Liquid Chromatography & Mass Spectrometry Flashcards
What is Chromatography?
Greek: Writing in Color
- physical method of component/analyte separation
- components interact with a stationary phase (not moving) and a moving (mobile) phase
- strength of interactions defines separation
What does a chromatographic system consist of?
- a mobile phase reservoir
- a flow device (e.g. pressure pump) moving the mobile phase across
- a stationary phase
- an injector setup loading components onto the stationary phase
- a detector detecting components after separation.
Many detection systems available:
UV/VIS light absorption, fluorescence, light scattering, thermal conductivity, ionization, mass detector (MS)
Choice of detector depends on physical properties of components.
What is LC?
Name three pumping LC systems.
Liquid Chromatography
Measurement of retention time. Separation based on polar interactions of analytes with:
- Stationary phase (column)
- Mobile phase
Pumping LC systems:
- FPLC: Fast Protein LC
- HPLC: High Performance LC
- U(H)PLC: Ultra-High Pressure LC
What are the three main characteristics of LC?
- Retention time (tR): is the elapsed time between start of separation and the appearance of the peak apex of a component at the detector, determined by strength of interactions and diffusion driven mobility of component
- Column dead time (t0): is the time taken for solvent molecules or other non-retained analytes to move through the column (without interaction with the stationary phase)
- Column diameter: defines optimal flow rate of mobile phase as a function of flow velocity (range μm to m).
What is RP-LC?
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REVERSED PHASE LIQUID CHROMATOGRAPHY
RP-LC is routinely used in combination with mass spectrometry for reasons such as:
- Volatile mobile phase without ions/salt -> no ion suppression during component ionization
- Therefore, column effluent is directly compatible with MS
- Sample cleanup during loading as salts and other hydrophilic substances are removed
- Fractionation of components during column development
In RP-LC we usually use a column of C18 (very hydrophobic) that is made of a chain of 18 carbon atoms. This will trap the hydrophobic molecules and the hydrophilic molecules will be detected first
What is the problem with chromatography?
The problem with chromatography is that one has to play between the pressure applied in the column and the flow rate (how was is the mobile phase going in the column). In fact, if the flow rate is too fast, there is a high change that we won’t be able to separate the different analytes very well. On the other hand, if the pressure is too low, it will result in a very broad peak in the data which is no good either.
It is all a game between the length of the column, the type of column used, the type of mobile phase, the pressure and the flow rate.
What is the basic concept of MS?
What are the major components of a mass spectrometer?
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Basic concept:
- A mass spectrometer is a scale.
- It mesures the weight of molecules based on their mass to charge ratio.
- A mass spectrometer generates gas-phase ions from a sample, separates them according to their mass-to-charge ratio (m/z) and produces a record of their abundances
- A MS is ALWAYS composed of an ion source, one or multiple mass analyzer and a detector.
Major components:
- Ion Source
- Mass Analyzer
- Detector
How does the ionization in an MS work?
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What are the two main techniques?
Generation and transfer of ions into the gas phase
In MS usually analyze positive ions only.
There are two types of ionization:
- *Molecular ion**: M+
- *Pseudo-Molecular ion**: [MH]+
Hard ionization techniques: processes resulting in almost total ion fragmentation
- Electron Impact (EI)
Soft ionization techniques: processes which impart little residual energy onto the subject molecule resulting in little ion fragmentation
- ElectroSpray Ionization (ESI)
- Atmospheric Pressure Chemical Ionization (APCI)
- Matrix-Assisted Laser Desorption/Ionization (MALDI)
What is a mass spectrum?
A mass spectrum displays the signal intensity vs. mass-to-charge ratio
The highest peak in the spectrum is called the base peak
What is the resolution of an MS?
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What differentiation is made concerning resolution?
In mass spectrometry, resolution is a measure of the ability to distinguish two peaks of slightly different mass-to-charge ratios ΔM, in a mass spectrum
–> One “bumpy” peak vs. multiple distinct peaks
Low-resolution:
- Quadrupole (Q)
- QQQ
- Q-Trap
- Q-TOF
- Q-Orbitrap
High resolution (HRMS)
- time of flight (TOF)
- Orbitrap
What is ELECTROSPRAY IONIZATION (ESI)?
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What are the ESI theories?
ELECTROSPRAY IONIZATION:
ELECTRICAL NEBULIZATION OF LIQUID AND ELECTRO-CHEMICAL OXIDATION
- ESI is a technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol.
- In ESI ion intensity is concentration dependent
- ESI is different from other ionization processes (e.g. matrix-assisted laser desorption/ionization (MALDI)) since it may produce multiple-charged ions, effectively extending the mass range of the analyser to accommodate the kDa-MDa orders of magnitude observed in proteins and their associated polypeptide fragments. This means that the same molecule can get multiple charges which will lead to a detection at different weights
- Electrospray ionisation is a “Soft” ionization technique
- Does not require derivatisation
There are two theories that explain how ions are formed in the ESI. Both theories are valid:
- Single Ion in Droplet Theory (SIDT) : droplets get smaller through a number of Coulomb-explosions/Coulomb-fission until only one ion per droplet remains
- Ion Evaporation Model (IEM) : single ions are ejected from droplets
What is MALDI?
