Mass spec1 Flashcards
Is ESI used for LC-MS or GC-MS?
LC-MS
Is EI and CI for LC-MS or GC-MS?
GC-MS
What is MALDI?
Matrix assisted laser desorption ionisation
What is EI?
Electron ionisation
How many volts are used in EI?
5000 V to 4030 V which makes it a difference of 70 V
How many eV should you use?
70 eV
If you have a fragment of 14 Da, what is it most likely?
CH2
Does EI or CI produce the M+1 molecular ion?
CI
What are the advantages of CI over EI?
Less fragmentation
Higher intensity of molecular ion
Does EI and CI use the same source?
Yes, in CI you just add gas into the EI source to run chemical ionisation
What is an adduct?
The molecule plus an ion such as NH4+ or C2H5+
Where do the adducts come from
Methane and ammonia, can come from other sources too
What is base peak?
The largest peak on the mass spectrum
What will the methane and ammonia adducts have as m/z ratio?
NH4+ is [M+18]
C2H5+ is [M+29]
Is EI or CI softer/milder?
CI
Does EI or CI produce more fragmentation?
EI
What is a challenge with EI?
The molecular ion is often not present and can be hard to identify molecules
What is a downside with CI?
The ion source cleaning is a downside, you have to clean it much more frequently
So in EI and CI the molecular ion is positive in both cases but only in one case does it change the m/z value of the molecule, explain why
Because in EI an electron is removed making it positive, whereas in CI a proton is added making it positive. A proton weights 1 Da but an electron doesn’t weigh much and the loss of an electron can not be detected
When you apply a voltage do you give the molecule kinetic energy?
Yes
What is the formula for kinetic energy?
Q(charge)*Vext(voltage) = 1/2Mv(velocity)^2
Will ions with different mass to charge ratio (m/z ratio) have different velocities
Yes
What is the formula for velocity?
v = sqrt(2QVext/Mass)
From KE = mv^2 = Q*Voltage
Give an example of a mass analyzer
Quadropole
Ion Trap
Time of flight (TOF)
What might be the source of the background ions?
Leaks (gas), contaminants, column bleeding, desorption from the walls
Explain MS in short
1) Ion created
2) Ion accelerated
3) Ion extracted (analysed by mass analyser and detected)
What is average mass?
Average mass is calculated from the natural abundance of isotopes (eg. 0.99x12+0.01x13 = 12.011)
What is nominal mass?
Mass calculated from the predominant isotopes with atom masses rounded to nearest integer
What is exact mass?
Mass of a molecule containing a given set of isotopes
How can you calculate the number of carbons in a molecule?
Use the equation based on the natural abundance of carbon
C = 90*(M+1)/(M)
Because the ratio between carbon 12 and carbon 13 is 100/1.1 = 90 (for every 100 carbon 12 there is 1.1 carbon 13)
What is monoisotopic mass?
Mass of a molecule containing only the most abundant isotopes
Describe the essential parts in an atmospheric pressure chemical ionisation source APCI. What are their roles?
Nebulising gas flow
Droplets heated and evaporated
Neutral analytes released into gas
Corona discharge needle (supplies electrons) ionises nitrogen
Nitrogen charge is transferred to solvent
Solvent charge is transferred to analyte
Describe the essential parts in an atmospheric pressure photo ionisation source APPI. What are their roles?
Using a UV lamp to excite electrons and charge the analytes.
Nebuliser making droplets
Vapouriser evaporating the droplets
UV light will charge the analytes
What are the three ionisation sources in LC-MS?
ESI (electron spray ionisation)
APCI (Atm pressure chemical ionisation )
APPI (Atm pressure photo ionisation)
Which is the most common ionisation source?
ESI is the most common for LC-MS
Is ESI concentration dependent or flow dependent? What about APPI and APCI?
ESI concentration dependent
APPI, APCI flow dependent
Does ESI or APPI/APCI work best for polar analytes? Which works best for non-polar?
ESI works best for polar and APPI/APCI for non-polar
How does the quadropole mass analyser work?
It has four metal rods and an alternating quadropolar electric field. It only lets through one m/z at a time.
How does the time of flight (TOF) analyser work?
The time we detect depends on the m/z ratio and the distance they need to travel. So m/z are separated/analysed based on flight time.
