2021

PHASE EQUILIBRIUM
A LEVEL CHEMISTRY

LAYAN
EXTRATERRESTRIAL
CONTENT

Compiled and Edited by Mr Magaba 0774 372 589

PHASE EQUILIBRIUM
• A phase is defined as a portion of matter which is homogeneous,
• A potion of a system is called homogeneous when it has the same physical properties and
identical chemical composition throughout the whole potion.
• Phase diagram of a substance shows the regions of pressure and temperature at which its
various phases are thermodynamically stable.

Phase Diagram

THE CONCEPT OF PHASE

• Various distinct phases in a system have distinct physical properties and is mechanically
separable.
• Physically distinct and mechanically separable means that the phase will have a definite
boundary surface.
• (e.g. water and ice) are separated from each other by definite phase boundaries therefore, a
phase may contain one or more components.

An Introduction to Saturated Vapour Pressure

• When a liquid is heated, the vapour pressure of the liquid increases until eventually it is
equal to the atmospheric pressure. Bubbles of vapour will form in the body of liquid.
• The bubbles then rise to the surface of the liquid, burst open and escape into the
atmosphere as a gas. The liquid boils.
• This happens because when a liquid is heated, the particles absorb energy until it is
sufficient to overcome the forces of attraction between them. The particles break away from
the fairly close arrangement of the liquid and boils.
• This process is called boiling. The temperature at which this process happens is called the
boiling point.

Factors Affecting Boiling point

i. Boiling point depends on external pressure. If a liquid boil under a pressure lower than the
atmospheric pressure (1 atm), it will boil faster.
ii. Conversely, if it boils at a pressure higher than the atmospheric pressure, it will boil slower.

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Condensation
• In condensation, the reverse happens, the particles lose energy and experience increasing
attractive force. They move slower and become closer together when temperature is
sufficiently low. The gas liquefies.

Evaporation and vapour pressure

• The energy distribution of particles in the liquid state follows a shape similar to the normal
distribution.
• The average energy is governed by the temperature. The higher the temperature, the higher
the average energy.

• Some particles have energy higher than the average while some have lower. The more
energetic particles at the surface of the liquid can be moving fast enough and eventually
overcome the attractive forces and escape into the atmosphere. They evaporate to form
vapour (Vapour is the gas form of a particle below its boiling point).
• Unlike boiling, evaporation only takes place on the surface of the liquid.
• In an open container, the liquid will evaporate until none is left.
• However, a different thing happens when the liquid is evaporated in a closed container.
• At first, liquid particles with higher energy escape from the surface of the liquid to become
vapour. The vapour particles will collide with the wall of container. The collisions exert a
pressure called vapour pressure.
• Vapour pressure of a pure substance is the pressure exerted by the substance against the
external pressure which is usually the atmospheric pressure.
• As more and more particles escape, the vapour particles become close together. Eventually
the particles with lower energy will not be able to overcome the attractive forces between
them. The vapour begins to condense and return to liquid.
• Eventually the vapour particles return to liquid at the same rate as the liquid particles
evaporate to form vapour. An equilibrium is reached. At this equilibrium, the concentration
of liquid particles and vapour particles remains constant.
• In this situation, the vapour pressure is maximum and is called the saturated vapour
pressure.

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IMMISCIBLE LIQUIDS
• Immiscible liquids are liquids which wont mix to give a single phase e.g. water and oil and it
explains the background to steam distillation.
• Examples of Polar and Non-Polar Solvents

Polar Solvents Non-Polar Solvents

Water H2O Tetrachloromethane CCl4
Ethanol C2H5OH Hexane C6H14
Liquid Ammonia NH3 Benzene C6H6

• Polar Dissolves in Polar and Non-Polar Dissolves in Non-Polar

Total Vapour Pressure of the mixture

• Assuming that the mixture is being agitated, then both of the liquids will be in equilibrium
with their vapours. The total vapour pressure is then simply the sum of the individual
vapour pressures:

• where po refers to the saturated vapour pressure of the pure liquid.

• Notice that this is independent of the amount of each sort of liquid present. All you need is
enough of each so that both can exist in equilibrium with their vapour.
• For example, phenylamine and water can be treated as if they were completely immiscible.
(That isn't actually true, but they are near enough immiscible to be usable as an example.)
• At 98°C, the saturated vapour pressures of the two pure liquids are:

Phenylamine 7.07 kPa

Water 94.30 kPa

• The total vapour pressure of an agitated mixture would just be the sum of these - in other
words, 101.37 kPa

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Boiling Point of the mixture
• Agitated mixtures of immiscible liquids will boil at a temperature lower than the boiling
point of either of the pure liquids. Their combined vapour pressures are bound to reach the
external pressure before the vapour pressure of either of the individual components get
there.

