1. LC-MS Lectures
Dr Mike Kinsella

2. Mass Analysers in LC-MS
Several Common Mass Analyser Types:
Ion trap
Single Quadruupole
Triple Quadrupole
Time-of-Flight (TOF), Q-TOF
Orbitrap
MALDI
Ions formed in that gas phase are subsequently separated in mass analyser
Remember: Mass spectrum of an analyte represented by a bar graph that plots relative intensity of each of ion against m/z. m/z measured by mass analyser
Mass Resolution: ΔM is the ability of a mass analyser to separate one mass from an adjacent mass
Mass accuracy: measurement of the closeness of the given measurement to the true mass of the analyte
Mass Range: Difference between highest and lowest measurable m/z which mass analyser can measure

3. Mass Analysers
All mass analysers require low vacuum environment-At atmospheric pressure, average distance gas phase ion will travel without colliding with a gaseous molecular species is 10-8 m, therefore unlikely to reach detector
Mean free path travelled by ion inversely proportional to pressure and pressure of 10-6 Torr increases mean free path to approx 10 m. Therefore, under vacuum, majority of ions won’t undergo collisions but will make it to detector 

4. Mass Analysers-Quadrupoles
Quadrupole mass analyser contains 4 circular, parallel rods or poles positioned equidistant from each other
In quadrupole mass analysing devices, electric fields used to separate ions according to their m/z ratio as they travel through central axis of quadrupole which has fixed DC and alternating RF voltages applied to them
DC current is constant flow of electric charge from high to low potential
RF: The range of electromagnetic frequencies above the audio range and below infrared ( 10 kHz to 300 GHz), typically 10 MHz used
Depending on magnitude of these voltages, can be arranged that only ions of certain masses allowed to pass the whole length of the quadrupole on a resonant or non-collisional trajectory. Can alter voltages to allow ions with different m/z values through to detector.
Mass Analysers-Quadrupoles

5. Pole assembly length 50-250 mm, pole diameter approx 5-15 mm
Pole assembly length mm, pole diameter approx 5-15 mm. Held in position with insulating rings. Tunnel radius r0 ~ 5 mm.
Extremely accurate machining and positioning of quadrupole rods essential for mass accuracy and resolution, hyperbolic shaped rods ideal but circular rods often used for improved manufacturing reporducibility
Each opposing rod pair is connected together electrically, and an alternating radio frequency (RF) voltage is applied between one pair of rods and the other. RF voltage cycles in a sinusoidal fashion, therefore each rod pair is successively +ve then –ve. In central axis, electric field = 0
Application of voltages creates a hyperbolic field – complex ion trajectory – must consider 3 dimensions/axes (X, Y & Z in previous diagram)
A direct current accelerating voltage (typically 5 V) is then superimposed on the RF voltage to accelerate ions in the Z direction – Ions travel down the quadrupole between the rods.
Quadrupole Design

6. Quadrupole-Hyperbolic Field
Rod pairs successively +ve and –ve, ions attracted and repelled from rods or central axis
Application of voltages creates a hyperbolic field within them given by:
Where X and Y are distances along the coordinate axes, r0 is distance from Z axis to surface of any of the poles, Φis oscillating electric field
When X=Y, Φ=0 giving rise to zero field strength in the quadrupole
As long as x and y (which determine position of rods from centre of rods < r0, ion pass through quad without colliding
When ion oscillates with trajectory whose amplitude >r0, ion will collide with rod and become discharged and ejected into vacuum

7. Stable vs unstable ion path
Equipotential lines for a quadrupole (hyperbolic field)

8. Ion Trajectory
For an ion entering quadrupole hyperbolic field, 2 important factors a & q determining regions of stable ion trajectory:
For any quadrupole, r0 and ω are fixed while voltages U and V can be varied to ensure the spatial X-Y coordinates of the ion never exceeds r0 – ion manipulated in non-collision trajectory-pass through quad.
Region A = 1st
stability region
Where:
z = charge on electron
m = mass of ion
ω = frequency of RF voltage
U = applied DC voltage
r0 = radius of quadrupole
V = amplitude of the RF voltage

