3.3.16 - chromatography Flashcards
thin layer chromatography (TLC) allows us too…
separate and identify unknown compounds
stationary phase of TLC and mobile phase
Stationary phase - doesn’t move - silica or alumina mounted on a glass/metal plate
a pencil base line is drawn and drops of mixtures added
Mobile phase - moves - solvent or eluent
what is done with stationary phase of TLC
placed in the solvent - the base line must be above the solvent level
leave until solvent has moved up to near the top of the plate
remove, mark the solvent front and allow to dry
how does TLC work
works by mixture spots dissolving in the solvent
some chemicals in the mixture may not dissolve as much and stick to the stationary phase quickly
what we are left with is a chromatogram
how can we identify chemicals
using their positions on the chromatogram
how do we see a colourless compound/ spot on a chromatogram
colourless compounds can be seen using iodine or fluorescent dues and uv light
how are fluroscent dyes and uv light used to see colourless compounds
adding a flurosecent dye to silica/alumina can be seen using a uv lamp
the colourless spots on the chromatogram will block any glow from the fluorescent dye
you can then draw round these spots to mark where they are
how is iodine used to see colourless compounds
place the chromatogram in a sealed jar with a few iodine crystals
the iodine vapour sticks to the chemicals on the plate dying them purple
iodine vapour when using iodine to see colourless compounds is known as a…
locating agent
Size of sample in chromatography and what can be done after separation
Only small sample
After seperation pure components can be further analysed
TLC adv
Simple and quick
TLC method
a) Wearing gloves, draw a pencil line 1 cm above the bottom of a TLC plate and mark spots for each sample, equally spaced along line.
b) Use a capillary tube to add a tiny drop of each solution to a different spot and allow the plate to air dry.
c) Add solvent to a chamber or large beaker with a lid so that is no more than 1cm in depth
d) Place the TLC plate into the chamber, making sure that the level of the solvent is below the pencil line. Replace the lid to get a tight seal.
e) When the level of the solvent reaches about 1 cm from the top of the plate, remove the plate and mark the solvent level with a pencil. Allow the plate to dry in the fume cupboard.
f) Place the plate under a UV lamp in order to see the spots.
Draw around them lightly in pencil.
g) Calculate the Rf values of the observed spots.
Why use a pencil line
Won’t dissolve in solvent
Tiny drop used bc
Too big a drop will cause different spots to merge
Depth of the solvent why
If solvent to deep it will dissolve the sample spots from the plate
Why use a lid
Prevent evaporation of toxic solvent
Dry in fume cupboard bc
Solvent is toxic
Uv lamp used bc
the spots are colourless and not visible
Rf =
Distance traveled by spot
—————————————
Distance travelled by solvent
TLC works dep on the
affinity of the component molecules for the mobile phase compared to the stationary phase.
The greater the solubility of the component molecules in the solvent…
the further the component molecules move up the TLC plate.
Solubility of the component molecules depends on the …
intermolecular forces between solvent and component molecules.
After you calc Rf value you …
Compare to a known reference value to identify substance
Known Rf value must be for same solvent
2 dimensional chromatography
In order to separate a complex mixture that has components of different solubility in solvents, it may be necessary to do chromatography with two different solvents.
A spot of the mixture on a TLC plate is first separated with one solvent.
Then the TLC plate is rotated 90° and the plate is placed in a second solvent for a second
separation to take place
Elute means
When a substance leaves a chromatography column
Elutant means
This is the solvent/gas that moves the sample through the chromatography column
Column chromatography
Column chromatography is a technique for purifying individual components from mixtures. It uses a glass column filled with a solid adsorbent as the stationary phase.
How column chromatography works
- The mixture is placed at the top of the column filled with the stationary phase. - solvent and silica
- A solvent is passed through the column, carrying the mixture components at different rates.
3.Components separate based on their solubility in the solvent and their adsorption to the stationary phase. - The separated components exit the column and are collected for analysis.
Gas chromatography is
Gas chromatography (GC) separates volatile liquid mixtures into individual substances
How gas chromatography works:
- A liquid sample is vaporised in a heated chamber.
- An inert carrier gas then transports the vaporised sample through a chromatographic column containing the stationary phase, consisting of either an immobilised solid or a solid coated with a non-polar high boiling liquid.
- The column has a stationary phase that interacts differently with each component of the sample.
- Components are separated based on how they partition between the mobile gas phase and the stationary phase.
- As they exit the column, a detector records the separation, producing a chromatogram with distinct peaks for each component.
Stationary phase of gas chromatography
Liquid supported on an inert solid
Mobile phase of gas chromatography
Unreactive gas like nitrogen or Argon
Gas chromatography mass spectrometry what is it
GC-MS - GC-MS combines the separation capabilities of gas chromatography with the identification power of mass spectrometry, making it a highly effective method for analysing complex mixtures.
How GC-MS works
- The mixture is first separated by gas chromatography.
- Each component is then introduced into a mass spectrometer, where it is ionised.
- A distinct mass spectrum for each component is generated.
- Computerised matching to spectral libraries then identifies each substance based on their unique fragmentation patterns.
Retention time
time taken for the corresponding substance to travel through the chromatographic column.
Can be shown on a a graph splay peaks that represent different substances in a mixture. The position of each peak is determined by its retention time,
To identify substances using retention time
To identify the substances in the mixture, compare the retention times of the peaks with those of known standards.
To identify substances using retention time
To identify the substances in the mixture, compare the retention times of the peaks with those of known standards.
Why add developing solvent to a depth of not more than 1cm in TLC
if too deep it will dissolve the mixture from the plate
