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1 Thin-Layer Chromatography
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Thin Layer Chromatography (TLC) is a technique used to analyse small samples via
separation
o For example, we could separate a dye out to determine the mixture of dyes in a
forensic sample
There are 2 phases involved in TLC - stationary phase and mobile phase
Stationary phase
o This phase is commonly thin metal sheet coated in alumina (Al 2O3) or silica
(SiO2)
o The solute molecules adsorb onto the surface
o Depending on the strength of interactions with the stationary phase, the
separated components will travel particular distances through the plate
o The more they interact with the stationary phase, the more they will 'stick' to it
Mobile phase
o Flows over the stationary phase
o It is a polar or nonpolar liquid (solvent) or gas that carries components of the
compound being investigated
o Polar solvents - water or alcohol
o Non-polar solvents - alkanes
If the sample components are coloured, they are easily identifiable
We can examine the plate under UV light using ninhydrin to identify uncoloured
components
Step 1:
Step 2:
On a TLC plate, draw a horizontal line at the bottom edge (in pencil)
Step 3:
Place a spot of pure reference compound on the left of this line, then a spot of the
sample to be analysed to the right of the baseline and allow to air dry
The reference compounds will allow identification of the mixture of compounds in the
sample
Step 4:
Place the TLC plate inside the beaker with solvent - making sure that the pencil
baseline is lower than the level of the solvent - and place a lid to cover the beaker
The solvent will begin to travel up the plate, dissolving the compounds as it does
Step 5:
As solvent reaches the top, remove the plate and draw another pencil line where the
solvent has reached, indicating the solvent front
The sample’s components will have separated and travelled up towards this solvent
front
A dot of the sample is placed on the baseline and allowed to separate as the mobile
phase flows through the stationary phase; The reference compound/s will also move
with the solvent
Rf values
Exam Tip
The baseline on a TLC plate must be drawn in pencil. Any other medium would interact with
the sample component and solvents used in the analysis process.
Interpreting & Explaining Rf Values in TLC
Retention times
Peaks represent different molecules from the sample - each roughly taking the shape of a
triangle
The area under each peak is the relative concentration of each component (the peak
integration value)
If the area under each peak is very small or too difficult to decipher, the height of peaks
are used for further analysis
To find the area under each peak, treat each peak as a triangle - see the examples shown
using blue triangles in the diagram
Percentage composition of a mixture
Retention time is the time taken for a sample molecule to travel through the column, from
the time it is inserted into the machine to the time it is detected
Molecules in the gaseous mixture travel at different rates, therefore giving rise to
different retention times
Longer retention times are associated with:
o Non-polar components in the mixture
o They are more attracted to the non-polar liquid in the stationary phase
o So non-polar molecules travel slower through the column
Shorter retention times are associated with:
o Polar components in the mixture that prefer to interact with the carrier gas
o They are less attracted to the non-polar liquid in the stationary phase
o So polar molecules travel faster through the column
o These molecules may have lower boiling points, therefore are vapourised more
readily
Chemical shift values (relative to the TMS) for 13C NMR analysis table
C NMR spectrum displays sharp single signals – there aren’t any complicated spitting
13
NMR spectra shows the intensity of each peak against their chemical shift
The area under each peak gives information about the number of protons in a particular
environment
The height of each peak shows the intensity/absorption from protons
A single sharp peak is seen to the far right of the spectrum
o This is the reference peak from TMS
o Usually at chemical shift 0 ppm
A low resolution 1H NMR for ethanol showing the key features of a spectrum
Molecular environments
1
H NMR peak splitting patterns table
8.1.6 Use of Tetramethylsilane (TMS)
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TMS gives a single sharp peak on the NMR spectrum and is given a value of zero
The molecular formula of TMS is Si(CH3)4
o There are 12 hydrogens in this molecule
o All of the protons are in the same molecular environment. Therefore gives rise to
just one peak
o This peak has a very high intensity as it is accounting for the absorption of energy
from 12 1H nuclei
When samples are analysed through NMR spectroscopy, they must be dissolved in a
solvent
Tetramethylsilane (TMS) is a commonly used solvent in NMR
Despite TMS showing one sharp reference peak on NMR spectra, the proton atoms can
still interfere with peaks of a sample compound
To avoid this interference, solvents containing Deuterium can be used instead
o For example CDCl3
o Deuterium (2H) is an isotope of hydrogen (1H)
Deuterium nuclei absorb radio waves in a different region to the protons analysed in
organic compounds
Therefore, the reference solvent peak will not interfere with those of the sample
When interpreting 1H NMR spectra of amines and amides, the same exchanging
phenomenon can be seen
Protons of these functional groups exchanging leads to changes in their chemical shift
ranges
This table shows the range of chemical shifts for -OH and -NH- protons
Their surrounding molecular environment has a direct impact on this range
The range of chemical shifts for -OH & -NH- protons table
Using D2O
Deuterium oxide (D2O) can be added to correctly identify -OH and -NH- protons
Adding a small quantity of this solvent ‘removes’ the peaks from the spectrum
The -OH proton and the -NH proton both undergo the same exchanging process as seen
before. This time with a deuterium atom (D) of D2O