9. Ion migration
The DC voltage (U) and RF voltage (V) are altered according to a linear relationship-often referred to as the ‘SCAN’ function. When U and V are ramped linearly-termed SCAN mode
Slope of SCAN function (rate of change of U against V) referred to as quadrupole GAIN
Stability regions of different mass ions often overlap.
To allow only specific m/z ions through, U and V are chosen to lie just inside apex of stability zone A
U is changed as a linear function of V (scan line)

10. Resolution & Sensitivity
Increasing (mass Gain) slope of scan line leads to enhanced resolution but lower sensitivity, high mass ions particularly affected
The intensity of all peaks within a spectrum are summed to give an overall signal intensity which can be plotted against time- TOTAL ION CURRENT (TIC)-similar to LC-UV chromatogram
Intensity of TIC governed by instrument scanning speed-faster scan rate, more data collected and ion intensity increases
For Selected/Specific ion monitoring (SIR/SIM mode)– set quadrupole to only certain values of U and V thereby only allowing certain mass ions through quadrupole. SIM experiment times faster than full scanning mode.
If only useful m/z values recorded, intensity of SELECTED ION CURRENT larger than TIC- much improved sensitivity for analyte

11. Quadrupole limitations
Limited mass accuracy – typical resolution of 300 (dotted blue line)-lysozyme and its oxidation product ion not resolved
Mass discrimination – peaks a higher mass give lower intensity due to non-efficient ion entry into tunnel radius due to DC fringing fields- can use additional entrance lens/filter to overcome
Scanning speeds must be sufficient in order to achieve data points across chromatographic peak- minimum required for good quantitation
Quadrupole limitations

12. Mass Analyser – Time Of Flight (TOF)
Fundamental principles of TOF mass analysers relatively straightforward – gas phase ions from ionisation source enter the TOF in pulses (or bursts) and are subjected to an acceleration voltage
The ions then ‘fly’ down an evacuated tube towards the detector
Time taken for ion to travel length of tube (d) is correlated to their m/z
Flight times correlated against known masses from an infused tune solution allowing conversion to obtain abundance vs m/z spectrum.
Ions from source

13. Equations of Motion
Important ions extracted in pulses into mass analyser since m/z distinguished based on flight time – ions start flight simultaneously
Kinetic energy of ions expressed as:
Where m = mass of ion, z = no. of charges on ion, e = charge of an electron, v = final velocity
Time taken (t) for ion to travel distance d from ion source to detector:
Combining equations shows flight time of ion (t) directly related to square root of m/z
Ion of m/z 100 takes twice as long as ion m/z 25 to travel distance d.

14. TOF Resolution
Resolution in TOF is limited by 2 important factors, especially for higher mass ions
1st problem inherent to technique itself – difference in flight times varies with mass. However the arrival time at the detector become smaller as the mass of ions arriving at detector increases and are difficult to differentiate
E.g. previously saw Ion of m/z 100 took twice as long as m/z 25 to reach detector.
Consider 2 heavier ions which differ in mass by 75 Da, m/z 1075 and m/z 1000
Represents a difference in flight time of just 3.7%

15. TOF-Resolution
2nd issue regarding resolution stems from method of ion production and acceleration
Not all ions of the same m/z arrive at detector at same time due to distribution of kinetic energies they acquire during acceleration process and are not all accelerated from same point in ion source
Therefore even for ions of same m/z, there is a distribution of arrival times at detector
Leads to reduction of spectral resolution
Particularly problematic for high mass ions
TOF resolution improved using a
reflectron device

16. TOF Resolution-Reflectron Device
Reflectron is a series of electrostatic lenses which create a homogenous electrostatic field at the end of the flight path/TOF tube where detector would reside.
Plates have same polarity as ions of interest
Ions will be slowed when they reach the electrostatic field of the electron and momentarily come to a standstill and then accelerated in opposite direction.
Reflectron often referred to as ion mirror-it reverses flight path
Ions with greater kinetic energy penetrate reflectron deeper and spend slightly longer in the device compared to lower KE ions
Isobaric ions can be caused to bunch together leading to reduction in distribution of flight times and enhanced resolution

17. Reflectron TOF Mass Analyser

18. TOF Resolution-Reflectron
Usual to expel ions on a slightly deviated path
The longer the flight tube, longer it takes for ions to traverse it – double flight path doubles time difference between sequential ions – enhanced resolution
Traditional linear TOF, Rs 1000
Reflectron TOF, Rs 10,000
Even with reflectron, becomes increasingly difficult to discriminate unit masses over m/z 3000 but ok for routine work
Typical mass accuracy for small ions m/z 2000 approx 10 ppm with internal calibration
Large ions m/z 10,000 – 80,000 mass accuracy of 100 ppm achievable
TOF Resolution-Reflectron

