Separation Science and Mass Spectrometry Flashcards
What is chromatography?
A physical method of separation, in which the components to be separated are distributed between two phases, one of which is stationary, while the other moves in a definite direction
What is the mobile phase?
The liquid or gas flowing through the tube
What is the stationary phase?
A substance that holds in place and coated on the wall of the tube
How fast will a molecule with a high affinity for the stationary phase move through the tube?
Slowly
How fast will a molecule with a low affinity for the stationary phase move through the tube?
Fast
Name four different types of liquid chromatography
Paper Chromatography
Thin Layer Chromatography(TLC)
Column / Flash Chromatography
High-Performance Liquid Chromatography (HPLC)
What property must the molecules have when using liquid chromatography?
They must be soluble
What property must the molecules have when using gas chromatography?
They must be volatile
This can also be used if the molecules are not themselves volatile, but can easily be derivatised (converted) into volatile versions of themselves
What does ‘partition’ refer to when talking about chromatography?
When both of the phases are liquids, the solute will dissolve more in one phase than the other
What does ‘adsorption’ refer to when talking about chromatography?
When the stationary phase is solid, different molecules will ‘stick on’ to it more or less
What does ‘electrostatic interaction’ refer to when talking about chromatography?
When the solute is ionic, it can form a strong affinity with an ionic phase
What does ‘molecular sieving’ refer to when talking about chromatography?
It uses a stationary phase made of gel particles, containing cavities of defined sizes. Molecules that are too big cannot get into the cavities, and so pass through the tube more quickly
Why do particles of the same type not all go at the exact same speed?
They are subject to random thermal motion and diffusion, and therefore will travel at slightly different speeds
Why does separation require an input of energy?
It is opposite diffusion (a passive process) and is therefore going against what is wanted by the substance
What happens when the column length increases?
The gap between the peaks get larger
What happens when the mobile phase flow rate increases?
The gap between the peaks get larger as there is less opportunity for thermal motion
What does the gap between the peaks measure?
They measure how effective the separation has been
Why is it important to load the compound in a small volume?
Every molecule will start at the same place in the medium
What is a chromatogram?
It is the name for the output of a chromatography instrument
What is the baseline of the chromatogram?
The signal recorded when no compound is being eluted
At what time is the sample injected?
t₀
What is the retention time (tᵣ)?
The amount of time it takes for a specific compound to elute, measured at the height of its peak
It depends on: the relative affinity of the analyte for the different phases, the length of the column and mobile phase flow rate
What is the retention time proportional to?
Retention time is directly proportional to the length of the column
Retention time is inversely proportional to the flow rate
What is the peak width (W)?
A measure of how long it takes for all of the analyte (of one type) to pass the detector
What is the column length (L)?
The length of the chromatographic equipment
What is the resolution (Rₛ)?
A measure of how good the chromatographic method is for separating two specific components of a mixture
It depends on the retention time, peak width and column length
What is the formula for resolution?
Rₛ = (2 x (tᵣ of A - tᵣ of B)) / (W of A + W of B)
What is resolution directly proportional to?
The square root of the column length
What gases usually make up the mobile phase in GC?
Nitrogen, hydrogen or helium
What type of material usually makes up the stationary phase in GC?
A very high-boiling liquid polymer, immobilised on the inner wall of a narrow tube
What are the two most common types of stationary phase in GC?
PDMS (polydimethylsiloxane) - low polarity
PEG (polyethyleneglycol) - medium polarity
How does GC work?
A small volume of sample is injected into the start of the column and the analytes in the sample are carried along by the gaseous mobile phase, separating as they travel through the column
At the end of the column, the analytes are detected and the a chromatogram is outputted
How does GC work?
A small volume of sample is injected into the start of the column and the analytes in the sample are carried along by the gaseous mobile phase, separating as they travel through the column
At the end of the column, the analytes are detected and the a chromatogram is outputted
How does packed column GC work?
The column is filled with small particles, which are coated in the stationary phase
The mobile phase moves through the gaps between the particles
How does capillary column GC work?
The capillary walls are coated with a thin film (1-5 micrometres) of the stationary phase
The mobile phase moves through the middle of the capillary
What are the three different types of compound GC can be applied to?
Are naturally volatile e.g. gases and liquids with low boiling points
Are not very volatile at room temperature, but become more volatile when heated e.g. liquids with higher boiling points, and some solids
Are not naturally volatile, but can be chemically converted (derivatised) into volatile compounds
What happens if the analyte in GC has a high affinity for the stationary phase?
It will elute more slowly
What happens in the analyte in GC is more volatile?
Ir will spend more time in the mobile phase, making it elute more quickly
How are the best separation results achieved in GC?
When there is a good match between the polarity of the stationary phase and the polarity of the analytes to be separated - if this is the case, the order the analytes elute will reflect their boiling point
How can high temperatures in GC reduce the resolution of the separation?