What are the two mechanisms of ion formation in ESI? Describe the mechanism. What type of analyte is preferred to be ionised?
Charged residue mechanism (solvent evaporates CRM) best for compact big molecules like folded proteins
Field induced ion desorption mechanism (ion evaporation mechanism IEM) best for small organics as well as unfolded large molecules like proteins
How does the field induced ion desorption mechanism work, IEM?
Electric field makes the large drop let go of a smaller drop containing ions, eventually the ion leaves the drop and is nolonger solvated, it is desolvated
Which ionisation methods make M+1 molecular ion?
ESI (for LC-MS) and CI (for GC-MS)
Which ionisation method makes the molecular ion M (not M+1)?
EI (for GC-MS)
How does ESI ion formation mechanism called charged residue mechanism CRM work?
Solvent molecules leave the ions (evaporate) which makes desolvated ions.
Is ESI a soft ionisation tecnique?
Yes
Does ESI work in both in positive and negative mode?
Yes
Does CI work in both positive and negative mode?
Yes
Does EI work in both positive and negative mode?
No
Does ESI work by adding a proton?
Yes
What does it mean when you run MS in negative mode?
You add negatively charged adducts H-, Cl-, Br-. CH3CO2-
How can adding more acid help to make the analyte protonate better?
Adding more acid makes more H+ available
Where are the ions coming from in ESI?
They are already in the solution but need to be extracted by the electrospray
Why is it called an electrospray if it is not spraying electrons?
It uses electrical energy to assist the transfer of ions from solution into the gaseous phase before they are subjected to MS
In positive mode electrospray ionization (ESI), how do you avoid matrix adduction of acetonitrile with a neutral analyte?
Add sodium acetate or potassium acetate which act as a buffer. To get adducts with Na+ (M+23) and K+ (M+39) instead of acetonitrile
What buffer is used in mass spectrometry?
Buffers composed of two buffering species are used commonly in HPLC systems. In particular, ammonium formate, ammonium acetate, and ammonium carbonate are used widely when HPLC is coupled to mass spectrometry (MS).
Describe the mechanism underlying ionisation in the EI source. Also describe design of the source and indicate at least three important components
What is the isotope pattern of halogens?
Cl has 3:1 (75% and 25%)
Br has 1:1 (50 % and 50 %)
Where does cleaving happen?
At branches and carbon next to heteroatom
How does the degrees of unsaturation rule work? What is the formula?
Degrees of Unsaturation (number of double/tripple bonds or rings) = x - y/2 + z/2 + 1
x= C and Si
y= H, F, Cl, Br, I, P
z = N
Tells you how many double bonds or rings are present in the molecule
What is the nitrogen rule and how does it work?
Because nitrogen has an even atomic mass (14) but an odd valence (3), compounds
containing an odd (and non-zero) number of nitrogen atoms will have an odd molecular
mass. In other words:
If the mass of a compound is even, then the compound contains either zero or an even
number of nitrogen atoms (0, 2, 4, 6, etc. nitrogens). If the mass of a compound is odd
then the compound contains an odd number of nitrogen atoms (1, 3, 5, 7, etc. nitrogens).
The “nitrogen rule” holds true for all neutral compounds containing any elements from
the first or second period, sulfur (but not phosphorus) and all of the halogens.
*The rule is typically only applied to the molecular ion signal in the mass spectrum because for each consecutive covalent bond that is broken or formed, the nitrogen rule again reverses making it complicated.
*An even nominal mass indicates that a net even number of covalent bonds have been broken or formed and an even number of nitrogen atoms are present, or that a net odd number of covalent bonds have been broken or formed and an odd number of nitrogen atoms are present. An odd nominal mass indicates that a net even number of covalent bonds have been broken or formed and an odd number of nitrogen atoms are present, or that a net odd number of covalent bonds have been broken or formed and an even number of nitrogen atoms are present.
Only true for organic compounds, doesn’t hold true for compounds like NO2
What are the components of the mass spec?
Ion source, vacuum chamber, mass analyzer and detector
Why is there a vacuum?
To have a collision free trajectory
How is the vacuum achieved?
With vacuum pumps
What is the base peak?
The peak with the highest intensity which is set to 100% since it is relative abundance
What is a challenge with MS that is solved by using chromatography?
