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Revision on chromatography and mass spectrometry
1. REVISION
ChromatographyMass spectrometry
2.
CHROMATOGRAPHYWhat is Chromatography?
a technique for separating and identifying the components of a mixture.
Mobile phase?
Moves and pushes
components
Paper
Solvent
Thin layer
Solvent
Gas-liquid
Inert gas
Stationary phase ?
Holds components
and causes
separation
H2O on
cellulose
Silica gel or
Aluminium
oxide
Non-volatile
liquid (either
polar or nonpolar)
3. Thin Layer Chromatography (TLC)
Saturday, 16 May 2026Thin Layer Chromatography (TLC)
Thin layer chromatography allows us to separate and identify compounds.
TLC uses a stationary phase of silica or alumina mounted on a
glass/metal plate. A pencil base line is drawn and drops of mixtures
added.
Place the plate in a 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.
It works by the 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.
We can identify the chemicals using the positions on the
chromatogram
Prevents solvent evaporating
Stationary Phase
(silicon dioxide or
aluminium oxide)
Mobile Phase (a
liquid solvent)
4. Thin Layer Chromatography (TLC)
Saturday, 16 May 2026Thin Layer Chromatography (TLC)
Colourless compounds can be seen using iodine or fluorescent dyes and UV
light
Fluorescent dyes and UV light
Adding a fluorescent dye to the 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.
Iodine
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.
The iodine vapour is
known as a locating
agent.
Iodine crystals
5. Calculating Rf values
Saturday, 16 May 2026Calculating Rf values
Compounds can be identified by calculating the Rf value from a
chromatogram.
The number of spots on the plate tells you how many
chemicals make up the mixture.
The chemicals can be identified by calculating the Rf
value and comparing these to a library of known Rf
values.
Rf = distance travelled by spot
distance travelled by solvent
Rf values are fixed for each chemical. HOWEVER – this
changes if the temperature, solvent or make up of the
TLC plate changes.
Solvent Front
Distance
travelled by the
spot
Base Line
Distance
travelled by the
solvent
6. Retention Time, Rt is the time taken by the component to travel from injection to detector
Retention factor, RF, is a ratio ofthe distance travelled by the
center of the spot to the distance
simultaneously travelled by the
mobile phase:
Rf = b|a
Retention Time, Rt is the time
taken by the component to travel
from injection to detector
Longer time in the column
shorter time in
the column
What are the factors that affect the retention time in a
gas-liquid chromatography?
Nature of sample, rate of gas flow, length of
column, temperature
7. Column Chromatography
Saturday, 16 May 2026Column Chromatography
Column chromatography is ideal for separating and purifying larger
quantities of a mixture.
TLC is useful for separating tiny quantities but it can’t be used to
separate larger volumes. Column chromatography is used instead.
A burette or another glass column is packed with silica or alumina
which is the stationary phase (just like in TLC).
Pure substances
being separated
The mixture and solvent (mobile phase) is run through the
column. The solvent is run through continuously.
The different compounds in the mixture run through the column
at different rates. This means they come out the bottom of the
column at different times.
As you have separated the chemicals you now have pure
chemicals
Each pure
substance can
be collected
separately.
8. Schematic of Gas Chromatograph
9. Gas Chromatography (GC)
Saturday, 16 May 2026Gas Chromatography (GC)
Gas chromatography is useful to separate a mixture of liquids that are
volatile and hence can be identified
In GC you have a very thin column that is wound up inside an
oven to save space. The column is lined with a solid or viscous
liquid (e.g. oil) that acts as the stationary phase.
The sample is injected into the machine and carried by an inert
gas (e.g. nitrogen) which is the mobile phase.
Each substance takes a different amount of time
to travel through the column and reach the
detector. The length of time it takes is called the
retention time.
The time it takes for the sample to travel through
varies as some molecules spend more time stuck
to the stationary phase and some spend more
time travelling in the mobile phase.
10. Gas Chromatography (GC)
Saturday, 16 May 2026Gas Chromatography (GC)
GC spectra show peaks of varying sizes and appearing at different times
Retention Time
Detector Response
Each peak in the spectra
represents a different
substance and each substance
has a different retention time.
We can compare retention
times with a library of known
substance times to help identify
substances in the mixture.
The area under the peaks tell us the amount of each substance.
The larger the area the more substance.
0
1
2 3 4 5
Time (mins)
6
7
8
GC can also be used to identify volatile compounds in
oil paints such as esters. Useful for restoration
experts of antique paintings.
GC is used to detect
the amount of alcohol
in urine and blood
which can be used as
evidence in court as
the results are reliable.