19. Orthogonally Assisted TOF Analysers
Direct coupling of API sources with TOF analysers can be problematic – API yields continuous ion beam, TOF requires pulsed ions
Continuous Ion beam emerges from ion source, accelerated through voltage V1. A second electrode is placed 90° to the continuous ion beam with an applied potential V2. This 2nd electrode can be switched on and off rapidly to form a pulse of ions at right angles to the main beam.
These pulsed ions enter flight tube simultaneously
Magnitude of pushing potential can be adjusted so that injected ion packet will enter axis of flight tube
Accelerating voltage V1
Accelerating voltage V2
Vacuum
Reflectron

20. TOF Overview ADVANTAGES Highest mass range of all mass analysers
Typical Specification:
Resolution: 10, Mass range m/z: 50 – 1,000,000
Mass accuracy: Da Detection Limit: 10-15g
Linear Dynamic range: Cost: >300,000
ADVANTAGES
Highest mass range of all mass analysers
Sensitivity
Good for high mass accuracy (molecular formula)
Rapid scanning of mass range
DISADVANTAGES
Expensive
Limited dynamic range
Difficult to couple to continuous sample sources
MS/MS not possible
Requires very high vacuum (10-6 Torr)

21. Ion Trap Mass Analysers
Ion trap mass analysers use oscillating electric fields (RF) to trap ions in a controlled manner
A typical quadrupole ion trap MS consists of a ring electrode with a hyperbolic inner surface and 2 electrically common hyperbolic end cap electrodes
The ring electrode acts as one pair of rods while the end-caps behave as the other pair of rods (similar to quadrupole systems)
RF and AC voltages applied to trap ions within a specific m/z range, trap ions of a specific m/z or eject ions of specified m/z
Ion Trap Mass Analysers

22. Ion Trap-Equations of Motion
Ion motion in ion trap can be described by Matthieu equations as studied previously for quadrupole
Stability of ion trajectories are determined by trap geometry, applied voltages and m/z of the ion (see equations of motion below). Ions resonate in figure of 8 trajectory. Can sequentially alter applied potential to selectively remove ions from trap.

23. Quadrupole Ion Trap – Space charging effects
The trap is limited in capacity to trap charges – operating conditions should be optimised to ensure only ions of interest introduced to the trap
Trap resolution decreases rapidly as ion density increases
As no. of ions entering trap increases, average distance between ions decreases and repulsive ‘space charging’ forces increase.
Ion Concentrations above space charge limit lead to ejection of ions, poor instrument performance- poor resolution, shifts in mass calibration, poor linearity
To avoid space charging, no. of ions within trap regulated to optimum through finding optimum time to fill trap to optimum level.

25. QIT-Scanning Experiments
Ion trap spectral experiments composed of several sequential steps including ion injection, isolation, excitation and analysis
Pre-scan often conducted to determine ideal injection time and minimise space charging effects
After ions injected into trap from source, suitable RF voltage applied to ring electrode to confine ion to stable trajectories.
Isolation scan can be performed to accumulate a specific ion or range of ions
Next optional step = ion excitation where voltage applied to cause trapped ions to oscillate with higher energy leading to collisions with background gas (usually He) and fragmentation.
Finally, ions analysed by ejection from trap to detector
If desired, can select specific m/z ions to be ejected from amongst all ions in the trap

26. Scanning Experiments 2
Most significant use of ion trap mass analysers involves sequential trapping and fragmentation of specific ions to produce highly specific MSn data – achieved through collision induced dissociation of precursor ions at increased gas pressure
Product/daughter ions are resonance ejected and monitored.
One or more of the fragment ions may be retained within the trap for further fragmentation. Can obtain MSn theoretical fragmentations

27. QIT Overview ADVANTAGES Compact and easy to operate MSn possible
Typical Specification:
Resolution: 5, Mass range m/z: 50 – 2,000
Mass accuracy: 0.1 Da Detection Limit: 10-15g
Linear Dynamic range: Cost: €150,000
ADVANTAGES
Compact and easy to operate
MSn possible
Relatively inexpensive
Sensitive
DISADVANTAGES
Poor for quantitation
Limited dynamic range due to space charging effect
Only capable of unit resolution

