13.

0 PHASE EQUILIBRIA
13.2 Separation techniques
 OBJECTIVES
By the end of the subtopic, you should be able to:
• Explain steam distillation of two immiscible liquids.
• Demonstrate an awareness of the applications of steam distillation.
• Explain: paper, high performance liquid, ion exchange, thin layer, and column and
gas/liquid chromatography in terms of absorption and/or partition, based on
appropriate practical experience.
• Demonstrate an awareness of the applications of these methods of chromatography in
industry and medicine.
• Describe the process of electrophoresis, and the effect of pH
• Describe the hydrolysis of proteins, separation and detection of the products by
electrophoresis.
• Outline the process of analysis of genes and genetic fingerprinting.
 13.2.1 STEAM DISTILLATION
Immiscible liquids
• Immiscible liquids do not dissolve into
each other for example oil and water.
• In other words, they don’t mix to give a
single phase.
• When left still, one liquid will float on
top of the other.
• It is possible to shake up the liquids and
get them to mix but they soon separate.

Fig 13.2.1: Example of immiscible liquids

Vapour pressure of immiscible liquids
• If two immiscible liquids are kept still in a flask then the vapour pressure measured will be of
the one on top.
• If the mixture is stirred or agitated, the vapour pressure measured will be of both or all liquids
in the mixture.
• The vapour pressure will increase.
• This is because it now becomes the sum of all the vapour pressure in the mixture known as the
Total Vapour Pressure.
• Both liquids will be in equilibrium with their vapours.
• The Total vapour pressure is the sum of their individual vapour pressures.
• 𝑇𝑜𝑡𝑎𝑙 𝑉𝑎𝑝𝑜𝑢𝑟 𝑃𝑟𝑒𝑠𝑠𝑢𝑟𝑒 = 𝑃𝐴𝑜 + 𝑃𝐵𝑜

Steam Distillation
• Separating immiscible liquids is done using steam distillation.
• Steam distillation is a type of distillation process in which water is used as one of the
immiscible liquids.
Fig 13.2.2: Steam Distillation
• Liquids boil when their vapour pressure becomes the same as the external pressure.

• Normal atmospheric pressure is 101.325kPa.

• Immiscible liquids combined vapour pressures reach the external pressure before the vapour pressure of either of the individual components gets there.

• Therefore, a heated and agitated organic mixture of immiscible liquids will boil at a temperature lower than the boiling point of either of the pure liquids.

• Steam is passed through the organic compound (Mixture of immiscible liquids) to be separated.

• The steam condenses inside the compound forming a mixture of steam and one of the immiscible liquids.

• The mixture gets heated further by more incoming steam.

• The mixture evaporates and due to reduced vapour pressure, the required organic compound evaporates together with the mixture.

• The extracted mixture is cooled, condensed and allowed to settle.

• Settling allows the required liquid to settle at the top and water at the bottom.

Experimenting on steam distillation

Title: Steam distillation

Materials:

Immiscible liquids (water and phenylalanine), thermometer, laboratory steam distillation column set up.

Procedure

1. Construct a steam distillation apparatus set up in the lab as in fig 13.2.2.

2. Add water and phenylalanine into the distillation column.

3. Heat the first test tube until the water boils.

4. Observe and record temperature change.

5. Collect the distillate at 25, 50 and 98 degrees Celsius.

6. Let the distillate settle and observe.

• Expected results
• At 25oC and 50oC, there will be no distillate formed.
• At 98oC a distillate is formed and it should have two layers. The top layer should be phenylalanine and the bottom one should be water.
• Table below shows the saturated vapour pressure of phenylalanine and water at different temperatures.
Table 13.2.1: Vapour pressure chart
Temperature/ oC Vapour pressure/ kPa
Water 25 3.169
50 12.344
98 94.301
Phenylalanine 25 1.76
50 -
98 7.07
• At 98 degrees Celsius

