Chromatographic

methods of separation

SARIKA CHANDOO

Cape chemistry unit 2

Objective 8
OBJECTIVES
Principles of chromatographic methods
 Chromatography- COLOUR WRITING
 Non-destructive separation of chemical substances by selective sorption ( solution and
adsorption)
 Analyte is passed in a dilute solution or as a vapour across a stationary material.
 Elution: Separating materials by washing a mixture.
 It is based on the principle where molecules in mixture applied onto the surface or into the
solid, and fluid stationary phase (stable phase) is separating from each other while moving
with the aid of a mobile phase.
 The factors effective on this separation process include molecular characteristics related to
adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their
molecular weights and technique used to separate the components of a mixture.
 Chromatography works by dividing the components of the mixture between two different
phases.
 We call this partitioning
 Eg. Water and hexane do not mix. They form two separate layers.
 If we mix some aqueous iodine with hexane, most of the iodine would go into the hexane
layer but some remains in the aqueous layer.
 This means that that the iodine has been partitioned between the two layers.
PHASES
 Stationary phase- solid eg silica (silicon IV oxide), alumina (
Aluminium oxide), cellulose, or liquid eg water trapped in cellulose
fibres. The stationary phase tends to hold back the components of the
mixture which are attracted to it. The greater the attraction, the
slower is the movement of the components during chromatography.
 The mobile phase can be a liquid or a gas. The greater the solubility
of a particular component in the mobile phase, the faster is the
movement of that component during chromatography.
 During chromatography, the mobile phase moves over the stationary
phase. The different components in the mixture are attracted to the
stationary phase to different extents. So some move faster than
others in the mobile phase and separation occurs.
Elution:
 Adsorption chromatography- Stationary phases is a solid.
 Adsorption is the process of forming bonds of varying strength with
a solid surface. As the mobile phase move over the solid, some of
the components are adsorbed more strongly to the solid than
others and separation occurs.
 A small volume of mixture is added in a suitable solvent to the top
of a column packed with stationary phase.

 Partition chromatography: The stationary phase is a liquid

surrounding the particles of a solid support. As the mobile phase
moves over the stationary phase, the components which are more
soluble in the liquid stationary phase will be held back and move
more slowly than those that are more soluble in the mobile phase.
This difference depends on the partition coefficient.
Types of
chromatography

 Paper chromatography

 Thin layer chromatography

 Column chromatography

 High performance liquid

chromatography

 Gas-Liquid chromatography
Experimental Conditions for
Chromatography
 Depending on the type of compounds present in the mixture, the stationary
and mobile phases must be chosen carefully.
 Standard paper chromatographic paper is cellulose that readily adsorbs polar
compounds.
 If a non polar solvent (hydrocarbon) is used, polar products will remain at the
start line and no separation will be attained.
 At the same time, in a highly polar solvent (such as water) polar products will
stay in the mobile phase and travel readily with the solvent.
 Therefore, most common solvents used for chromatography must have a
balance between polarity and non-polarity.
 Partitioning Coefficients
 The principle of partition of a solute between two solvents helps us
to understand more fully how the components in a mixture are
separated in chromatography.
 In paper chromatography the different partition coefficients of the
components in a mixture correspond to their relative solubilities in the
two solvents.
 The mobile phase is the solvent chosen. The other solvent is the
water trapped in the paper’s structure, which is the stationary phase.
 Figure 29.5 shows solute molecules partitioned between the mobile
phase and a stationary liquid phase on a solid support.
 The solutes in the mixture being separated are partitioned to different
extents between the solvents in the mobile and stationary phases.
 The greater the relative solubility in the mobile phase, the faster the
rate of movement as the mobile phase passes over the stationary
phase.
PAPER CHROMATOGRAPHY
 This is a form of partition chromatography.
 The stationary phase is water absorbed onto the cellulose of the
paper.
 The mobile phase is usually an organic liquid or a mixture of solvents.
 As the solvent moves up the paper, the components partition
themselves between the water (stationary phase) and the
solvent(mobile phase).
 Due to capillary action, the solvent rises up the paper and eventually
reaches the spots.
 The samples is partitioned between mobile and stationary phase
according to their affinities for the solvent and the chromatographic
paper.
 Higher solubility…more time in mobile phase and more up faster than
less soluble compounds with a greater tendency to adsorb on the
stationary phase.
Same/ Similar Rf values?- Two way
Chromatography
 If two or more components have similar Rf values (overlap in spots and poor separation) in a
particular solvent, the experiment can be repeated by rotating the paper through 90º and
using a different solvent , pH or separation method.
Thin layer chromatography
 This is a form of adsorption chromatography.
 Silica, alumina or cellulose (stationary phase) are made into a paste
and spread in a thin even layer over a glass or plastic plate.
 In thin-layer chromatography, referred to as TLC, the stationary
phase is a solid that adsorbs solute molecules onto its surface.
 A small spot of the mixture to be separated is put near the bottom of
the plate.
 The plate is placed in a solvent (mobile phase) and the solvent
allowed to move up the stationary phase.
 As the solvent moves up the stationary phase, the spot separates
into its components.
 Thin layer chromatography
PREOCEDURE:
The procedure is the same for both TLC and paper chromatography.
• Place a spot of the mixture to be separated on the base line.
• Dip the paper (or TLC plate) into a solvent. The solvent level must be
below the base line.
• Allow the solvent to move up the paper (or TLC plate).
• When the solvent front is near the top, mark its position
Gas- Liquid Chromatography

 This is a form of partition chromatography.