It reduces the retention time of the species as they elute more quickly, therefore making the peaks closer to each other
High column temperatures also increases the random thermal motion that the analytes experience, leading to broader peaks
What is isothermal elution?
When there is a single temperature throughout the whole elution process
What is programmed temperature elution?
When there is an increase or decrease in temperature as the elution process goes on - this is useful when there are analytes with significantly different boiling points
Name two methods of GC detection
Flame Ionisation Detector (FID)
Mass Spectrometry (GCMS)
How does FID work?
The mobile phase carrying the analytes with it exits the column and is combusted in a hydrogen flame, generating a supply of CHO⁺ ions which cause a measurable current in the FID
How does the number of CHO⁺ ions impact the magnitude of the peaks?
The more CHO⁺ ions there are, the greater the peak and can therefore help to see the amount of analyte in the species
Why must FID be standardised when determining concentrations of analytes?
The constant of proportionality (or response factor) can differ significantly from one analyse to another
For what substances can Liquid Chromatography (LC) be used?
Compounds that are soluble
Why is LC very useful and can be applied to almost all substances?
The mobile phase and stationary phase can be changed greatly, reflecting any substances with differing polarities
Describe Paper Chromatography
Typically uses a strip of paper as a stationary phase, which is then dipped in a solvent acting as the mobile phase
The solvent moves up the paper, carrying the different analytes up it at different rates
Describe TLC
Typically uses silica powder as the stationary phase which is absorbed onto a thin strip of aluminium
The plate is then dipped in solvent (mobile phase) and then the solvent carries the different compounds up at different rates
This is usually used for monitoring the progress of the reaction
Describe Column Chromatography
Typically uses silica powder as a stationary phase, which is supported in a vertical glass column
A mixture can be placed at the top of the column, and then a solvent can be poured on top
The analytes then elute down the column at different rates, allowing for large scale separations and can purify reaction mixtures
Describe Flash Chromatography
It is the same as column chromatography, with the only difference is that is done under elevated pressure to speed up the elution rates
Describe HPLC
A small-scale, instrumental method for analysing and separating mixtures
It typically uses silica or chemically-modified silica as a stationary phase, with almost any type or combination of solvents being used as the mobile phase
The mobile phase is typically kept at a very high pressure (up to 400 atmospheres)
Why is a pre-column usually used in HPLC?
It is the same as the main column, but is smaller and cheaper so is easier to replace if something has a very strong affinity to the stationary phase
Describe the main column in HPLC
Usually made from stainless steel in order to withstand the high pressure
Typically 10-25 cm long, with an internal diameter of around 75 micrometres
What material is usually used if a polar stationary phase is required in HPLC? Why?
Silica is a common choice as the surface has many polar Si-O and O-H bonds, allowing it to strongly retain polar analytes
What material is usually used if a non-polar stationary phase is required in HPLC? Why?
Chemically modified silica is a common choice, with long carbon chains (such as C18) bonded to the surface
This allows it to strongly retain non-polar analytes by ‘dissolving’ them in the organic groups
What is the mobile phase in HPLC?
Almost any solvent, or combination of solvents, can be used as long as they are fully miscible
Buffered aqueous solutions can be used, and salts can be added
What are some common solvents in HPLC?
Hexane
Ethyl Acetate
Isopropanol
Methanol
Acetonitrile
Water
What is ‘normal phase’ HPLC?
A polar stationary phase (e.g. silica) is used with a non-polar mobile phase (e.g. hexane)
What is ‘reverse phase’ HPLC?
A non-polar stationary phase (e.g. C18-silica) is used with a polar mobile phase (e.g. water)
What is isocratic elution?
The composition of the mobile phase is set, and does not vary
What is gradient elution?
The ratio of solvents used changes over time, giving a high resolution and a quick time, making the best of both problems
What are two types of detection when using HPLC?
UVvis
HPLC-MS
What is UVvis (Ultraviolet-Visible absorption)?
It relies on analytes absorbing electromagnetic radiation (i.e. light) in the UVvis region, 200-800 nm, and is quantified in the Beer-Lambert Law
What is the equation for the Beer-Lambert Law?
A = ε x c x l
A = absorbance
ε = molar absorption coefficient (dm³ mol⁻¹ cm⁻¹)
c = concentration (mol dm⁻³)
l = path length (m, dm, cm, mm…)
What is the molar absorption coefficient?
A constant for a specified compound at a specified wavelength
It describes how much light the compound absorbs with a high number meaning that it absorbs a lot of light
What is the path length?
The distance through the solution that the light beam passes, during which it is being absorbed by the analyte
Increasing the path length will result in a higher absorbance
What are two potential problems of UVvis?