• 𝑇𝑜𝑡𝑎𝑙 𝑉𝑎𝑝𝑜𝑢𝑟 𝑃𝑟𝑒𝑠𝑠𝑢𝑟𝑒 = 𝑃𝐴𝑜 + 𝑃𝐵𝑜

• = 94.301 + 7.07 = 101.371 kPa

• 101.371 kPa is greater than the atmospheric pressure (101.325kPa)

• This mean that the mixture of water and phenylalanine will boil at a temperature less than 98 oC, which is way less than the boiling points of
water and phenylalanine which are 100oC and 184oC respectively.
• Thus a distillate of phenylalanine is formed at 98oC.
 Application of steam distillation
• Steam distillation is used in separation of oils such as those used in perfume
manufacturing.
• In industries, steam distillation is used to extract natural oils such as eucalyptus oil
from eucalyptus plants and citrus oils from orange and lemon peels.
• Steam distillation is also used in petroleum refineries commonly known as steam
stripping.
• Is also applied in separating fatty acids from mixtures.
 13.2.2 CHROMATOGRAPHY
• Chromatography is the partition of components of a mixture between a stationary and a
mobile phase.
• These different components experience a slightly different adsorption forces with the
stationary phase.
• They have different solubility in the mobile phase.
• The technique of chromatography exploits these differences and brings about separation.
• Adsorption is the attraction and temporary adhering of molecules of a gas or liquid on to
a solid surface.
• Chromatography is therefore a surface process unlike ion exchange.
• The partition equilibrium involved is between the adsorption of a component on to the
surface of the stationary phase and the dissolving of that component in the mobile phase.

13.2.2.1 Paper chromatography

• Paper chromatography is an analytical method used to separate coloured chemicals or
substances.
• An adsorbent paper is used where coloured substances are
spotted.
• The paper is dipped into the solvent and the solvent separates
the components in the mixture.
• The paper is the stationary phase also known as the
chromatogram.
• If the stationary phase is a solid, then the type of
chromatography is known as adsorption.
• The solvent is the mobile phase and it is the one that travels
up the paper (stationary phase) due to capillary action.
• The different components of the mixture are attracted to
different extents and they get partitioned between the two
phases.
• The components of the sample will start to move along the
paper at the same rate as of the solvent.
• Components of the sample with a stronger attraction to the Fig 13.2.4: Paper Chromatography
paper than to the solvent will move more slowly than that of
the components with a strong attraction to the solvent.
• This rate difference at which of each component in the
mixture travel along the paper leads to their separation.
• The factors influencing the reproducibility of paper chromatography are: types of extracted chemical
compounds, type of paper materials used and type of solvent used.

Application of paper chromatography

• Among all the chromatography methods paper chromatography is an inexpensive and rapid method that
provides graphic and clear results.
• Used as a qualitative method for identifying the components in a mixture.
• The separated spots on the finished and dried chromatogram can be cut out and re-dissolved to obtain a
pure sample of component of the sample mixtures.
• Used in several scientific studies in identification of unknown organic and inorganic compounds from a
mixture.
• In forensic studies paper chromatography is used in crime scene investigation and DNA and RNA
sequencing along with other studies.
• Paper chromatography is used as an analytical chemistry technique for identifying and separating
coloured mixtures like pigments.
• Sugars, amino acids, lipids and nucleic acids and other bio molecules can be easily identified by spraying
with appropriate reagents to detect these specific compounds.
• Paper chromatography can be reproduced easily as long as the conditions are controlled and maintained.
 13.2.2.2 Thin layer
• Thin layer chromatography (TLC) is chromatography technique similar to paper chromatography and is used to
separate non-volatile mixtures.
• TLC has advantages over paper chromatography in that its results are fast and that separations are very efficient
because of the much smaller particle size of the stationary phase. It also gives clear separation of the spots.
• Thin-layer chromatography is performed on a plate or sheet of glass, plastic, or metal foil.
• The plate is coated with a thin layer of adsorbent material usually silica gel, aluminium or cellulose.
• This layer of adsorbent is known as the stationary phase.
• The sample plate is the place in a closed vessel containing the solvent so that the liquid level is below the spot.
• This technique is usually done in a closed vessel to ensure that the atmosphere is saturated with solvent vapour
and that evaporation from the plate is minimised before the run is complete
• The solvent is the mobile phase.
• The solvent ascends the plate by capillary action, the liquid filling the spaces between the solid particles.
• As the solvent rises up the plate, the components get partitioned between the solid silica and the liquid solvent.
• Since the different components travels along the plate at different rates separation is achieved.
• The plate is removed when the solvent front approaches the top of the plate and the position of the solvent front is
recorded before it is dried.
• To quantify the results, the distance travelled by the
substance being considered is divided by the total
distance travelled by the mobile phase. (The mobile
phase must not be allowed to reach the end of the
stationary phase.)
• This ratio is called the retention factor(𝑅𝑓 )
• 𝑅𝑓 values are a way of measuring how far a component
has travelled relative to the solvent front.

