Lecture 5 Flashcards
What does a mass spectrometer do?
- It measures mass better than any other technique.
- It can give information about chemical structures
What are mass measurements good for?
To identify, verify, and quantitate: metabolites, recombinant proteins, proteins isolated from natural
sources, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers.
How is mass defined?
- Based on using carbon-12 as a reference point
- One unit of mass 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
m/z
Mass is given as m/z which is the mass of the ion divided by its charge
Monoisotopic mass
The mass of an ion for a given empirical
formula calculated using the exact mass of the most abundant isotope of each element.
Average mass
Average mass is the mass of an ion for a given empirical formula calculated using the average exact mass for each element
Nominal Mass
Nominal mass is the mass of an ion for a given empirical formula calculated using the integer mass of the most abundant isotope for each element
Can Mass Spectrometers “see” isotopes?
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.
When is Monoisotopic Mass used?
When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate measurement.
What does the average mass correspond to?
- Average mass corresponds to the unresolved peak cluster.
- When the isotopes are not resolved, the centroid of the envelope corresponds to the weighted average of all the isotope peaks in the cluster, which is the same as the average or chemical mass.
Mass Resolution
- Measure of the ability of a mass spectrometer to distinguish between ions of slightly different mass to charge ratios.
- Resolution must be quoted with a qualifier.
What are the three ways in which mass is defined in?
Monoisotopic, Average and Nominal.
How is Mass Resolution Calculated?
R = m/Δm
R = Mass resolution
m = Mass of the peak (usually the peak of interest)
Δm = Full width at half maximum (FWHM) of the peak
What does mass measurement accuracy depend on?
- Mass Resolution accuracy depends on resolution
- High Resolution means better mass accuracy
Measuring Mass Accuracy
(Measured Mass- Known Mass)/Overral Mass?
See Slide 16
What are the two ways in which mass accuracy can be stated in?
ppm and percentage
What are the main principles of Mass Spectrometry?
- Charge the molecule of interest (ionise)
- Place the charged molecule into a magnetic field/electro field and measure its speed or radius of curvature relative to its mass-to-charge ratio (analyse)
- Detect the ion using micro-channel plate or photo-multiplier tube (detection)
Components of a Mass Spec
- Source - Analyser - Ion Detector - Data System
- See slide 20
To measure the characteristics of individual molcules they need to be converted to….
Sample introduction - Source
To measure the characteristics of individual molecules they need to be converted to ions in order for them to be manipulated by external magnetic and electric fields.
Why must the formation and manipulation of ions be carried out under vaccum?
- Ions are very reactive and short lived.
Atmospheric Pressure of the ions that are handled
Atmospheric pressure is roughly 760 torr and the pressure that ions are generally handled is between 10-5
to 10-8 torr
Name some of the Ionisation Methods
- Atmospheric Pressure Chemical Ionisation (APCI)
- Chemical Ionisation (CI)
- Electron Ionisation (EI)
- Electrospray Ionisation (ESI)
- Fast Atom Bombardment (FAB)
- Matrix Assisted Laser Desorption Ionisation (MALDI)
Positive Ionisation
If the sample has functional groups that readily accept a proton (H+) then positive ion detection is used e.g. amines R-NH2 + H+ = R-NH3+ as in
proteins or peptides.
Negative Ionisation
If the sample has functional groups that readily lose a proton then negative ion detection is used e.g. carboxylic acids R-CO2H = R-CO2- and alcohols
R-OH = R-O- as in saccharides or oligonucleotides.
Matrix Assisted Laser Desoroption Ionisation
What does it work well with? What is it used for?
- Works well with thermolabile, non-volatile organic compounds.
- Used in the analysis of
proteins, glycoproteins, peptides, oligonucleotides and oligosaccharides. - Sample ionisation is carried out by its bombardment with a
laser light usually a pulsed nitogen laser at 337nm
wavelength. - The sample is mixed with a highly absorbing matrix which
converts the laser energy into excitation energy for the
sample (sinapinic acid for proteins, α-Cyano-4-hydroxycinnamic acid for peptides)
Check Slides
What is the Electrospray Ionisation suited to?
Polar Molecules
How does Electronspray Ionisation work?
Slide 27-28
Nanospray Ionisation
- Nanospray Ionisation is a very low flow rate version of ESI
- Sample volume and concentrations tends to be small (2-10µl sample at concentrations in the pmol/µl range)
- Flow rates from 100-100nl.min of sample and solvent are passed through very narrow spray needles applying voltages of 1-2kV for sample ionisation.