Each analyte gives rise to multiple fragments. You reduce complexity by using chromatography since the analytes are separated before.
When you apply a _____ you give kinetic energy
voltage
What is resolving power?
Ability to separate ions of one mass from another - how detailed a spectrum the mass spec can generate
Quadropole is a low mass resolution which can separate based on one unit
What is a BPC?
Base peak chromatogram, only the most intense ion in each scan plot. Background is reduced.
What is TIC?
Total ion current, all ions are plotted over time and it looks very complex
What is an EIC?
Extracted ion chromatogram. One one or a few ions are plotted
Does the temperature of the column have to be above boiling point?
No, it is about the vapour pressure. The gas used is in the vapour pressure.
Are alkanes split into carbocations and neutral radical leaving groups?
Yes
Which carbocation is more stabile tertiary, secondary or primary?
Tertiary most stable and primary least
Does an even mass give odd fragments?
Yes
Does fragmentation usually happen at branches?
Yes
Is it common that H2 is eliminated as a neutral molecule after a carbocation has been formed?
Yes
If a compound seems to “like to fragment”, is it usually branched?
Yes
When you see a fragment that is -2 eg. 43 and 41 or 29 and 27, what is it likely to be caused by?
H2 gas being removed
Why is the molecular ion peak high in a cyclic alkane?
Because you need to fragment twice to get a different m/z value (closed ring and open ring both have same m/z value)
What is a heteroatom?
Any atom that is not C or H
Does fragmentation occur at the carbon closest to or furthest from the heteroatom?
Closest to
Will the leaving group be as large or as small as possible?
As large as possible
What is a McLafferty ion?
m/z values that are common for
aldehyde (44), methyl ketone (58), acid (60), methyl ester (74), ethyl ester (88) and amide (59).
Only occurs if you have a carbonyl with a gamma CH
How do you achieve efficient ionisation if you have analytes containing a lot of basic sites?
Positive mode ESI, lower the pH. Acetic acid or formic acid. Good solvents are ethanol, methanol, acetonitrile in H20
How do you achieve efficient ionisation if you have analytes containing a lot of acidic sites?
Negative mode ESI, raise the pH. Ammonium hydroxide. Good solvents propanol, isopropanol and butanol in H20
How do you achieve efficient ionisation if you have neutral analytes?
Ionised by forming adducts. Add potassium acetate or sodium acetate or formiate
When you connect LC to MS, what do you do if the flow rate is too high?
Split off some flow
When you connect LC to MS what do you do if the mobile phase pH hives neutral analytes?
Tee-in additives that promote ionisation such as acids, bases, Na)
What is ionisation suppression?
When the matrix or another substance co-elutes with the analyte it can cause competetion for charge which means your analyte of interest doesn’t get ionisased. It can be caused by contaminants/plastisizers, proteins in the sample, phospholipids in the sample and eluent additives.
What is CID?
Collision induced dissociation. Ions collide with gas molecules N2 or Ar to cause fragments
What is an issue with centroid data? Why is it used?
It is difficult to distinguish noise and it may appear as results in discrete masses. It may be that some peaks actually contain two peaks but only one shows up as centroid data.
It is used because high resolution profile MS data takes up far too much space
What happens if your analyte carries more than one charge? (in LC-MS, because in GC-MS you don’t have large enough molecules for this)
You need to figure out the charged state because it divides the mass. z= 1 Da / (change in m/z). Four peaks may appear that are actually the same analyte but with four different charged states.
What is the rule of 13?
13 is the mass of CH
If you divide a molecular ion by 13 it predicts how many carbons you have in the molecule (the quotient). The remainder + quotient gives you the number of hydrogens. Only works if you have hydrocarbon. If you have heteroatoms you need to subtract them.
Why is the vacuum needed?
For analytes to have a collision free trajactory
To reduce ion molecular interactions
To reduce the background interference
What is a reflectron? Which a TOF analyser is often equipped with
Ions of same m/z but with different Ek penetrate to different depth in the reflectron
Hence, those with higher Ek (having an initially higher velocity) are delayed.
To improve resolving power ions are turned around by the reflectron. It is a series of hollow rings at increasingly positive potential. Ions entering it are slowed down, stopped and reflected back. All ions of the same mass reach this grid (at the detector) at the same time regardless of their initial kinetic energies