28. Magnetic Sector Mass Analyser
Based on principle that ions can be deflected in magnetic & electric fields
Degree of ion deflection is proportional to square root of the m/z and the potential through which they are accelerated prior to mass analysis – HIGHLY ACCURATE m/z determination
Sector instruments historically extensively used for accurate mass but developments in other mass analysers mean they have been superseded
Electric & magnetic fields used to focus beam of ions created in API source according to the KE of each ion, allowing each m/z to be sharply focussed prior to deflection in the magnetic field
This improves resolution, measurements can be made on ions which differ by only a few ppm.

29. Equations of Motion-Magnetic Sector
The KE of an ion mass m, charge z accelerated by a voltage Vs can be determined from:
If this ion enters a magnetic field which is applied perpendicular to the velocity v, the ion is deflected in a circular path where r = arc radius, z = charge on ion& B = magnetic field strength
Combining these equations gives:
For ions with a single charge and a constant field strength and accelerating voltage, the radius of the arc depends on mass.
Can vary m/z of ions which reach detector through varying magnetic field (B) or applied voltage Vs, therefore a scan of desired m/z range can be easily performed

30. Magnetic Sector Mass Analyser
Can separate ions of different m/z values by changing magnetic field (B) or applied voltage (Vs)
In practise, more convenient for all ions to arrive at a single point for detection so r is kept constant.
Magnetic scanning allows entire mass range to be scanned, however high m/z ions may appear close together
Quadratic dependence of m/z on value of B, therefore high mass peaks appear closer together than low mass peaks

31. Electrostatic Analyser
An electrostatic Analyser (ESA) is a directional focussing device which focusses ions with the same m/z but different kinetic energies
In an electrostatic analyser the degree of the deflection is not dependant on the mass or charge and is only dependant on the velocity of the ion and the ESA voltage
By manipulating the ESA voltage, possible to bring ions of differing velocity but same m/z to the same focal plane prior to entering into the mass spec

32. Magnetic Sector ADVANTAGES Excellent sensitivity High resolution
Typical Specification:
Resolution: 50, Mass range m/z: 2 – 15,000
Mass accuracy: Da Detection Limit: 10-15g
Linear Dynamic range: Cost: €500,000
ADVANTAGES
Excellent sensitivity
High resolution
High mass accuracy-can determine molecular formula
Wide linear dynamic range-ideal for quantitation
DISADVANTAGES
Expensive
High resolution decreases sensitivity
Needs very high vacuum (10-6 Torr)
MS/MS not possible

33. Selection of Mass Analyser

35. Practical LC-MS Considerations

36. Tandem Mass Spectrometry
Triple Quadrupole (QqQ)
Two mass filtering quadrupoles bracket an Rf only collision cell.
Ion Trap (IT)
A single ion trap serves as mass analyzer and collision cell.
Hybrids (e.g. LIT)
Instrument is in the QqQ geometry, but one quadrupole can also trap and store ions.

37. Triple Quads v. Ion Traps
Triple Quadrupole
Advantages
Very sensitive. (SIM)
Good for quantitation
Some useful MS scanning modes
Limitations
No MSn
Expensive
Limited to unit mass resolution.
Less sensitive in full scan mode.
Advantages
Higher full scan sensitivity
Higher mass resolution
MSn
Limitations
Not as good for quantitations.
Space Charge Effects
1/3 cut-off rule.
Cannot perform certain MS experiments.

38. Triple Quad Configuration
Scanning
RF/DC
RF only Collision Cell
Scanning
RF/DC
RF only
Q1 and Q3 are standard mass filter quadrupoles.
The can scan masses sequentially (e.g. 50 to 500 amu)
The can be used to select a single mass.
Q2 is an RF only quadrupole that is in a gas filled chamber.
Q2 is the “collision cell” where mass fragmentation occurs.
Q2 does not filter ions. It accepts all ion sent to it by Q1 and passes all ions formed by collision to Q3 to be sorted.