STEAM DISTILLATION
• It is a convenient method for the separation and purification of organic compound solid or
liquid from non-volatile organic and inorganic impurities.
• This method is only applicable to those compounds which are volatile in steam, insoluble in
water, and possess a vapour pressure of about 10-15mmHg at 373K and contain non-
volatile impurities.
• Steam distillation is particularly valuable when the substance to be purified boils above
373K at 760mmHg and decomposes below its boiling point. This is due to the fact that
steam distillation makes the high boiling substances to distill at low temperature and hence
avoid their decomposition.

Carrying out Steam Distillation

Preparation of Phenylamine
• Phenylamine is produced as a part of a mixture containing solution of all sorts of inorganic
compounds and it is removed from this by steam distillation.
• Steam is blown through the mixture. Water and Phenylamine turn into vapor. The vapor can
be condensed and collected.

• As the hot steam passes through the mixture it agitates, condenses, releasing heat. This will
be enough to boil the mixture of water and phenylamine at 98°C provided the volume of the
mixture isn't too great.
• For large volumes, it is better to heat the flask as well to avoid having to condense too much
steam and increase the volume of liquid in the flask too much.
• The condensed vapour will consist of both water and phenylamine. If these were truly
immiscible, they would form two layers which could be separated using a separating funnel.
• In fact, the phenylamine has a slight solubility in water and various other techniques have to
be used in this particular case to get the maximum yield of phenylamine. These aren't
relevant to this topic.

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Advantages of Steam Distillation
• It enables us to extract and purify substances which decomposes before reaching their
boiling points.
• It enables to purify substances with very high boiling point which will be difficult to achieve
in the lab

Effect of Pressure During Steam Distillation

Consider Low Pressure
Advantages
• Boiling will be achieved at a low temperature

Disadvantages
• Low pressures are difficult to maintain

Efficiency of the distillation process

• To check for the efficiency of the distillation process the substance that will be purified and
extracted
1. Check for its boiling point and its value is compared with the standard theoretical
boiling point of the pure substances.
2. Mass Spectrometry and results are compared with the theoretical one.

Applications of Steam Distillation

• Steam distillation can be used to extract some natural products - for example, to extract
eucalyptus oil from eucalyptus, citrus oils from lemon or orange peel, and to extract oils
used in perfumes from various plant materials.

Questions

1. At one atmosphere, pure water boils at 1000C.

a) State and explain how the boiling point of water is affected by adding CCl4.
b) Suggest with a reason how the boiling point of the H2O/CCl4 mixture is affected
by lowering the pressure in the environment.
c) State the best method for extracting sea water solution.
d) Explain why steam distillation is used to extract thermally unstable perfume
from crushed flower petals.

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DISTRIBUTION BETWEEN PHASES
Partition Coefficient
• If a solute, S, is shaken with two immiscible solvents X and Y (which forms two phases), the
solute dissolves in both solvents.
• An equilibrium is achieved in which the ratio of the concentrations of the solute S in the two
solvents is equal to a constant.
• This constant K, is called the partition co-efficient for the particular solute and the solvents.

• This relationship is known as the partition law and it has no units.

• The partition coefficient of a solute between two immiscible solvents is defined as the ratio
of the concentration of the solute in one solvent to the concentration of the solute in the
other solvent at a given temperature.
• The partition coefficient varies with temperature.
• The value of the partition coefficient is independent of the total solute concentration.
• The magnitude of the partition coefficient shows the relative solubility of a solute in the two
immiscible solvents.

Factors Affecting the Partition Coefficient

Temperature
• Kpc is constant only for a particular system at a particular temperature.
• Kpc varies with a change in temperature.

Changing the Solvent

• The volumes of solvent or mass of solute is used does not affect the value of Kpc and Kpc
remains constant.
• If one of the solvents is changed, e.g. hexane is replaced by ether, this becomes a different
system, and the partition coefficient will change.

The Partition Law only holds when:

• When there is no association of the solute particles in one solvent and not in the other e.g.
CH3COOH in H2O and ether. In Water CH3COOH it forms hydrogen bonds in water and in
Ether it dimerizes by forming Hydrogen bonds as follows:

• When there is dissociation of the solute particles in one solvent but not in the other e.g. HCl
in water and in ether. In ether HCl does not dissociate but in water it completely dissociates
realizing H+ and Cl-.
• When solute particles are different in in the two solvents, the ratio of the two
concentrations varies now with concentrations.

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Application of Partition Law
• The Partition law can be used to explain the process of solvent extraction.

SOLVENT EXTRACTION
• It is a process whereby a non-polar solvent is used to obtain a solute from the dilute
aqueous solution of that solute.

Procedure of Solvent Extraction

1. Place a dilute aqueous solution of a solute in a separating funnel and the add an
extracting solvent.
2. Close the separating funnel with a stop cork and shake the two solvents for some time
until an equilibrium is established.
3. The extracted solute is then obtained by evaporating the solvent, leaving behind the
pure organic compound.