The analyte does not absorb in the UVvis region
The sensitivity of the detector may be too low (it is impractical to reliably measure values of A below 1 x 10⁻⁴)
What can we do if the analyte does not absorb in the UVvis region?
It can potentially be overcome by derivatising the analytes e.g. by attaching a group which absorbs or fluoresces
What can we do if the sensitivity of the detector is too low?
If the values of c and ε are low, the path length can be increased by using a ‘Z-cell detector’
What is a ‘Z-cell detector’?
A way of increasing path length by having the incident light coming at an angle that maximises length
What does the MS do after HPLC?
It yields molecular mass and structural data that can be analysed
What are the three main principles of Mass Spectrometry (MS)?
Ionisation
Separation of ions by mass/charge ratios
Detection
What are three methods of ionising a sample for MS?
Electron Impact (EI)
ElectroSpray Ionisation (ESI)
Matrix Assisted Laser Desorption Ionisation (MALDI)
What is the principle of EI?
Involves colliding electrons with gas-phase sample molecules, and generally produces a molecular ion (a radical cation) with numerous fragment ions
What is the principle of ESI?
A ‘soft’ ionisation technique in which ionisation takes place from the solution phase, making it useful for fragile or involatile species
It typically generates a protonated molecule MH⁺ ions, and does not generate fragment ions
What is the principle of MALDI?
A ‘soft’ ionisation technique in which ionisation takes place from a condensed phase (solution or solid), making it useful for fragile or involatile species
Typically generates MH⁺ ions and does not generate fragment ions
What are four different types of mass analysers?
Magnetic Sector
Quadrupole
Quadrupole Ion Trap (QIT)
Time of Flight (ToF)
What is the principle of Magnetic Sector analysis?
Involves passing ions through a magnetic field in which the ion light path is deflected proportionally to its m/z value
It is familiar, but is less commonly used
What is the principle of a Quadrupole mass analyser?
Ions pass between four parallel metal rods to which electric currents are applied which allows ions with selected m/z ratio to pass through
It is cheap and compact
What is the principle of QIT as a mass analyser?
Ions pass into a cavity with metal walls to which electric currents are applied causing the ions to orbit in the space
Altering the currents allows ions with selected m/z stations to be detected from the ion trap and detected
It is cheap and very compact
What is the principle of ToF as a mass analyser?
This involves accelerating ions with an electric field, and then measuring the time taken for the ions to reach a detector
It typically has an excellent mass accuracy
What are the x and y axes of an MS spectrum?
Ion intensity on the y-axis
M/Z value on the x-axis
What is the most stable ion called?
The base peak, and this sets the top of the scale at 100%
How does the stability of the ions have an impact on the spectrum?
The higher the stability, the higher the intensity as there are more of them
Where do very unstable ions appear in the spectrum?
They do not appear on the spectrum
How do isotopes impact the MS spectrum?
They leave clusters of small peaks near larger peaks
What is an isotopologue?
Molecules/ions which have the same formula, but differ in isotopic composition
Where does the intensity in the clusters arise from?
They arise from differences in the abundance of the isotopes which contribute to the formula of the ion
What are the main steps for EI ionisation?
The sample is either already in the gas phase, or is heated to volatilise it
Electrons are ejected from a heated filament and accelerated towards the electron trap
The high-energy electrons interact with the sample vapour, causing ionisation
The ionisation process creates a radical cation and two electrons
The ionised sample molecules are guided out of the ionisation chamber by the ion repeller
Why do EI spectra not always contain a peak for the molecular ion?
They typically have excess energy, leading to their fragmentation
What molecules does EI work well for?
Molecules that are relatively volatile as they need to be in the gas phase
What are the main steps for ESI?
The sample is in solution
A small volume of the solution is sprayed through a capillary maintained at a high voltage, creating droplets containing ions in solutions
The droplets evaporate under a flow of warm, inert gas ‘curtain gas’, reducing their size
As the droplets shrink, increasing electrostatic repulsion causes the ejection of ions e.g. [MH]⁺ or [MH₂]²⁺
The ions are guided out of the ionisation chamber by electrodes into the mass analyser
How much fragmentation occurs in ESI?
Very little fragmentation occurs
What substances does ESI work on?
Solutions can be used, making it useful for biomolecules such as protein and DNA fragments
This is especially useful for species that are involatile, and species that would undergo decomposition if heated
What are the main steps in MALDI?
The sample is in solution and mixed with a solution of matrix and dried onto a metal target
A laser pulse is directed at the target, vapourising the matrix and analyte - the matrix helps to prevent thermal decomposition here
A vaporised matrix molecule transfers a proton to a vapourised analyte molecule, forming [MH]⁺
The ions are guided out of the ionisation chamber by electrodes into the mass analyser
How much fragmentation occurs in MALDI?
Very little
What is MALDI suitable for analysing?