39. Triple Quads… In scanning mode 99% ions lost between the rods.
Poorer full scan sensitivity
In SIM mode 100% of selected ion reaches detector.
Makes them highly sensitive and great for quantitation!
Mass resolution typically limited to “unit” (+/- 0.2 amu)
Fragmentation is controlled by the energy ions have when they enter the collision cell.
Higher energy >> greater fragmentation.

40. Collision Cell LINAC (linear accelerator) Collision Cell
Filled with N2 gas at roughly 3x10-5 torr.
Drives ions out, reducing “cross-talk”
The analyte molecules undergo collision activated disassociation by energetic collision with the N2 molecules.
The N2 also acts to “cool” fragments, facilitating transport to the detector.

41. Ion Traps
Ions fill the space between a ring electrode and a pair of end-cap electrodes.
Mass analysis and fragmentation occur in the same space.

42. Ion Traps…
In full scan mode: Ions fill and are trapped in space then masses are scanned out of the trap sequentially.
Ions are not lost, so full scan sensitivity is better, but filling/closing cycles make them poorer at quantitation.
Mass resolution is controlled by the “speed” at which masses are scanned out of the trap.
slower scanning = better mass resolution.
In MS/MS mode: Ions trapped. Fragmentation occurs when the selected ion is excited by a so called “tickle” voltage and collides with bath gas (He). This process can occur recursively thus MS/MS/MS/MS….

43. Hybrid Instruments
Applied Biosystems 3200 Qtrap System Q0 Q1 Q2 Q3
Typically in the same configuration as a triple quadrupole instrument.
On the Qtrap Q3 is the hybrid quadrupole dubbed a “linear ion trap” or LIT.
Q3 can function as a quadrupole OR an LIT.

44. Advantages of LIT vs. IT
Has a larger “volume” so it can be filled with more ions before exhibiting space charge effects.
Ions are formed outside the trap, so it is not limited by the 1/3 rule.
Can perform MS/MS/MS experiments by selecting an ion and fragmenting it using the spillover collision gas. (1/3 rule applies here…)

45. Modes of Operation H Y B R I D S Triple Quads and Ion Traps
Full Scan (LC/MS)
MRM (Multiple Reaction Monitoring)
Product Ion Scan (PI)
Exclusively Triple Quad
Constant Neutral Loss
Precursor Ion Scan
Exclusively Ion Trap
MSn

46. Multiple Reaction Monitoring (MRM)
Steps MS2: &4
Exit lens
Ion accumulation Q0 Q1 Q2 Q3
Precursor ion
selection
Fragmentation
N2 CAD Gas
Q1 Selects an [M+H]+
Q2 fragments the selected ion.
Q3 monitors only one daughter ion

47. MRM Only the daughter ion reaches the detector.
Steps MS2: &4
Exit lens
Ion accumulation Q0 Q1 Q2 Q3
Precursor ion
selection
Fragmentation
N2 CAD Gas
Only the daughter ion reaches the detector.
Sensitivity of MRM is a function of how much of the daughter ion is produced.
The parent ion fragmentation to daughter ion is commonly referred to as a “transition”

48. Example MRM Data Many transitions can be stacked together in a method.
Oxycodone: (316.2241)
Parent : 316.2
Daughter : 241
Result of one MRM cycle of 130 drugs.
Many transitions can be stacked together in a method.
The instrument will monitor each pair for a short time.
MRM is analogous to SIM on a GC/MS only more compound specific.

49. Multiple Reaction Monitoring –Instrument Optimisation

50. Product Ion Scanning Q1 selects a parent ion.
Steps MS2: &4
Exit lens
Ion accumulation Q0 Q1 Q2 Q3
Precursor ion
selection
Fragmentation
linear ion trap
3x10-5 Torr
N2 CAD Gas
Q1 selects a parent ion.
Q2 fragments the selected ion
Q3 traps then scans out all fragment ions.

51. Product Ion Scan
Steps MS2: &4
Exit lens
Ion accumulation Q0 Q1 Q2 Q3
Precursor ion
selection
Fragmentation
linear ion trap
3x10-5 Torr
N2 CAD Gas
Selection of a single parent ion by Q1 allows separate product ion scans for coeluting compounds to be easily generated.
Provided they don’t have the same mass…

52. Acknowledgements
The WINSS project has been part-funded by the European Regional Development Fund through the Ireland Wales INTERREG 4A programme