Precautions
• The process has to be done in a fumes cardboard because most of the extracting solvents
are carcinogenic.
• Naked flames are must be avoided because most extracting solvents are very flammable.

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Continuous Extraction of a solvent
• This is a more efficient and improved way of performing solvent extraction method.
• If the solvent is a volatile one, and if the solute is involatile and stable to heat, it is possible
to ‘automate’ the process by using a continuous extraction.

How Separation is achieved

• The vapour travels through the tube connecting the flask and tube where it is taken to the
condenser in the tube.
• After condensation at the condenser, the liquid solvent is then collected in the funnel and it
pushes down the funnel resulting in the slow release of drops.
• After being released from the funnel the drops move up and they collect the solute from the
dilute aqueous solution.
• As the organic solvent (ether) rises through the aqueous layer, it dissolves and carries
solute particles with it to the top. This transfer of solute from the aqueous solution layer to
the ether layer occurs as a continuous process, as long as there is still ether vapour being
delivered from the reservoir (flask). The solvent is therefore used in small amounts in one
continuous process.
• This greatly increases the efficiency of the separation process.

Characteristics of a good solvent

• A good solvent must immiscible with water.
• A good solvent must have high solubility for the solute of interest or solute to be extracted.
• The extracting solvent must be inert.
• The extracting solvent must be volatile that is it should vapourised at very low
temperatures.

Examples of Good Solvents

• Ethoxyethane
• Tetrachloromethane
• Hexane

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Applications of Solvent Extraction
• Extraction of crude oil from Soya Beans
• Oils from crushed rose petals
• To extract natural products e.g. caffeine from tea leaves

Improving the efficiency of Solvent Extraction

• The efficiency of the solvent extraction process can be improved by the use of smaller
portions of the extraction solvent instead of one bulky one e.g. two 15cm3 potions of the
extracting solvent instead of one 30cm3 of the extracting solvent.

Calculations involving Solvent Extraction

1. When 20cm3 of ether was shaken with 30cm3 of an aqueous solution which contained
10g of solute S. The solute dissolves in ether given that the temperature remained
constant at 250C and the partition coefficient was 10
a) Calculate the amount extracted by ether
b) In a separate experiment two 10cm3 of ether was used to extract the same
solute. Calculate the amount extracted.
c) Give a conclusion and comment on the two different methods used

2. The Distribution of constant between hexane and water is 9.6. Calculate the final
concentration of the X ion given that 25cm3 of 0.05mol/dm3 of X is extracted with two
25cm3 of hexane.
3. A solution of iodine in trichloroethane (TCE) was shaken with water. The iodine content
of the two layers was determined by titration with aqueous sodium thiosulphate. A
25cm3 portion of the aqueous layer required 9.5cm3 of 0.02 moldm-3 thiosulphate to
reach the end point, whereas 5cm3 portion of the TCE layer required 17.5 cm3 of 0.2
moldm-3 thiosulphate. Calculate the Kpc for iodine between TCE and water.

CHEMICAL ANALYSIS
The analysis of samples and analytes involves
• Detection of the components present.
• Separation of the components in the analytes.
• Identification of the components present in the analytes.

METHODS OF CHEMICAL ANALYSIS

• Mass Spectrometry
• Chromatography
• Electrophoresis

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Chromatography
• It is the separation of mixtures by distributing the mixture between two immiscible phases,
which are stationary phase and mobile phase.
• The separation of analytes during chromatography is achieved by Adsorption and Partition.
• Adsorption is the adhesion of atoms, ions or molecules from a gas, liquid or dissolved solid
to a surface.
• Analytes are separated because they have different adsorptivity on the stationary phase
hence they move at different speeds.
• Analytes can also be separated due to their differences in solubility in the mobile phase as a
result they move with different speeds.
• Partitioning of analytes (separation by distribution between two phases) varies between
the mobile phase and the liquid stationary phase.

Stationary Phases
• It is an inert material coated with a non-volatile liquid where the sample to be analyzed
adsorb on. E.g. Silica, Alumina and an example of a non-volatile liquid is oil.
• The stationary phase can be a solid or a thin layer of a liquid supported on the surface on an
inert solid.

