mass of a single carbon-12, 12C, atom is 1.99266×10-26 kg
• One unit (u) of mass (atomic mass unit, amu) is defined as a Dalton
(Da).
• One Dalton is defined as 1/12 the mass of a single carbon-12 atom.
• Thus, one 12C atom has a mass of 12.0000 Da - 5Calculation:• Mass of a peptide is 5685 Da
• m/z of double protonated peptide?
• Mass of the peptide in kg? - 6Calculation: • Mass of a peptide is 5685 Da • m/z of double protonated peptide?
- 7Calculation: • Mass of a peptide is 5685 Da • Mass of the peptide in kg?
- 8Resolution & mass accuracy on mellitin
- 9Mass resolution– High Resolution Mass Spectrometry
• If sufficient resolution (R > 5000) exists, mass numbers can be
recorded to precise values (6 to 8 significant figures)
– Resolution:
• Ability to distinguish between ions of adjacent m/z values
- 10Mass resolution For example,
- 11The Analysis of isotope patternsis helpful
in element composition determination - 12Interpretation: IsotopesMost elements have more than one stable isotope.
For example, most carbon atoms have a mass of 12 Da, but in nature, 1.1%
of C atoms have an extra neutron, making their mass 13 Da.
Why do we care?
Mass spectrometers can “see” isotope peaks if their resolution is high
enough.
If an MS instrument has resolution high enough to resolve these isotopes,
better mass accuracy is achieved. - 13Isotopes• Atoms of the same element can have different numbers of
neutrons
• The most common isotope of hydrogen has no neutrons at all;
there's also a hydrogen isotope called deuterium, with one
neutron, and another, tritium, with two neutrons.
- 14Isotopes cont.
- 15Definitions for Monoisotopic ion mass Nominal ion mass Average ion mass• Monoisotopic ion mass
Mass of ion for a given empirical formula calculated using the
exact mass of the most abundant isotope of each element, as
accurate as possible (e.g. 1H = 1.007825 and 12C=12.000000).
• Nominal ion mass
Mass of ion calculated using the integer mass (mass number
of most abundant isotope of each element (e.g. 1H =1 and
12C=12).
• Average ion mass
Mass calculated with the average of the isotopic mass of each
element, weighted for isotopic abundance (e.g. H=1.00797
and C=12.01115). - 16Interpretation: isotopes
- 17Interpretation: isotopes
- 18Mass spectrum of peptide with multiple Catoms
- 19Mass spectrum of insulin (257 C-atoms)
- 20The nitrogen rule (use nominal mass)• If the molecular mass of an unknown compound to the
nearest integer value is an odd number, the compound
contains an odd number of nitrogens in its molecular formula.
Correspondingly, if the molecular mass is an even number, the
compound contains zero or an even number of nitrogens in its
molecular formula - 21The nitrogen rule (use nominal mass)
- 22Isotopes - summary• Mass spectra will show isotopic patterns of ions for the elements present
• For n Carbon atoms, 13Cn will contribute to
M+1 ion (n x 1.1%)
• If one chlorine is present a 3:1 pattern will be
seen for M and M+2
• Hydrogen and oxygen have low abundances for their isotopes 2H and 18O and do not give significant signals for small molecules - 23summaryMass Spectrometry can give structural evidence
from:
1. Isotope distribution and accurate mass
leading to elemental composition
2. Fragmentation; tandem mass spectrometry
3. When combined with HPLC – retention time data
4. Derivatization of functional groups - 24Liquid Chromatography-Mass Spectrometry• Interfacing HPLC with mass spectrometer
- 25Advances of LC-MS detection• A mass spectrometer is
more sensitive and far
more specific than all
other LC detectors
• Analysis of compounds
without a suitable
chromophore
• Identification of
components in
unresolved
chromatographic peaks - 26Why Liquid Chromatography - Mass Spectrometry?• Two-dimensional abundance data
and three-dimensional mass
spectral data from a mass
spectrometer
• Mass spectral data add specificity
that increases confidence in the
results of both qualitative and
quantitative analyses
• Information gained: molecular
weight, structure, identity, quantity,
and purity of a sample - 27Interfacing LC with MS Fundamental issues:• LC eluent – limited acceptable LC flow rate ~ 200
uL/min (ESI)
• Ionisation under high vacuum
• Temperature differences
• No non-volatile salts, e.g. phosphate, borate.
• Ion competition (matrix effect)
• LC is the time limiting step in high through put
analysis - 28Interfacing LC with MS contChanges to LC methods required for modern LC-MS
systems generally involve changes in sample
preparation and solution chemistry to:
• Ensure adequate analyte concentration
• Maximise ionisation through careful selection of
solvents and buffers
• Minimise the presence of compounds that compete
for ionisation or suppress signal through gas-phase
reactions - 29Ionisation chemistry (ESI)Formation of analytes ions in solution is essential to
achieving good ESI data
• Select more volatile buffers to reduce the buildup of
salts in the ion source
• Adjust solvent pH according to the polarity of ions
desired and the pH of the sample
• Use solvents that have low heat of vaporisation and
low surface tension to enhance ion desorption
• Make sure that gas-phase reactions do not neutralise
ions through proton transfer or ion pair reactions - 30HPLC–ESI–MS/MS applications:• Now considered the benchmark for
measurement of drugs and their metabolites
in biological matrices
• The method of choice for screening for
inherited metabolic disorders
• Drug quantification and therapeutic drug
monitoring
• Toxicological investigations - 31Gas Chromatography-Mass Spectrometry• Identification and quantitation of (semi) volatile
organic compounds in complex mixtures
• Determination of MW/elemental composition of
unknowns
• Quantitation of drugs and their metabolites (blood,
urine) for pharmacological and forensic applications
• Environmental analysis – quantitation of pollutants in
drinking and waste water
• Explosive investigation
• Steroids
• ‘Specific tests’ in Forensic science - 32Gas Chromatography-Mass Spectrometry
- 33The column• Mobile phase: gas (helium or nitrogen)
• Stationary phase: 5% phenyl-, 95% dimethylsiloxane
• 25 m x 0.5 mm i.d. (capillary column of glass or
stainless steel)
• The column within the oven can be programmed to
increase the temperature gradually (ramped up to ca.
400°C by 1°C/min to 40°C/min) better separation:
as the temp. increases, low bp analytes elutes faster
than high bp analytes two separation forces:
temperature and the stationary phase interactions
• DETECTOR: MASS SPEP: Electron ionization (EI) and
quadrupole mass analyze - 34Sample preparation• Injection volume 0.5 - 2.0 µL
• Analytical sensitivity of 1 – 100 pg per component
• Derivatives
In order to make the molecule both resistance to the high temperatures
used in the GC and to selectively improve the mass spectra, functional
groups can be ‘derivatised’ i.e. chemically modified. Reaction with a
silylation reagent is most common
Why Derivatize:
• To permit analysis of compounds not directly amenable to analysis due to,
for example, inadequate volatility or stability
• Improve chromatographic behaviour or detectability - 35The range of functional groups which can be derivatised is large and includes:COOH
OH
NH2
C=C
The essential aspect of the
chemistry is that the
reaction must be fast,
efficient, and the reagents
and by-products should be
volatile and easily removed - 36—
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