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Summary Notes 3.3.6 - Organic analysis, Chromatography and NMR
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Summary Notes 3.3.6 - Organic analysis, Chromatography and NMR
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Taylor’s Notes Organic Analysis, Chromatography and NMRChromatography
Chromatography is an analytical technique that separates components in a mixture between a mobile phase and a stationary phase
Any form of chromatography uses a fixed material (the stationary phase) and moving substance (the mobile phase)
Separation occurs due to the equilibrium between the components in the mixture and the stationary and mobile phases – the degree of
movement depends upon the strength of the interaction of the material with the stationary phase and its solubility in the mobile phase –
the more attracted to the mobile phase, the further it travels
If the component is colours, it is clear how far it is moved, it can be collected, and the solvent evaporated to obtain the pure substance
If the substance is colourless, there are other ways of detecting their presence:
If colourless amino acids are present, ninhydrin can be used which is a contrast agent which makes them visible
Other organic compounds can be detected using UV light
The Four Different Types of Chromatography
Thin Layer Chromatography (TLC):
The sample is placed on a very thin support (the stationary phase) with the chromatography being carried out in a suitable solvent (the
mobile phase)
distance travelled by solute
The Rf value is used to identify substances: Rf =
distance travelled by mobile phase
The number of components present on a TLC plate can be determined by counting the number of dots
Thin-layer Chromatography Method
1. Wearing gloves, draw a pencil line 1 cm above the bottom of a TLC plate and mark spots for each sample along the line
2. Using a capillary tube, add a tiny drop of each solution to a different spot and allow the plate to air dry
3. Add solvent to a chamber or large beaker with a lid so that it is no more than 1 cm in depth
4. Place the TLC plate into the chamber, making sure that the level of the solvent is below the pencil line and then replace the lid to
get a tight seal
5. 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 before allowing the plate to dry in the fume cupboard
6. Place the plate under a UV lamp in order to see the spots and draw around them lightly with a pencil
7. Calculate the Rf values of the coloured spots
Safety and practical technique:
Always wear plastic gloves to prevent contamination from the hands to the plate
The pencil line will not dissolve in the solvent
Make sure a tiny drop of the solution is added as too big a drop will cause different spots to merge
If the solvent is too deep, it will dissolve the sample spots from the plate
The lid prevents evaporation of a toxic solvent
A more accurate result can be calculated if the solvent is allowed to rise to near the top of the plate, but the Rf value can be
calculated if the solvent front does not reach the top of the plate
Dry in a fume cupboard as the solvent is toxic
A UV lamps are used if the spots are colourless and not visible
Column Chromatography
The sample is placed on the top of an absorbent solid (the stationary phase) and a suitable solvent (the mobile phase) is then run down the
column – the components in the reaction mixture pass down the column at different speeds and are collected one at a time
Column Chromatography Method
1. A glass tube is filled with the stationary phase, usually silica or alumina in powder form to increase the surface area
2. A filter or plug is used to retain the solid in the tube and a solvent is added to cover the powder
3. The mixture to be analysed is dissolved in a minimum of a solvent and added to the column
4. A solvent or mixture of solvents is then run though the column
5. The time for each component in the mixture to reach the end of the column (retention time)
Separation by column chromatography depends on the balance between solubility in the moving phase and
retention in the stationary phase
HPLC – High Performance (pressure) Liquid Chromatography
The sample is dissolved in an inert solvent and passed over the stationary phase using high pressure – volatile molecules move quickly
along with the solvent and come out of the HPLC first and the less volatile molecules come out last, a detector gives a peak for each
molecule that comes out with the time taken for the molecule to come out being the retention time (the more volatile the compound/the
lower boiling point, the lower the retention time) – the area under the peak recorded is proportional to the amount of compound present
, Taylor’s Notes Organic Analysis, Chromatography and NMR
Gas Chromatography
This is very similar to HPLC, but the sample is heated, and an inert gas (nitrogen) is passed over the stationary phase
This form of chromatography can be used to separate mixtures of volatile liquids
The time taken for a particular compound to travel from the injection of the sample to where it leaves the column to the detector is known
as its retention time, however, some compounds have similar retention times so they will not be distinguished – basic gas liquid
chromatography will tell us how many components there are in the mixture by the number of peaks as well as telling us the abundance of
each substance
It is also possible for gas-liquid chromatography machines to be connected to mass
spectrometers, IR or NMR machines which enables all the components in a mixture to be
identified
Most commonly, a mass spectrometer is combined with GC to generate a mass spectra which
can be analysed or compared to a database by a computer for positive identification of each
component in the mixture – this is called a GC-MS machine which is used in analysis, in
forensics, environmental analysis, airport security and space probes
Nuclear Magnetic Resonance Spectroscopy (NMR)
Some nuclei have ‘spin’ (1H and 13C) although many do not 12C, any nucleus with spin generates a magnetic field – when a nucleus with spin
is placed in a magnetic field, the field generated by the nucleus aligns with or against the magnetic field
There is a small difference in energy between the two alignments which corresponds to the energy or radio waves – this means that if radio
waves are passed through the substance, some frequencies of radio waves are absorbed in ‘flipping’ the nuclei from one spin direction to
the other, therefore, this characteristic absorption allows chemists to determine important information about nuclei in a structure and
their surrounding nuclei
The Solvent in NMR
Solvents with 1H atoms in their structure are generally avoided in NMR spectroscopy because the 1H atoms can interfere with the scan
Therefore, solvents often contain deuterium (D or 2H) instead of 1H atoms as D atoms have no spin and so won’t be visible in an NMR scan –
deuterium is an isotope of hydrogen as it has an extra neutron which 1H does not have
The typical solvents used in NMR are CCl4, CDCl3 AND C6D6 – CCl4 and C6D6 are non-polar and so are
used to dissolve non polar substances whereas CDCl3 is polar and so is used to dissolve polar
substances
Calibration
To calibrate an NMR spectrum, a small amount of a standard is added which produces a signal which
can be used to compare other peaks
The most common standard is tetramethylsilane (TMS) because it is non-toxic, inert and has a low BP
because it has dispersion forces
1
H NMR Spectroscopy
Chemical Shift
NMR spectra are recorded on a scale known as chemical shift (), which is how far from the signal is away from the signal for TMS – this is
measured in parts per million (ppm)
For 1H NMR, the signals usually fall between 0 and 10 ppm away from the signal for TMS which is defined as being = 0 ppm
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