Mobile Phase
• It is an inert gas or liquid which carries analytes (samples being analyzed) in a
chromatography technique.
• The process in which the analyte passes over the stationary phase is known as elution.
When the analyte comes out through the other end of the chromatographic column, it is
known as the eluant.
• The “eluting power” of a solvent is largely a measure of how well the solvent can "pull"
an analyte off the adsorbent to which it is attached
• A liquid mobile phase can be ethanol
• A gas mobile phase can be nitrogen

Types of Chromatographic Techniques

1. Paper Chromatography
2. Thin Layer Chromatography
3. Column Chromatography
4. High Performance Liquid Chromatography
5. Gas Liquid Chromatography
6. Ion Exchange Chromatography

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Summary of Chromatographic Techniques
Technique Mobile Phase Stationary Phase Basis of Separation
Paper Liquid Thin film of water Partition
Chromatography
Thin Layer Liquid Solid Adsorption
Chromatography
Column Liquid Solid Adsorption
Chromatography
High Performance Gas Solid/Liquid Adsorption/Partition
Liquid
Chromatography
Gas Liquid Gas Thin film of involatile Partition
Chromatography liquid

Basis of Separation of Chemical Analysis

Adsorption
This is the process whereby particles of the solute stick onto the surface of the stationary phase
either by the formation of intermolecular forces or temporary bonds [Do not confuse with the term
‘absorption’ in which particles pass through a substance].

Partition
This involves differential dissolution of the solute in the mobile and stationary phase.

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Paper Chromatography

The Retardation Factor Rf

• Retardation factors are used for the identification of dyes
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒖𝒕𝒆
Rf =
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒗𝒆𝒏𝒕

For the Set Up

• The solvent (mobile phase) is going to move up by capillary action.
• Capillary action is the ability of a liquid to flow in narrow spaces without the assistance of
external forces like gravity (Cohesion, adhesion and surface tension)
• As it moves up it carries with it a mixture of the unknown dye

Basis of Separation
• The stationary phase is a thin film of water held through hydrogen bonding with the –OH
groups in cellulose fibres found in the paper.
• This film of water is present even when the paper looks or feels dry.

• The mobile phase moves up the chromatographic paper with analytes by capillary action.
• This allows some polar solutes to dissolve into the fibres of the paper (often by means of
ionic polar interactions with the stationary phase or by hydrogen bonding to the O-H
groups of the cellulose fibres, or more likely to the H2O molecules that are still associated
with cellulose.
• The solvent is usually non-polar or less polar than the cellulose surface and its associated
thin film of water layer.
• Non-polar molecules or less polar compounds in the analyte dissolve poorly in the
stationary phase, but dissolve well in the mobile phase. Polar substances in the analyte

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 therefore travel a small distance, whereas the less polar and less hydrogen bonded
compounds in the analyte move more rapidly up the paper and travel a larger distance.
• The tendency of a compound to dissolve in two immiscible solvents is known as partition
paper chromatography.
• Solutes in the mobile phase may or may not dissolve well in the stationary phase.
• After some time, the chromatogram is obtained.

The Chromatogram
• This is the information obtained from a chromatographic technique

Identification Methods
• After separation has occurred as shown by the chromatogram, the identification of
unknown dyes can be done in two ways:
1. The use of Rf values
2. The use of standard dyes
• When Rf values are used, dyes of the same Rf values will be of the same composition.

The use of standard Dye

• When standard dyes are being used to identify unknown dyes, the separated substance
comparison are made as shown on the chromatogram.
• The dyes which would have moved with same speed with a standard known dye can be
identified.

Uses of Chromatography
• Used to separate a mixture of dyes
• To analyze plant pigments
• To analyze amino acids and polypeptides

TWO-WAY PAPER CHROMATOGRAPHY

• Used to separate complex mixtures of pigments i.e. different compounds can have can have
very similar RF values in a particular solvent so it is not easy to separate them. If a new
solvent with different polarity is used the compounds are likely to have different RF values
thus separation is achieved.
• The process is done in two stages thus chromatography is done twice using two different
solvents.
• After obtaining chromatography in the first step a new start line is drawn and the
chromatogram is rotated 900.The chromatogram is again performed with a different and
this results in better separation of the complex mixture.

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Applications of Two-Way Chromatography
• It is used to identify the amino acids obtained from the hydrolysis of proteins

Questions
1. The diagram shows the results of two-way paper chromatograph

i. How many spots were there after the first solvent has been used. [1]
ii. Circle the spot that moved very little in solvent 2, but moved a greater distance in solvent 1.
[1]
iii. Draw a square around the spot that can be separated from the rest by using only solvent 1.
[1]

THIN LAYER CHROMATOGRAPHY (T.L.C)

• TLC is similar to paper chromatography when we are to consider the basic principle, the
basic principle of separating and even the to identify the separated substances.
• TLC is an improvement of paper chromatography because it uses a better stationary phase.
• The solid stationary phase is a thin layer of a finely divided adsorbent (usually alumina
(Al2O3) or silica SiO2 ) supported on a glass plate or aluminium foil or insoluble plastic,
which is made into a slurry with water and spread onto a microscope slide (a thin flat piece
of glass used to hold objects for examination on a microscope). This is then put into an oven,
where it dries out into a solid white coating on the glass. A chromatogram is then made in a
similar way to paper chromatography.

