Lecture 3 - Chromatography Flashcards
Chromatography
Chromatography is a physical method of separation in which the components to be separated are distributed between 2 phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a distint direction.
It is an analytical and preparative technique
The mobile phase moves relative to a stationary phase in a defined direction. Components have different affinities for mobile and stationary phase resulting in separation
Requirements of a chromatographic system
Stationary phase - Solid/gel/immobilised liquid held by support matrix
Chromatographic bed - Configuration containing stationary phase e.g. column or plate in the lab usually have aluminium or plastic for TLC and for gas chromatography its the tubing
Mobile phase
Liquid or gas which carries sample through stationary phase (acts as a solvent)
Mobile phase delivery system
Passes the mobile phase through chromatographic bed Eg for TL. its a capillary tube
Detection system
Allows visualisation of the analytes
Light source / ionisation / mass spec
Chromatography separations
Chromatography is subdivided according to mechanism of interaction of solute with stationary phase
Adsorption: silica and alumina are the main 2 materials to give adsorption chromatography
Partition: Most widely used - partitioning between 2 (l) phases. Solid support - silica sphere and then stationary phase is bound onto the surface - usually a long hydrocarbon phase
Ion-exchange: Used to separate ions - eg cations stick to a negative plate whereas the neutral solute and anions pass through etc negative charge anion resin
Size exclusion: polymer analysis. Small polymers will get sucked into the pores of the resin.
Gel permeation
Affinity: Enzyme immobilised on stationary phase, antigen / antibody interaction
What are the 2 chromatography modes
Normal phase
- polar stationary phase eg silica gel or alumina
- non-polar mobile phase eg hexane, ethyl acetate
Something that has a low affinities to stationary phase will have a low retention time eg in this case something non polar
Reversed phase
- stationary phase is non-polar eg C18, C8, C4 hydrocarbon chains
- mobile phase is polar eg methanol
Will give revered order to compare to normal
Elution techniques
Isocratic
- a single solvent (or solvent mixture) used through analysis
-solvent composition constant through analysis eg same % make up of each solvent in mixture
Gradient
- solvent composition changes over time so all species are eluded from column in reasonable time
- uses tow or more solvents that vary in polarity
- solvent ratio varied in a programmed way eg high % of solvent A and low % of solvent B at start, then low % of solvent A and high % of solvent B at end ( if solvent A is less polar it would be normal phase)
TLC
Used for reaction monitoring and method development for purification by column chromatography
Helps to decide if something needs to be quenched
Inert plate (chromatography bed) is coated in a thin layer of stationary phase - make sure not to touch as that would contaminate
Mobile phase ascends the plate by capillary action
Equipment:
Glass beaker with glass lid containing solvent reservoir at bottom, spotted mixture on plate on pencilled line
TLC examples for stationary phase
What is the sheet made of?
Usually aluminium or plastic sheet with a silica coating
Silica is a stationary phase containing Si-OH groups (which is polar) therefore polar molecules elute slowly as interact strongly with silica. Lipophilic molecules interact weakly and elute quickly.
TLC method
Cut TLC to size
5 cm long and wide enough for 0.5 cm between spots
Draw a line with a soft pencil 0.5 cm from the end of the plate
Dissolve the sample in a volatile solvent (dichloromethane)
Use a glass capillary to spot the sample on to the baseline, keep the spots as small as possible
When analysing a reaction mixture, it should be run in comparison with the starting material and a co-spot should also be run
Place the TLC plate in a tank lined with filter paper containing the chosen solvent system (ca. 0.3 cm depth)
Allow the solvent to rise up the TLC plate until it is about 0.3 cm from the top, then remove it and mark the level of the solvent front
TLC solvent system
Relative distance travelled up plate also depends on the polarity of the solvent system
Compounds travel further up the plate if a more polar solvent is used as the mobile phase
Ranking solubility in water of solvents
From least soluble to most
Heptane, hexane, cyclohexane, toluene , carbon tetrachloride, M ethyl-t-butyl ether, benzene, diethyl ether, dichloromethane, 1,2-dichloroethane, iso-popanol, tetrahydrofuran, chloroform, ethyl acetate, acetone, methanol, ethanol
Use literature values and trial-and-error in order to work out best mix of solvents. Usually 1 polar and 1 non-polar solvent
TLC visualisations: line 1 is visualisation technique and line 2 is what it visualises
UV light - conjugated systems
Iodine - unsaturated systems
Ninhydrin - amino acids
Dinitrophenylhydrazine (DNP) - aldehydes and ketones
Vanillin - general stain, giving a range of colors for diff spots
Phosphomolybdic acid (PMA) stain - universal stain, all compounds appear as dark green spots on heating
Retardation/ retention factor - Rf
Formula
Rf = (distance from center of spot to baseline) / (distance from baseline to solvent front)
Remember to quote the solvent system and visualisation method
Should ALWAYS get a value below 1
Preparative column chromatography - diagram on slide 20
Purification technique using stationary phase (usually
SiO2) packed in glass tube
Mobile Phase is determined by TLC
Difference of Rf value 0.3 required (usually 20% error in Rf value)
- Put a glass wool stopper down in the bottom of the
column - not rammed in or too loose - Half-fill with solvent - the solvent composition has already been worked out in advance - use a solvent system that gives the best separation of components on TLC
- Add a 1 cm layer of sand
- Add the silica/alumina (~25g of absorbent per 1g of sample) and while it settles gently agitate the column
- When settled add a 1 cm layer of sand to the top to have a level surface
Never allow the silica to become dry
pack dewn- no air bmobus - homogeneous - Stop clock to control flow
How to load the column in PCC
- Run the solvent down to just above the level of sand
- Take the crude product and dissolve in a minimum amount of the solvent and gently pipette the solution down the side of the column
- Open the tap and let the solvent run out until the solvent level is just above the sand - not dry !
- Add solvent to the top of the column
Types of solvent systems for PCC
- Isocratic eg same solvent composition throughout
Is slower and uses more solvent - Gradient
Gradually increase the % of the polar solvent until all components have eluted
Generally faster but risk of products co-eluting
Remember solvent system should be chosen so product should have a Rf value of approx 0.3 by TLC
PCC running the column
- Open the tap and collect the elutant in fractions
- Sit there all afternoon and collect lots of fractions ! (test
tubes/sample bottles/conical flasks) - Analyse each fraction by TLC using the same solvent system.
- Combine the fractions that consist of the same component and evaporate the solvent to leave a pure product
- If solid recrystallise etc….
Remember to keep small amount of crude material
Flash column chromatography
- same set up and loading procedure as for gravity column chromatography
- solvent is forced through pressurised air/bellows
Top has a rotaflow valve to regulate pressure
Flash adaptor for connection to bellows or low pressure pump
Connects to reservoir
Tubing with sand etc and cotton wall at bottom
Tap at very bottom
Automated flash chromatography
Uses a computer with detector response (y axis) and time (x axis) as well as a UV spectrum
It comprises of a solvent pump, inline detector, fraction collector, various column chemistries
It saves time solvent and stress