11. Gas Chromatography – Mass Spectrometry (GC-MS)
Saturday, 16 May 2026Gas Chromatography – Mass Spectrometry (GC-MS)
We can combine gas chromatography and mass spectrometry together to
help identify a mixture of substances
Mass spectrometry is better suited to identifying
unknown compounds via their mass to charge ratio
(m/z). However when analysing a mixture of
compounds this can produce a confusing spectrum.
Gas chromatography is not very good at
identifying unknown compounds but is very
good at separating a mixture of substances.
Combining the 2 together (GC-MS) allows us to use the benefits of both to produce a powerful analytical
tool for chemists.
The separated compounds run through the
MS machine which produces spectra to
identify the individual substances.
The mixture of substances is fed through the GC
machine and this separates them out however instead
of a detector it feeds through into a mass spectrometer
The substances can be positively identified as the mass spectra produced are compared with a library of
spectra stored on a computer. This also makes GC-MS an efficient process.
12.
MASS SPECTROMETRY• First the sample is vaporized then ionized by being bombarded
by high-energy electrons to produce positive ions.
• These pass on through a magnetic field where they are
deflected and separated according to m/z (mass : charge ratio)
and later detected.
• When a sample of propane (C3H8) is introduced into a mass
spectrometer, molecular ion of formula C3H8+. is produced.
+.
Equation C3H8(g) + e− → C3H8 (g) + 2e−
13.
FRAGMENTATION• Molecular ion fragments due to covalent bonds breaking:
[M]+. → X+ + Y.
• Much information from fragmentation patterns – can often
work out structure.
• The more stable the ion, the greater the peak intensity.
• M+1 peak is formed due to presence of C-13 isotope
• M+2 peak formed if the molecule contains Cl or Br atom
with Cl the ratio of M:M+2 peaks is 3:1
while with Br the ratio of M:M+2 peaks is 1:1
14. Mass Spectrometry
Saturday, 16 May 2026Mass Spectrometry
Mass spectrometry
is used to find the
relative molecular
mass (Mr) of a
compound
Relative Abundance
100
80
60
Peaks show fragments of the
original molecule.
The last peak is the M+ peak OR
the molecular ion peak.
This is the same as the relative
molecular mass of the molecule!
40
M+
20
0
35
45
40
50
Mass/charge OR m/z
m/z is just the mass of a
fragment divided by charge. As
most have just a +1 charge this is
the same as the fragment mass.
15. M+1 Peak
• Due to C-13 isotope organiccompounds have M+1 peak.
• The size of the M+1 peak
depends on the number of
carbon atoms in the
compound
C-12 : C-13
100 : 1.11
• Number of carbon atoms in
the organic molecule can be
calculated from the peak
heights.
M+
M+1
16.
Calculating the # of C atoms• An unknown compound has a molecular ion peak, M+ , with a relative
abundance of 54.5% and has an [M + 1]+ peak with a relative
abundance of 3.6%. How many carbon atoms does the unknown
compound contain?
17. M+2 Peaks Molecules with Heteroatoms
• If Cl is present, M+2 is one-third of M+64
66
Note that the heights are
in the ratio of 3:1
18. M+2 Peaks Molecules with Heteroatoms
• If Br is present, M+2 is almost equal to M+.108
110
Note that the heights
are in the ratio of 1:1
Bromoethane
Br-79 and Br-81
1:1
19.
Fragments for heteroatomsThe mass
spectrum of
chlorobenzene
is given below.
List the ions
responsible for
the peaks at
77, 114 and
112.
20.
butaneC4H10 [C4H10]+. + e–
m/z 58
CH3CH2CH2CH3
[C4H10]+. [CH3CH2CH2]+ + .CH3
m/z 43
[C4H10]+. [CH3CH2]+ + .CH2CH3
m/z 29
[CH3CH2CH2]+
43
• Peak with greatest m/z value =
MOLECULAR ION
[CH3CH2]+
• Other peaks due to molecular
ion breaking into fragments
[C4H10]+.
29
58
10
20
30
40
50
60
m/z
70
80
90
100
110
21.
CH3CH237Cl [CH3CH237Cl]+. + e–m/z 66
chloroethane
CH3CH2Cl
CH3CH235Cl [CH3CH235Cl]+. + e–
m/z 64
28
[CH3CH237Cl]+. [CH237Cl]+ + .CH3
m/z 51
29
[CH3CH2]+
64
[CH3CH235Cl]+. [CH235Cl]+ + .CH3
[CH3CH235Cl]+.
m/z 49
[CH3CH2Cl]+. [CH3CH2]+ + .Cl
m/z 29
[CH237Cl]+
[CH235Cl]+
49
66
[CH3CH237Cl]+.
51
10
20
30
40
50
60
m/z
70
80
90
100
110