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Basis of Separation
• This separation involves adsorption of the solute particles on to the stationary phase. The
solvent, as in paper chromatography, moves up the plate through capillary action by filling
in the spaces between the particles of the stationary phase.
• Particles of the solute adsorb to the stationary phase via polar attractions.
• Acid-base interactions are also possible, for example, if the stationary phase is made of
alumina which is amphoteric, then it will be able to interact with both acidic and basic
solutes in the mobile phase.
• Hydrogen bonds are also possible when silica gel is used because of the hydroxyl group on
the surface of silica gel particles.
• Separation occurs because different substances in the sample have different adsorptivity for
the stationary phase and solubility for the mobile phase. If a solute has a strong
adsorptivity for the stationary phase, it easily leaves the mobile phase and adsorbs strongly
to the stationary phase. Such a solute move slowly up the chromatographic column.
• The purpose of the lid is to establish a saturated atmospheric pressure with solvent vapour
and that evaporation from the plate is minimized before the run is complete.

Detection after separation

• Solutes are located on the chromatogram and identified by comparing with standard known
substances or by calculating Rf values.
• UV Spectroscopy
• Colour developing reagents are also used e.g. amino acids and amines are detected by
spraying the plant with a solution on ninhydrin which is converted to a blue or purple
colour.

Advantages of Thin Layer Chromatography over Paper Chromatography

• It has high reproducibility of results.
• It can analyze very small samples, making it used in forensic science where it can be used to
identify drugs and explosives residue.
• TLC produces better separation because of the very small particle size of the stationary
phase.

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Disadvantages of Thin layer over Paper Chromatography
• It is more expensive

Uses of T.L.C
• To analyze plant pigments.
• To analyze amino acids (since amino are not colored a locating agent can be employed so
that the separated substances can be visible) e.g. using ninhydrin.
• Identify drugs and explosive residue.

Questions
a) TLC can separate mixtures of components mixtures of components.What do we call the
mechanism of seperation usually at work in TLC.
b) A mixture of propanone and hexane was seperated on a TLC chromatogram using Alumina
as the stationary phase and methylbenzene as the solvent.Which substance would you
expect to rise further up the chromatogram.Explain why?

COLUMN CHROMATOGRAPHY
• Column chromatography is a useful technique that uses liquid solid adsorption.
• Column chromatography is similar to Thin Layer Chromatography but it has the advantage
that it forms more product than the TLC but it takes longer.
• In Column chromatography, the mixture is dissolved in a small amount of solvent that is
then placed on top of the column of a finely packed solid adsorbent e.g. silica coated with a
non-volatile liquid that acts as the stationary phase.

• The eluting solvent is allowed to move down the column by gravity and it takes with it the
mixture of the unknown dyes.
• The rate of movement of each compound depend on the affinity of the compound on the
stationary phase and the solubility of the dyes on the mobile phase.
• The separation of the substances appears as bands shown by the diagram above.

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Identification of Analytes
• The compound that elutes first is collected first and separated from the solvent by heating.
The compounds can be identified by other techniques such as mass spectrometry.
• Identification of analytes can also be done by comparing the retention time
• Retention time is the time spent by the substance in the column i.e. from the point of
introduction in the column to the point where the analyte is eluted from the column.
• A constant flow rate should be maintained.

Advantages of Column Chromatography

• It forms more product than Thin Layer Chromatography
• Identification of compounds can also be obtained by other accurate techniques mass
spectrometry.

Disadvantages of Column Chromatography

• The process is very slow

Uses of Column Chromatography

• Analysis of plant pigments
• Analysis of Drugs

GAS LIQUID CHROMATOGRAPHY

• It is an automated chromatography technique that uses a carrier gas mobile phase and a
liquid stationary phase.
• The carrier gas must be dried first to remove water vapour that would otherwise interfere
with the separation.
• The gas can be dried by passing it over anhydrous copper (II) sulphate or silica impregnated
with cobalt (II) chloride.
• This technique is very useful when the mixture of compound is very volatile.
• The mixture to be separated has to vapourised at the sample injection point. The mobile
phase which is an inert gas mixes with the mixture and carries along with it along the
column.
• The column is coated with a high molecular weight and high boiling liquid that adsorbs to
the solid support of the column and this serves as the stationary phase.
• The column also contains a computer-controlled oven which maintains the temperature at
which the mixture is vapourised.

Basis of Separation
• The process involves the partitioning process of analytes between the carrier gas and the
liquid stationary phase. Since different substances have different solubilities in the mobile
and the stationary phase, they will move through the column at different speeds.
• As the mixture of gases moves along the stationary phase, the gas that forms stronger
intermolecular bonds to the liquid coating will move slower than those that form weaker
bonds.
• The separated substances are then taken to the detector which provides a quantitative
measurement of the components of the mixture as they elute in combination with the
carrier gas.