𝑅𝑓 𝑓𝑜𝑟 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒 𝑃
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒 𝑏𝑒𝑖𝑛𝑔 𝑐𝑜𝑛𝑠𝑖𝑑𝑒𝑟𝑒𝑑(𝑋)
=
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒(𝑌)
𝑋
𝑅𝑓 =
𝑌
• In general, a substance whose structure resembles the
stationary phase will have low Rf, while one that has a
similar structure to the mobile phase will have high
retention factor.

Fig 13.2.5: 𝑹𝒇 for substance P

 Application of TLC
• Applied in industry in determining the progress of a reaction by studying the components
present;
• Used to check the products of a reaction.
• To determine whether the product contains impurities.
• Can be used for separating reaction intermediates.

13.2.2.3 Column chromatography

• Column chromatography involves similar principles to TLC only that the stationary phase
(adsorbent solid) is packed in a column instead of being coated on a plate.
 13.2.2.3 Column chromatography
• Column chromatography involves similar
principles to TLC only that the stationary
phase (adsorbent solid) is packed in a
column instead of being coated on a plate.
• The mobile phase is the solvent poured at
the top.
• The separated components flow out and
are collected at the bottom
• Application of Column
Chromatography
• Used in biochemical and hospitals for
identification of components such as
amino acids, peptides and nitrogen bases.

Fig 13.2.6: Column Chromatography

 13.2.2.4 High Performance Liquid chromatography (HPLC)
• This technique is an improved version of column chromatography.
• The efficiency of a separation increases if the particles in the stationary phase are made smaller.
• This gives a larger surface area and the solute can move more rapidly between the two phases.
• Therefore in this technique, the solid usually silica is powdered into very small particles
• However, if the particles are made smaller, capillary action increases and it becomes more difficult to drain
the column under gravity.
• Consequently high pressure has to be applied therefore a pump is needed to force the solvent through the
densely packed column.
• Separated components are detected as they leave the column at different times.
• The time each component spends in the column before it reaches the detector is known as retention time.

Application of HPLC
• Used for rapid identification of components for example identification of suspected stimulants, drugs or
doping that may be present in people, athletes and racehorses.
• Used in hospitals and industries for identification and separation of small quantities of pure compounds from
mixtures.
 13.2.2.5 Gas/liquid chromatography
• There are three types of chromatography that involves gas and or liquid which are:
• Gas-liquid chromatography
• Gas chromatography and
• Liquid chromatography
• Gas-liquid Chromatography (GLC)
• This method uses a column longer than HPLC.
• The column is packed with inert powder
• In this technique, the mobile phase is the gas
• The inert carrier gas mainly nitrogen flows through the column inside a heated oven.
• The inside of the column is coated with a thin layer of a stable non-volatile liquid such as silicone oil.
• GLC is a valuable for separation of gaseous mixtures and volatile liquids.
• It is very sensitive and can be used to detect tiny quantities.

