8.1.1 Thin-Layer Chromatography
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Thin Layer Chromatography: Basics
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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 (Al2O3) 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
Conducting a TLC analysis
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Step 1:
Prepare a beaker with a small quantity of solvent
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Step 2:
On a TLC plate, draw a horizontal line at the bottom edge (in pencil)
This is called the baseline
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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
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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
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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
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A TLC plate can be used to calculate Rf values for compounds
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These values can be used alongside other analytical data to deduce composition of
mixtures
Rf values can be calculated by taking 2 measurements from the TLC plate
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
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The less polar components travel further up the TLC plate
o Their Rf values are higher than those closer to the baseline
o They are more soluble in the mobile phase and get carried forwards with the
solvent
More polar components do not travel far up the plate
o They are more attracted to the polar stationary phase
The extent of separating molecules in the investigated sample depends on the
solubility in the mobile and stationary phases
Knowing the Rf values, of compounds being analysed, helps to compare the polarity of
various molecules
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8.1.2 Gas/Liquid Chromatography: Basics
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Gas/Liquid Chromatography: Basics
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Gas-Liquid Chromatography (GLC) is used for analysing:
o Gases
o Volatile liquids
o Solids in their vapour form
The stationary phase:
o This method uses a column for the stationary phase
o A non-polar, long-chain, non-volatile hydrocarbon with a high boiling point is
mounted onto a solid support
o Small silica particles can be packed into a glass column to offer a large surface
area
o Sample gas particles travel through this phase and are able to separate well due to
the large surface area
The Mobile phase
o An inert carrier gas (eg. Helium, Nitrogen) moves the sample molecules through
the stationary phase
Retention times
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Once sample molecules reach the detector, their retention times are recorded
o This is the time taken for a component to travel through the column
The retention times are recorded on a chromatogram where each peak represents a
volatile compound in the analysed sample
Retention times are then compared with data book values to identify unknown molecules
A gas chromatogram of a volatile sample compound has six peaks. Depending on each
molecule’s interaction with the stationary phase, each peak has its own retention time
8.1.3 Interpreting Rf Values in GL
Chromatography
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Interpreting Rf Values in GL Chromatography
Features of a gas-liquid chromatogram
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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)
Area under the peak = ½ x base x height
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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
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We can calculate the amount of a particular molecule in a sample by using an expression
If a chromatogram shows peaks for alcohols A, B, C and D, to calculate the %
composition of alcohol C, use this expression:
Explain Retention Times
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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
8.1.4 Interpreting & Explaining Carbon-13
NMR Spectroscopy
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Interpreting & Explaining Carbon-13 NMR Spectra
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Nuclear Magnetic Resonance (NMR) spectroscopy is used for analysing organic
compounds
Atoms with odd mass numbers usually show signals on NMR
o For example isotopes of atoms
o Many of the carbon atoms on organic molecules are carbon-12
o A small quantity of organic molecules will contain the isotope carbon-13 atoms
o These will show signals on a 13C NMR
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In 13C NMR, the magnetic field strengths of carbon-13 atoms in organic compounds are
measured and recorded on a spectrum
Just as in 1H NMR, all samples are measured against a reference compound –
Tetramethylsilane (TMS)
On a 13C NMR spectrum, non-equivalent carbon atoms appear as peaks with different
chemical shifts
Chemical shift values (relative to the TMS) for 13C NMR analysis table
Features of a 13C NMR spectrum
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C NMR spectrum displays sharp single signals – there aren’t any complicated spitting
pattern as seen with 1H NMR spectra
The height of each signal is not proportional to the number of carbon atoms present in a
single molecular environment
CDCl3 is used as a solvent to dissolve samples for 13C NMR
o On spectra, a single solvent peak appears at 80 ppm caused by 13C atoms in the
CDCl
o This can be ignored when interpreting 13C spectra
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Identifying 13C molecular environments