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Identification of Analytes
• Substances in the sample are eluted out at the end of the chromatographic column, where
they are detected by a suitable detector such as the flame ionization detector.
• The substances in the sample are eluted at different times therefore retention times are
used to determine the substances separated.
• GLC are also coupled with mass spectrometer which helps to identify analytes based on
their fragmentation pattern giving the relative abundances thereby quantifying the
analytes.

Factors that influence the Retention time of any gas

Vapour Pressure
• The lower the boiling point is, the higher the vapour pressure of the compound and the
shorter retention time.
• The compound will spend more time in gaseous phase.

Different Solubility of gas on the stationary phase

• If the polarity of the stationary phase and the compound is similar, the retention time
increases because the compound interacts stronger with the stationary phase

Temperature of the column

• An excessively high column temperature results in very short retention time but also in a
very poor separation because all components mainly stay in the gas phase.
• The best separations are usually observed for temperature gradients, because the
difference in polarity and boiling points are used here.

Flow rate of mobile phase

• A higher flow rate reduces retention times but a poor separation would be observed as well.
• The components will have very little time to interact with the stationary phase and are just
pushed through the column.

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Length and diameter of the column
• The longer column generally improves the separation because it increases the interaction of
the components with the stationary phase.

Limitations of GLC
• The major limitation of GLC is that the sample must be gaseous, or it must easily turn to a
gas.
• Moreover, some substances cannot easily be analyzed by this method, for example
carboxylic acids. These substances are too polar and tend to bind rather too firmly to the
stationary phase.

Applications of Gas Liquid Chromatography

• GLC is routinely used to analyze gaseous or liquid mixtures of organic compounds. It is a
very sensitive technique, that is, it can be used to detect minute quantities of a substance,
for example, pesticide residue in food.
• The technique also has a high resolution, that is, the ability to distinguish between
compounds with similar chemical and physical properties.

GLC has been used in the following ways:

• Detection of explosive residues on the skin or clothing
• Analysis of exhaust gases
• Analysis of beverages
• Analysis of medicines

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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
• The principle behind HPLC, and the instrumentation, is similar to that of GLC.
• There are some differences though.

Difference between HPLC and GLC

HPLC GLC
➢ the mobile phase is a liquid of high ➢ In GLC, the mobile phase is a gas.
purity, which also acts as the solvent
for the substances in the sample
➢ the chromatographic column is shorter ➢ In GLC the column is typically between
(typically 10 – 30cm) 1 and 3m long.

➢ the components are usually detected by ➢ In GLC the components are detected by
UV spectroscopy. This involves a mass spectrometer
measuring UV absorbance of the
separated components.

• High Performance Liquid Chromatography uses either liquid-solid adsorption or liquid-

liquid partition chromatography to separate and identify the components in a mixture.
• The stationary phase in the liquid-solid adsorption chromatographic column consists of
porous particles of silica of a uniform diameter. The particles are very small, typically with a
diameter of 1 x 10-6m. The very small size results in a very large surface area over which
adsorption may occur. The stationary phase is therefore solid, just as in TLC.
• However, in liquid-liquid partition chromatography, a thin layer of involatile and chemically
stable liquid is coated on the solid silica layer. In that case, the stationary phase can be said
to be both liquid and solid, because the solutes in the sample will be attracted to the polar
groups on both the liquid and the solid particles.
• The mobile phase from the mobile phase source is going to pass through the high-pressure
pump where it is given a high pressure.
• The high-pressure pump in the set up provides a constant flow rate of the mobile phase
through the column. It also forces the mobile phase to move through the tightly packed solid
particles in the column.
• The solvent chosen for the mobile phase is usually polar e.g. methanol/water solvent
• The sample to be analyzed is then mixed with the mobile phase at high pressure at the
sample injection point.
• Components in the sample will distribute themselves differently between two immiscible
phases.
• The separated analytes are moved to the detector which gives signal when there are
components eluted from the column.

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Identification of Substances
• The separated substances can be identified using their retention time.
• Substances exiting at the end of the column are mainly detected by UV spectroscopy. This
involves collecting the substance in a microcell at the end of the column and then measuring
UV absorbance of the substance. Different substances give different absorption patterns.

Advantages of HPLC
• It is a fast process
• It has a high reproducibility of results

Applications of HPLC
• HPLC can be considered to be an improvement of GLC. Many substances that can be
analyzed by GLC can also be analyzed by HPLC. Many other substances which cannot be
analyzed successfully by GLC can be analyzed by HPLC.
• HPLC is typically used for the separation and analysis of organic substances.
• HPLC has been useful in the identification of drugs in the blood or urine of athletes and
horses.
• HPLC allows separation and analysis of a wider range of substances than any other
chromatographic technique, including residual pesticides in food and detection of residual
chemicals from explosives.