Gas chromatography
• This technique uses a gas as the mobile phase, and the stationary phase can either be a solid or a non-volatile liquid (in which case
small inert particles such as diatomaceous earth are coated with the liquid so that a large surface area exists for the solute to equilibrate
with).
• If a solid stationary phase is used the technique is described as gas-solid adsorption chromatography, and if the stationary phase is
liquid it is called gas-liquid partition chromatography.
• The stationary phase particles are coated onto the inside of the column.
• In gas chromatography, the mobile phase is a carrier gas, usually an inert gas such as helium or an inert gas such
as nitrogen
• The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece
of glass or metal tubing called a column.
Application of Gas chromatography
• Typical uses of GC include testing the purity of a particular substance.
• Can be used for separating different components of a mixture (the relative amounts of such components can also
be determined).
• In some situations, GC may help in identifying a compound.
• In preparative chromatography, GC can be used to prepare pure compounds from a mixture.

Liquid Chromatography
• Liquid chromatography (LC) is a chromatographic technique in which the mobile phase is a liquid.
• The stationery phase is the inert solid, silica.
• The adsorbing properties of silica and alumina are reduced if they absorb water, but this reduction is reversed by
heating to 200–400 °C.
Application of Liquid Chromatography
• This makes LC more useful in the separation of many biological compounds, synthetic or natural polymers, and
inorganic compounds.
 13.2.2.6 Ion exchange
• Ion exchange chromatography is similar to partition chromatography in that it has a coated solid as the stationary phase.
• The coating is referred to as a resin, and has ions (either cations or anions, depending on the resin) covalently bonded to it.
• The ions of the opposite charge are electrostatically bound to the surface.
• When the mobile phase (always a liquid) is passed through the resin the electrostatically bound ions are released as other ions are
bonded preferentially.
• Ion exchange allows separation of ions and polar molecules based on their affinity to the ion exchanger.
• Exchange of ions is the basic principle for this technique.
• There are two types of exchangers in this process which are cationic and anionic exchangers.
• Cationic exchangers will attract positively charged cations.

Application of ion exchange

• Used in laboratory for both analytical and preparative purposes such as in analysis of amino acids.
• Applied in separation of proteins.
• Used to determine the base composition of nucleic acid.
• This is most effective method for water purification. Complete deionization of water (or) a non-electrolyte solution is performed by
exchanging solute cations for hydrogen ions and solute anions for hydroxyl ions. This is usually achieved by method is used for
softening of drinking water.
• Ion exchange can also be used for separation of inorganic ion, organic acid vitamins and bases.
 13.2.3 ELECTROPHORESIS
• Electrophoresis is from Greek word (electro = electron; and phoresis = carrying).
• Electrophoresis is the process of migration of charged molecules through solutions in an applied electric field.
• Different types of electrophoresis are shown below.

Electrophoresis

Free Zone
Electrophoresis Electrophoresis

Micro Moving Cellulose Paper Gel

Electrophoresis Electrophoresis Electrophoresis Electrophoresis Electrophoresis

Fig 13.2.8: Types of Electrophoresis

• Table 13.2.2: Types of Electrophoresis in Detail
Type of Electrophoresis
Micro • It is a type of electrophoresis used to separate dispersed particles using optical
microscopy. It is carried out in a closed medium with critical observations by focusing and
adjusting the lens of microscope.
Moving • In moving boundary electrophoresis the ions move through a simple liquid. Think of a
beaker containing buffer and two electrodes. If an electric field is induced and charged
molecules are added to the buffer solution, those molecules will move.
Paper • It is the form of electrophoresis that is carried out on filter paper. This technique is useful
for separation of small charged molecules such as amino acids and small proteins

Cellulose • Cellulose Acetate Electrophoresis is an important technique in clinical diagnostics. ...

These have the advantage that they can be used for a wide variety of
clinical electrophoresis applications including haemoglobin, serum proteins for
monoclonal gammapathies, urine proteins, isoenzymes, lipo and glycoproteins.