 A gaseous sample enters the chamber
 Sample must be volatile.
 The stationary phase is a high-boiling point liquid, e.g. a
long-chain hydrocarbon oil supported on a porous inert solid
such as silica or alumina.
 The mobile phase is an unreactive gas, e.g. nitrogen,
helium, argon.
 This is called the carrier gas.
 The mixture to be separated is injected into the apparatus.
 As the mixture is carried through the apparatus by the gas,
those substances that are more soluble in the oil will travel
more slowly and those that are less soluble will travel faster.
 Procedure for GC
 The mixture to be separated is injected into the gas which flow through a
long spiral tube containing the stationary phase.
• The substance injected must be able to form a vapour easily, e.g. it has to
be a gas, liquid or volatile solid.
• The time of injection is recorded.
• The components of the mixture separate in the tube.
• The components leaving the tube are detected (usually by measuring
changes in thermal conductivity of the gas coming out from the apparatus).
• The separated components leave the tube at different times. The time
between injection and detection is called the retention time.
• We can identify a component by matching its retention time with known
retention times for particular substances under the same conditions
Column Chromatography

 This is a form of adsorption chromatography.

 Silica, alumina or a resin(stationary phase) is mixed with a solvent such

as alcohol or water (mobile phase).

 The mixture is packed into a column. A mineral wool or sintered glass

plug at the bottom of the column keeps the stationary phase in place.

 The mixture to be separated is added to the top of the tube and then
allowed to soak into the stationary phase.

 Solvent (mobile phase) is then continuously added to the top of the

tube. The solvent moves through the tube separating the components of
the mixture
Column Chromatography
• After the column has been packed, the procedure is:
• Add the mixture to be separated to the top of the column.
• Allow the mixture to soak into the column (open the tap at the
• bottom).
• Keep adding solvent (mobile phase) to the top of the column and let the
solvent drain through carrying the components of the mixture with it.
• Don’t let the column run dry.
• Collect fractions of appropriate volume in test tubes at the bottom of the
column.
Visualising Agents/ Locating agents.
 When the components of a mixture are not coloured, we
spray the finished chromatogram or TLC plate with a
visualising agent (locating agent).
 This reacts with the colourless components.
 A coloured compound is formed.
 The colour may sometime need to be ‘developed’ by
warming the treated paper.
 Different types of visualising agents are used for different
types of compound, e.g. ninhydrin reacts with amino acids
to give purple coloured spots
 0.2% Ninhydrin in propanone produces a purple colour with
amino compounds when heated gently
 Ammonia vapour forms a deep complex with Cu 2+ ion
 Halogen anions are detected using 10% solution of silver
nitrate with a little fluorescein dissolved in ethanol added to
it.
 Spots containing a fluorescent substance become visible
when viewed in UV light.
Retention Factor
 We can identify the components on a chromatogram by comparing how far
the spots have moved from the base line compared with how far the
 solvent front has moved. We call this the retention value or retention factor,
Rf
 The chromatogram can be analysed by comparing the distances travelled by
the components of the mixture to the distances travelled by known
substances.
 If two substances travel the same distance then they are identical.
 Each component has a characteristic Rf value for a given solvent provided
that conditions of chromatography remain constant.
 These conditions are temperature, amount of material spotted, thickness of
absorbent, type of absorbent and solvent systems.
 Applications of Chromatography

Column:
 This method has the advantage that fairly large amounts of material can be
separated, e.g. mixtures of amino acids or mixtures of proteins
 Columns can be made of various sizes. Larger-sized columns can be used for
purifying natural products, e.g. plant oils such as limonene or for purifying drugs.
 Each fraction collected from the bottom of the column can be analysed
automatically, e.g. proteins can be analysed quantitatively by
 UV-spectrometry by measuring the absorbance at 280 nm.
 Amino acids can be analysed quantitatively by reaction with ninhydrin and
measurement of the colour intensity using visible-spectroscopy.
Applications of Chromatography

Thin layer and paper :

• These methods can be used to separate small amounts of compounds such as

amino acids, plant pigments and food colourings.
• We cannot, however, completely separate compounds with similar Rf values.
• We can use two-dimensional chromatography to help overcome this problem.
• After the initial chromatography, we allow the paper to dry and then turn it 90o and
carry out chromatography in a different direction using a different solvent.
• If colourless compounds are separated, the paper or TLC plate must be sprayed with a
suitable visualising agent. These methods are useful when small amount of materials are to
be identified but are less useful when quantification of large amounts of materials are
required.
• Quantification can, however, be made by cutting out the spots, removing the compound in
the spot with a solvent, then quantitatively analysing the solution using UV-visual
• spectroscopy or mass spectrometry
Applications- Gas Chromatography

 The components arising from GLC can be fed directly into a mass
 spectrometer or IR spectrometer for identification.
 The method can be very sensitive and is used to separate and identify
traces of substances in foodstuffs and analyse pesticide concentrations
in the environment.
 It is often used in forensic analysis to separate and identify particular
compounds, in medicine to determine gas concentration in blood
samples and in analysing fuels.
 A well-known use is in testing the blood or urine of athletes for
performance-enhancing drugs.
 The retention times of the components are matched with those of
known substances using the same flow rate, carrier gas and stationary
phase.
 Additional confirmation is given by mass spectroscopy.
 One limitation of GLC is that similar compounds have similar retention
times.
Videos on Chromatography

 https://www.youtube.com/watch?v=rMGQavOMAmc
 https://www.youtube.com/watch?v=pnTGNAfu6GE
 https://www.youtube.com/watch?v=TdJ57SQ6GAQ
 https://www.youtube.com/watch?v=UmWMlKJAdSk
 https://www.youtube.com/watch?v=yig3QCfBTzc
 https://www.youtube.com/watch?v=RX9Nl-SVHW8