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On an organic molecule, the carbon-13 environments can be identified in a similar way to
the proton environments in 1H NMR
For example propanone
o There are 2 molecular environments
o 2 signals will be present on its 13C NMR spectrum
There are 2 molecular environments in propanone
The 13C NMR of propanone showing 2 signals for the 2 molecular environments
8.1.5 Proton (1H) NMR Spectroscopy
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Interpreting & Explaining Proton (¹H) NMR Spectra
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Nuclear Magnetic Resonance (NMR) spectroscopy is used for analysing organic
compounds
Atoms with odd mass numbers usually show signals on NMR
In 1H NMR, the magnetic field strengths of protons in organic compounds are measured
and recorded on a spectrum
Protons on different parts of a molecule (in different molecular environments) emit
different frequencies when an external magnetic field is applied
All samples are measured against a reference compound – Tetramethylsilane (TMS)
o TMS shows a single sharp peak on NMR spectra, at a value of zero
o Sample peaks are then plotted as a ‘shift’ away from this reference peak
o This gives rise to ‘chemical shift’ values for protons on the sample compound
o Chemical shifts are measured in parts per million (ppm)
Features of a NMR spectrum
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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
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Hydrogen atoms of an organic compound are said to reside in different molecular
environments
o Eg. Methanol has the molecular formula CH3OH
o There are 2 molecular environments: -CH3 and -OH
The hydrogen atoms in these environments will appear at 2 different chemical shifts
Different types of protons are given their own range of chemical shifts
Chemical shift values for 1H molecular environments table
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Protons in the same molecular environment are chemically equivalent
Each peak on a NMR spectrum relates to protons in the same environment
Low resolution 1H NMR
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Peaks on a low resolution NMR spectrum refers to molecular environments of an organic
compound
o Eg. Ethanol has the molecular formula CH3CH2OH
o This molecule as 3 separate environments: -CH3, -CH2, -OH
o So 3 peaks would be seen on its spectrum at 1.2 ppm (-CH3), 3.7 ppm (-CH2) and
5.4 ppm (-OH)
A low resolution NMR spectrum of ethanol showing 3 peaks for the 3 molecular environments
High resolution 1H NMR
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More structural details can be deduced using high resolution NMR
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The peaks observed on a high resolution NMR may sometimes have smaller peaks
clustered together
The splitting pattern of each peak is determined by the number of protons on
neighbouring environments
The number of peaks a signal splits into = n + 1
(Where n = the number of protons on the adjacent carbon atom)
High resolution 1H NMR spectrum of Ethanol showing the splitting patterns of each of the 3
peaks. Using the n+1, it is possible to interpret the splitting pattern
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Each splitting pattern also gives information on relative intensities
o E.g. a doublet has an intensity ratio of 1:1 – each peak is the same intensity as the
other
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In a triplet, the intensity ratio is 1:2:1 – the middle of the peak is twice the
intensity of the 2 on either side
H NMR peak splitting patterns table
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8.1.6 Use of Tetramethylsilane (TMS)
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Use of Tetramethylsilane (TMS)
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In NMR spectroscopy, Tetrametylsilane (TMS) is used as a reference compound
The organic compound is dissolved in TMS before being introduced to the magnetic field
of the spectrometer
It is an ideal chemical to use as a reference
o TMS is inert and volatile
o This reduces undesirable chemical reactions with the compound to be analysed
o It also mixes well with most organic compounds
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
Tetramethylsilane (TMS) – Si(CH3)4
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When peaks are recorded from the sample compound, they are measured and recorded by
their shift away from the sharp TMS peak
This gives rise to the chemical shift values for different 1H environments in a molecule
H NMR spectrum for TMS showing it’s signal at 0 ppm
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8.1.7 Deuterated Solvents in Proton NMR
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Deuterated Solvents in Proton NMR
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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
Use of D20 in Identifying O-H & N-H Protons
Identifying -OH & -NH signals on a 1H NMR spectrum
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The proton in -OH is not affected by its neighbouring molecular environments
As a result, the hydrogen atom of -OH group appears as a singlet
This is due to the hydrogen atom readily exchanging with hydrogens atoms of water
molecules or any acid that may be present
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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
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The range of chemical shifts for -OH & -NH- protons table
Using D2O
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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