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ELECTROPHORESIS
• Electrophoresis involves separation of substances in an electric field, based on their charge
and size.
• This is the method used to identify, purify and separate charged particles in biochemical
analysis particularly useful in the separation of amino acids, nucleotides, DNA fragments
and proteins.
• During the process of electrophoresis, charged particles are placed on an electric field
created on an on absorbent paper or on a gel electrophoretic plate.
• Depending on the PH of the buffer on the electrophoretic plate, the amino acids will be
charged.
• pH greatly influences the total charge of molecules.
• When electricity is applied to the medium containing amino acids, depending on their net
charge and molecular size, they migrate differentially, thus different amino acids can be
separated.
• Positively charged amino acids will be attracted to the cathode and negatively charged
amino acids will be attracted to the anode when the switch is closed
• The speed of the movement of amino acids depend on the molecular size and the charge of
the amino acids as a result separation is achieved.
• A series of lines or bands is observed on the paper or gel once a chemical is applied.
• Sometimes ultraviolet light is used to show the bands up. The series of bands is called an
electropherogram.

The factors which affect separation of ions by electrophoresis

Type of charge
• The general direction of movement of the ions depends on whether they are positively or
negatively charged.
• If an uncharged substance is also present in the mixture, it does not move in the electric
field. It remains at the starting point, and thus it is separated and distinguished from
charged substances.

Size of charge
• Ions of the same type of charge will move in the same direction.
• However, those ions with the larger charge will move faster.
• In this way, it is possible to separate ions of the same type of charge. For example, M⁺ and
M2+ ions will both move towards the cathode. However, M2+ ions will move faster.

Size (relative mass) of the ions

• Heavier ions move more slowly than lighter ones, even when they have the same size of
charge.

pH of the buffer
• This affects ionization of the species present in the mixture. In turn, this affects the direction
in which the ions move.

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Strength of the electric field
• The greater the potential difference between the anode and the cathode, the faster an ion
will move.

Shape
• A highly branched molecule experiences greater resistance to movement by interfering with
other molecules. Such a molecule moves more slowly compared with an unbranched
molecule.

Temperature
• Increasing temperature increases the speed of movement of ions, but high temperatures
may cause denaturation of proteins in a sample.

Consider separation pattern for the three amino acids

Glycine

• At pH 7, glycine undergoes self-neutralization, with a hydrogen ion from the carboxyl group
protonating the amine group.
• The result is the formation of zwitterions with one centre of positive charge and one centre
of negative charge. The net charge in the molecule is therefore 0. Consequently, glycine
molecules remain stationary at the starting point.

Glutamic acid

• Glutamic acid has two carboxylic acid groups. One of the groups protonates the amine
group.
• This self-neutralization creates a positive charge on the amine group and a negative charge
on the carboxylic acid group. These two charges, being equal but oppositely charged, cancel
each other out. The second carboxylic acid loses its proton into solution, forming a
carboxylate ion.
• Glutamic acid therefore has a net charge of -1 at a pH of 7. Thus, it will move towards the
anode.

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Lysine

• Lysine has two basic amine groups. One of these neutralizes the carboxyl group, resulting in
the formation of an -NH₃⁺ and an –COO⁻ group.
• The effects of these two groups cancel each other out, so they do not contribute to the
movement of the amino acid in the electric field.
• The second –NH₂ group on the amino acid accepts a proton from the solution, even from
other amino acids, and becomes the –NH₃⁺ group.
• Lysine has therefore a net charge of +1 at pH 7 and it will migrate towards the cathode.

Questions
1. Predict the direction, if any, in which lysine glutamic acid and glycine will move during
electrophoresis at pH 8.
2. Predict the direction, if any, in which lysine, glutamic acid and glycine will move during
electrophoresis at pH 6.

Solution
1. Lysine will have a net negative charge of -1 at pH 8, so it will move towards the anode.
However, the amino acids can still separate due to the fact that one of the ions, glutamic
acid, has the larger magnitude of charge of -2, so it will be attracted more rapidly to the
anode. However, there is also the issue of molecular mass. Glycine, even though it has only a
charge of -1, will also move fast because of its small size.

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GENETIC FINGERPRINT
• This is a process used to analyze DNA fragments using electrophoresis.
• The technique of DNA fingerprinting involves obtaining a pattern of base sequences in the
minisatellite regions of an individual’s DNA. Each person in the whole world has a unique
pattern of bases in these satellite regions.