Matrix-assisted laser desorption/ionization
- A laser beam will be sent onto a matrix (looks like a resine) where our sample is. The energy sent by the laser will be absorbed by the matrix and sent to our sample. This energy is high enough to ionize the sample.
- MALDI creates mostly singly charged ions
Matrix function:
- ”Solvates” the analyte
- Absorbs the laser pulse (the energy)
- Performs analyte desorption
- Ionises the analyte (photo-ionised matrix)
What types of mass analyzer exist?
+ Examples
1. Beam type analyzers
Ions are accelerated through electric and/or magnetic fields and are separated in time and space
Examples:
- Sector field
- Time of flight (TOF)
- Quadrupole (Q)
- Q-TOF
2. Trapping analyzers:
Ions are trapped in an electric or magnetic field and detected by ejection from the field or
while oscillating in the field.
Examples:
- 3D iontrap (Paul trap)
- 2D linear quadrupole iontrap
- Fourier transform ion cyclotron resonance (FTICR)
- Orbitrap
What is a linear time-of-flight (TOF) mass analyzer?
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Beam type analyzer
When an ion enters an electric field, it will accelerate. With basic physics it is possible to know the m/z ratio by knowing the distance that the ions has travelled, the time when it was detected and the voltage applied in the electric field.
When one has a linear time of flight, there is problem. The problem is that if an ion has the same mass but different charges, it will be detected at two different times. This can be avoided when we add a reflector in the TOF. This reflector will make ions go back and forth in the TOF. If two ions with different charges but the same mass are in a flight, the one with the smaller charge will be reflected earlier than the second one. This allows them to arrive at the same time in the detector.
Linear: One row resolution peak
Reflector: several high resolution peaks
What is the quadrupole mass filter?
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Beam type analyzer
Quadrupoles are basically 4 tubes that have a charge. This charge will change at a certain frequency. If an ion is between these 4 rods, it will be attracted by 2 of the rods and repulsed by 2 others. This will create an oscillating motion of the ions. Depending on the voltage applied on the rods, the trajectory and the oscillating motion of the ions can be stable or unstable. When one applies a specific voltage to the rods, only a range of ions with a specific mass can be stabilized. All the others will crush onto the rods. This means that the ions detected in the detector, can only be in that range.
The physical equations that describe these stable/unstable trajectories are called Mathieu function
What does a quadrupole scan show?
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The stability regions/stable oscillations of ions in quadrupole mass analyzers
They can be calculated using the Mathieu Functions
How do the efficiency and mass range differ depending on the quadrupole used?
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How are ions detected after TOF mass analyzers?
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With a secondary electron multiplier
What is a tandem mass spectrometer?
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MS system with multiple mass analyzer
Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyse chemical samples. A common use of tandem MS is the analysis of biomolecules, such as proteins and peptides.
The molecules of a given sample are ionized and the first spectrometer (designated MS1) separates these ions by their mass-to-charge ratio (often given as m/z or m/Q). Ions of a particular m/z-ratio coming from MS1 are selected and then made to split into smaller fragment ions, e.g. by collision-induced dissociation, ion-molecule reaction, or photodissociation. These fragments are then introduced into the second mass spectrometer (MS2), which in turn separates the fragments by their m/z-ratio and detects them. The fragmentation step makes it possible to identify and separate ions that have very similar m/z-ratios in regular mass spectrometers.
Recently, the most common tandem mass spectrometer are Quadrupole-Time of Flight hybrid instrument (Q-TOF) and Quadrupole-Orbitrap hybrid instrument
What main data acquisition modes are there?
Usually one will send a “packet” of ions in the MS and measure their weight. Each detected ions in MS1 will be resent in the MS for a second analysis where the they will be fragmented. The fragments of each ion will be measured. This allows to have a detailed information about each ion that was present in the MS1.
- Full scan, MS1: Detection of who is there –> Non-targeted
- Fragment or MS2 scanning: Get to know, who is there (characterization/identification)
What is Selected reaction monitoring?
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Targeted acquisition
- usually done with triple quadrupole (QQQ)
- one single transistion from precursor to fragment
- Most sensitive MS scan type for detection of known components
- With Multi Reaction Monitoring (MRM) several SRM’s on same precursor but different product ions are doneàIncreasing specificity (correctness) with concomitant decrease in number of possible precursors quantifiable in one LC-MS run
- Absolute protein quantification possible with use of stable isotope-labeled internal marker
What are the advantages and disadvantages of the three different metods to acquire data?
DDA, SRM/MRM, DIA
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What types of chromatography are there?
How does Hydrophilic Interactions LC (HILIC) compare to Reverse-phase LC (RP-LC)?
What is precursor ion scan?
Targeted acquisition
- Effective method to determine m/z of precursor ions, which are structurally related,
e. g. bearing a special PTM (peptides) or headgroup (lipids) - Difficult to implement on a chromatographic time scale à infusion of component mixture
What is neutral loss scan?
Targeted acquisition
- An alternative scanning method to determine precursor ions with a common structural motif.
- In opposition to precursor ion scan, we use the fact that the structural motif is readily lost as a non charged (neutral) fragment N upon CID, leaving a molecular ion of same charge as precursor but mass reduced by massN