Thermolabile and involatile species, such as biomolecules and polymers
Which detection is MALDI often paired up with and why?
ToF mass detection as the ionisation time can be calculated very accurately
What is the purpose of the matrix in MALDI?
To absorb the laser radiation
What are the main features of the quadrupole mass analyser?
Separates ions by passing them through the space between four electrically-charged metal rods
It can accept and analyse a constant steam of ions
The ions move into the space between the four parallel metal rods with opposing poles having a voltages of opposite polarity
Variable AC and DC currents are applied, with the combination of these currents constraining the direction of the ion movement in a way that depends on m/z values
How does a QIT mass analyser work?
Once the ions are admitted into the ion trap, variable currents are applied to the end caps and ring electrodes, keeping the ions circulating inside the ion trap
Varying the electric currents influences the speed and direction of the ions in a way that reflects their m/z ration
Changing the currents, therefore, allows ions of a particular m/z ratio to be removed from the ion trap and detected
What is the difference between QIT and the quadrupole mass analysing technique?
It uses an electronically controlled gate which opens to accept ions from the ionisation source and then shuts, making it different from the standard quadrupole
How does the ToF mass analyser work?
Admits ions for the ionisation source, and then accelerates the ions with an electric field
The ions then enter the ‘flight tube’, where there is no electric field
In the flight tube, they drift towards the detector with a fixed speed, with the time taken to reach the detector depending on their m/z values
This gives very accurate results
What is the key equation that underpins ToF MS?
m / z = 2ev x (t / d)²
What different techniques can be used to promote fragmentation when doing ESI?
Increase the capillary voltage
Increasing the temperature of the curtain gas
Altering the curtain gas composition
Adding different additives (containing different ions) to the solution before ionisation
What is Tandem Mass Spectrometry?
The ions generated from a soft ionisation method are selected using one mass analyser, and then subsequently fragmented with the fragment ions being analysed using a second mass analyser
The fragmentation is usually achieved by Collision Induced Dissociation - accelerating the ions and colliding them with unreactive neutral species (e.g. Argon)
Why are there clusters in the mass spectrum?
Different isotopes of an element have different masses from each other, therefore impacting the values of m/z
What does monoisotopic mean?
Elements that only have one isotope
Using algebra, draw the mass spectrum for a sample of Cl₂
Assume that the ³⁵Cl : ³⁷Cl ratio is 3 : 1
(3a + b) represents the Cl chances for one atom, and so we can square this to get the algebraic ratios
This gives us a 9 : 6 : 1 ratio compared to the base peak
What is an accurate way to measure the different peak intensities when considering isotopes?
We can calculate individual isotope abundance probabilities to calculate relative peak intensities
This calculates it very accurately but is time-consuming and the level of detail is not usually required
Computers are mainly used for this work
Why does fragmentation occur?
Gas phase ions are put with a sufficient vibrational energy that they will fragment to produce different ions with a naturally occurring loss of uncharged species
What type of molecular ion does EI produce?
Radical cations
What type of molecular ion is generated in soft ionisation?
Typically protonated molecules, but can be ‘sodiated’ molecules
Which properties change the nature and intensity of ion clusters in a mass spectrum?
Mainly on the stabilities of the fragments they produce
The relative rates of the fragmentation processes
What ions are unfavourable to form?
Very small ions e.g. H⁺ and CH₃⁺
What do Even Electron (EE) species not usually fragment into?
Two Odd Electron (OE) fragments
Can fragment ions further fragment?
Yes
How can ion stabilise itself?
By undergoing a rearrangement reaction e.g. McLafferty rearrangement
Which molecules can get lost in fragmentation?
Stable, neutral molecules can get lost in fragmentation e.g. H₂O, CO₂, C₂H₄
What does an even-mass molecule usually produce?
Two odd-mass fragments
What must an odd-mass molecule produce?
An even-mass fragment, and an odd-mass fragment
What is the nominal mass number?
The mass number of element of its most abundant isotope
What is the Nitrogen rule?
If there is an odd number of nitrogen atoms in a molecule, the molecule will have an odd nominal mass
If the compound contains an even number of nitrogen molecules, the compound will have an even nominal mass
Why does the Nitrogen rule take place?
Its principal isotope has an even mass, but an odd valence
What do we know about the number of Nitrogen atoms if a molecular ion has an odd m/z value and is OE species?
It has an odd number of Nitrogen atoms
What do we know about the number of Nitrogen atoms if a molecular ion has an even m/z value, and is an OE species?
It has an even number of Nitrogen atoms
What do we know about the number of Nitrogen atoms if a soft ionisation technique is used and there is an odd m/z value?
It must contain an even number of Nitrogen atoms
What do we know about the number of Nitrogen atoms if a soft ionisation technique is used and there is an even m/z value?
It must contain an odd number of Nitrogen atoms