Steps in obtaining a genetic fingerprint

1. Extraction of DNA from sample (blood, inner cheek cells, skin, semen etc.)
2. Formation of DNA fragments by restriction enzymes.
3. Polymerase chain reaction (PCR) to multiply the amount of DNA. This is only necessary if
the amount of DNA obtained from the source is much smaller than 25mg.
4. Separation of fragments by gel electrophoresis

The use of restriction enzymes

• Certain enzymes, obtained from bacteria, are capable of slicing DNA into pieces. These
enzymes are known as the restriction enzymes.
• The restriction enzymes are highly specific i.e. a particular restriction enzyme can only cut
DNA at a specific point. The use of restriction enzymes enables the DNA to be cut in such a
way as to obtain fragments of DNA from the minisatellite regions.
• Several restriction enzymes are used together since one enzyme is only able to cut the DNA
at one point.

The polymerase chain reaction (PCR)

• This technique is used to amplify tiny quantities of DNA to get amounts that are sufficient
for use in gel electrophoresis.
• The amplification process is carried out by an enzyme known as DNA polymerase. the
process requires heating, an ordinary enzyme would fail to function due to denaturation.
The enzyme
used comes from an unusual source; Thermusaquaticus, a bacterium that lives in hot
springs.

Separation of the fragments by gel electrophoresis

• Fragments of DNA bear a negative charge at the phosphate end groups at the pH which is
usually used for the separation.
• The DNA fragments are now placed in wells near the cathode of a gel plate.
• The DNA fragments are repelled by the cathode and migrate to the anode. This is because
the fragments carry a negative charge at the dissociated phosphate group.
• Separation of fragments occurs because they move at different speeds. The smaller
fragments move faster.

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Development of the DNA pattern
• The separation pattern of the fragments could be obtained on the gel plate by spraying with
a suitable dye.
• Radioactive isotope of phosphorus 32 (32P) is used to darken photographic or X-ray film to
make DNA fragments visible

The applications of genetic fingerprinting

• Forensic investigations to determine the criminal among suspects.
• Paternity testing.

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ION EXCHANGE CHROMATOGRAPHY
• It is a chromatography process that separates ions and polar molecules based on their
affinity to the ion exchanger.
• The equilibrated stationary phase consists of an ionizable functional group were the
targeted molecules of a mixture to be separated and quantified can bind while passing
through the column.
• The mobile phase will go down the column by gravity
• A cationic stationary phase separate anion and an anionic stationary phase separates cation.
• The bound molecules can be eluted and collected by running an eluant which contains
anions or cations through the column or changing the PH of the column.
• It works on almost any kind of charged molecules e.g. large proteins, small nucleotides and
amino acids

Characteristics of resin beads

i. The resin beads allow ions to exchange on their surfaces due to the presence of ionic
exchange groups
ii. They are microporous to allow the mobile phase to pass through and as we as to
increase the surface area for the ion-dipole attraction to take place
iii. They allow the formation of temporary bonds on their surface
iv. They are insoluble in an aqueous environment.

This technique can be done in two different ways

i. Cation exchange
ii. Anion exchange

Cation exchange chromatography

• Cation exchange chromatography is used when the molecule of interest is positively
charged e.g. Water soluble and charged molecules such as amino acids, proteins and
peptides.
• During his method, a glass column packed with a negatively charged resins beads (cationic
exchangers) and positively charged molecules of interest are loaded to be attracted to the
stationary phase forming ionic interactions.
• Commonly used cationic exchangers are Suphates(S-Resin) and carboxylate (CM-Resin
derivatives.

• In cationic exchange, molecules with cations elute out last because they are bound strongly
by ionic interaction with negatively charged stationary phase resins beads and molecules
with anions elute out first since they are repelled with negatively charged stationary phase
resins beads.

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 • The bound molecules can be eluted and collected by running an eluant which contains
cations through the column which compete to bind on the surface of the negatively charged
stationary phase resin beads with the bound molecules. e.g. Na+ to displace the solute of
interest from negatively charged stationary phase resins beads.

• The resins are made of a polymer with groups which facilitates the exchange of cations on
its surface.

Anion resin exchange

• Anion exchange chromatography is used when the molecule of interest is negatively
charged e.g. Water soluble and charged molecules such as amino acids, proteins and
peptides.
• During this method, a glass column is packed with a positively charged resins beads (anion
exchangers) is used and negatively charged molecules of interest are loaded to be attracted
to the stationary phase forming ionic interactions with the anion exchanger.
• Commonly used anionic exchangers are quaternary amines (Q-Resin) and diethylamino
ethane (DEAE-Resin)

• In anionic exchange, molecules with anions elute out last because they are bound strongly
by ionic interaction with positively charged stationary phase resins beads and molecules
with cations elute out first since they are repelled with positively charged stationary phase
resins beads.

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 • The bound molecules can be eluted and collected by running an eluant through the column
which contains cations to compete to bind on the surface of the positively charged
stationary phase resin beads with the bound molecules. e.g. Cl- to displace the solute of
interest from positively charged stationary phase resins beads.

• It is often used in the purification of water analysis, protein purification and in quality
control

Applications of ion exchange Process

• To remove heavy metal ions from the industrial liquid waste
• To make hard water soft

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