Topic 15E - Spectroscopy and Chromatography - PART I

Spectroscopy and Chromatography
Title: Simple chromatography

C Grade: know that chromatography separates components of a mixture using a mobile phase and a stationary phase
B Grade: calculate Rf values from one-way chromatograms in paper and thin-layer chromatography (TLC)
A Grade: understand the reasons for differences in R f values
Chromatography
Chromatography
How does chromatography work?
 All forms of chromatography work on the same principle.

 They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile
phase (a liquid or a gas).
 Each compound in the mixture is attracted to both phases, but more strongly to one
than the other:
 A compound that is weakly attracted to the stationary phase but strongly
attracted to the mobile phase will travel a long way
 A compound that is strongly attracted to the stationary phase but weakly
attracted to the mobile phase will not travel a long way
Chromatography
 Paper chromatography
 Thin layer chromatography (TLC)
 Column chromatography
 High performance liquid chromatography (HPLC)
 Gas chromatography (GC)

Paper Chromatography
 Stationary phase: the water trapped in the fibres of the chromatography paper
 Mobile phase: the solvent
The reason for

covering the beaker is
to make sure that the
atmosphere in the
beaker is saturated
with solvent vapour
(stops the solvent
from evaporating as it
rises up the plate).
Paper Chromatography
 Most organic compounds are colourless! Chromatography can still be used, but the
components on the chromatogram must be visualised using ultraviolet radiation
(UV light) or by spraying with a locating agent (ninhydrin) that will react with the
component to form a coloured product.
 If you have synthesised na organic compound, chromatography is a good way to

check whether it is pure and, if not, what impuruties are present
Thin layer chromatography (TLC)
 Stationary phase: sheet of glass or plastic coated in a thin layer of a solid such
as silica or alumina
The apparatus and method used are very similar to those of paper chromatography
Measuring Rf values
Measurements are often taken from the plate/paper in order to help identify
the compounds present. These measurements are the distance travelled
by the solvent, and the distance travelled by individual spots
Measuring Rf values
If you could repeat this experiment under exactly the same conditions, then
the Rf values for each dye would always be the same.
However, if anything changes (the temperature, the exact composition of

the solvent, and so on), that is no longer true.
Column chromatography
 Stationary phase: silica or alumina packed into a tube (e.g. burette) and soaked in a solvent
Mixture is placed on top of the stationary phase and

more solvent (the mobile phase) added on top
Advantage: larger quantities of material can be separated

Suggest a disadvantage
DNA
Page 135
Title: Determining structure using mass spectra
Be able to use data from mass spectra to:
C Grade: know calculate accurate Mr and suggest possible structures of a simple organic compound
Mass spectrometry
High resolution mass spectrometry (HRMS)
In AS you have learned about 2 uses of Mass Spectrometry:
 Determine the relative atomic masses of elements from isotopic abundances
 Use fragmentation patterns to determine the structures of organic compounds
Determining the molecular formula (although not always the structure or

identity) of na organic compound from a precise relative molecular mass
Uses: forensic and environmental analysis


Mass spectrometers can provide a value for Mr to 4 or more decimal places
Values with this degree of precision sometimes enable a compound to

be positively identified from the relative molecular mass alone

DNA Page 137

Title: HPLC and GC
B Grade: know that HPLC and GC are types of column chromatography that separates substances because of different
retention times in the column and may be used in conjunction with MS, in applications such as forensics or drug
testing in sport
High performance liquid chromatography (HPLC)

Title: HPLC and GC
testing in sport

Title: HPLC and GC
testing in sport
Retention time: time

from injection to detection
depends on
• the nature of the solvent

• the pressure used
• the temperature inside the
column
Title: HPLC and GC
testing in sport

 Stationary phase: silica or alumina packed into a tube
• Improved form of column chromatography.

• Instead of a solvent being allowed to drip through a column under gravity, it is forced through
under high pressures of up to 400 atmospheres. That makes it much faster.
• It also allows you to use a very much smaller particle size for the column packing material
which gives a much greater surface area for interactions between the stationary phase and
the molecules flowing past it. This allows a much better separation of the components of the
mixture.
• The other major improvement over column chromatography concerns the detection methods
which can be used. These methods are highly automated and extremely sensitive.
Title: HPLC and GC
testing in sport
Normal phase HPLC
The column is filled with tiny silica particles, and the solvent is non-polar - hexane, for
example. A typical column has an internal diameter of 4.6 mm (and may be less than
that), and a length of 150 to 250 mm.
Polar compounds in the mixture being passed through the column will stick longer to the
polar silica than non-polar compounds will. The non-polar ones will therefore pass more
quickly through the column.
Title: HPLC and GC
testing in sport

Reversed phase HPLC
In this case, the column size is the same, but the silica is modified to make it non-polar by
attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms in
them. A polar solvent is used - for example, a mixture of water and an alcohol such as
methanol.
Polar molecules in the mixture will spend most of their time moving with the solvent.
Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups
because of van der Waals dispersion forces. They will also be less soluble in the solvent
because of the need to break hydrogen bonds as they squeeze in between the water or
methanol molecules, for example. They therefore spend less time in solution in the solvent and
this will slow them down on their way through the column.
That means that now it is the polar molecules that will travel through the column more quickly.
Reversed phase HPLC is the most commonly used form of HPLC.

Title: HPLC and GC
testing in sport
Do now Page 141

Topic 15E - Spectroscopy and Chromatography - PART I

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Topic 15E - Spectroscopy and Chromatography - PART I

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Spectroscopy and Chromatography

Title: Simple chromatography

How does chromatography work?

 All forms of chromatography work on the same principle.

 Thin layer chromatography (TLC)

 High performance liquid chromatography (HPLC)

 Gas chromatography (GC)

The reason for

 If you have synthesised na organic compound, chromatography is a good way to

Thin layer chromatography (TLC)

However, if anything changes (the temperature, the exact composition of

Mixture is placed on top of the stationary phase and

Advantage: larger quantities of material can be separated

High resolution mass spectrometry (HRMS)

In AS you have learned about 2 uses of Mass Spectrometry:

 Determine the relative atomic masses of elements from isotopic abundances

 Use fragmentation patterns to determine the structures of organic compounds

High resolution mass spectrometry (HRMS)

Determining the molecular formula (although not always the structure or

Uses: forensic and environmental analysis

High resolution mass spectrometry (HRMS)

Values with this degree of precision sometimes enable a compound to

High resolution mass spectrometry (HRMS)

DNA Page 137

High performance liquid chromatography (HPLC)

High performance liquid chromatography (HPLC)

High performance liquid chromatography (HPLC)

Retention time: time

• the nature of the solvent

High performance liquid chromatography (HPLC)

• Improved form of column chromatography.

High performance liquid chromatography (HPLC)

Normal phase HPLC

High performance liquid chromatography (HPLC)

Reversed phase HPLC is the most commonly used form of HPLC.

Do now Page 141

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