Gel • It is a technique used for the separation of Deoxyribonucleic acid, Ribonucleic acid or
protein molecules according to their size and electrical charge using an electric current
applied to a gel matrix
• There are different types of electrophoresis based
• In electrophoresis, an electric current is applied across a supporting medium such as gel so that one end of the
medium has a positive charge and the other end has a negative charge.
• Movement of charged molecules is called migration.
• Molecules will migrate to opposite charge thus a negatively charged molecule will be pulled towards a positive end.
• The supporting medium also has tiny pores present that acts as molecular sieve.
• Small molecules therefore move quickly through whereas larger molecules move much more slowly.
• As a result, smaller molecules migrate more quickly therefore they travel further than large molecules which migrate
slowly and therefore will travel a shorter distance.
• As a result, the molecules are gets separated.
• This is the double principle of electrophoresis separation which separation by size and charge.
• Electrophoresis of positively charged particles (cations) is called cataphoresis, while electrophoresis of negatively
charged particles (anions) is called anaphoresis.
• Electrophoresis is used for separation charged molecules such as DNA, RNA and proteins in labs.
• Electrophoresis is affected by pH.
• Degree of ionisation is pH dependent, therefore if pH increases, ionisation of an organic acid also increases.
• pH is maintained by use of buffers of different pH.
• Buffers has two functions which are they carry the applied current and they set the pH at which electrophoresis will
be carried out.
 Hydrolysis of Proteins by Electrophoresis
• Protein hydrolysis is the breakdown of proteins into smaller peptides and free amino acids.
• The separation of proteins by electrophoresis is based on the fact that charged molecules will migrate through a matrix upon application of
an electrical field.
• The most commonly used technique for the separation of proteins is Gel electrophoresis using Sodium dodecyl sulphate-polyacrylamide
(SDS PAGE).
• The gel matrix used for protein electrophoresis separation is polyacrylamide.
• Polyacrylamide is used to form a gel matrix of pores which allow the molecules to migrate at different rates.
• The size of pores is determined by the concentration of acryl amide.
• The higher the concentration, the smaller the size of pores
• The gel exists in two different forms which are non-dissociating (non-denaturing) and dissociating (denaturing).
• Non-dissociating (non-denaturing) system is designed to separate native protein under conditions that preserve protein function and
activity.
• In contrast, a dissociating system is designed to denature protein into their constituent’s polypeptides and hence examines the polypeptide
composition of samples.
• Sodium dodecyl sulfate (SDS) is used to denature and linearise the proteins
• SDS coats the proteins with negative charge.
• SDS- PAGE uses two types of buffer systems: the continuous buffer system and the discontinuous buffer system.
• In the continuous buffer system the pH of the gel matrix remains constant throughout the separation.
• In discontinuous, buffer system consists of a narrow layer of stacking gel (of large pore size and acidic pH) above the main separating or
resolving gel matrix of alkaline pH (pH 8.8).
• The stacking gel concentrates the protein sample before entering the separating gel.
• SDS-PAGE with a discontinuous buffer system is the most popular electrophoresis technique used to analyze polypeptides.
• In SDS-PAGE, the protein mixture is denatured by heating at 100°C in the presence of excess SDS and a reducing reagent is employed
to break disulfide bonds.
• The SDS-protein complex forms a rod with its length proportional to the molecular weight of the protein.
• All proteins are now negatively charged and when current is switched on, the proteins will move at different rates.
• The negatively charged protein-SDS complexes will move towards the anode.
• As they pass through the separating gel, the proteins separate, owing to the molecular sieving properties of the gel.
• When the proteins reach the bottom of the gel, the current is turned off.
• Gel is removed from between the glass plates and shaken in an appropriate stain solution.

Genetic Fingerprinting
• It is a technique used to distinguish between individuals of the same species using only samples of their DNA.
• DNA fingerprinting is a laboratory technique used to establish a link between biological evidence and for a example, a suspect in a
criminal investigation.
• A DNA sample taken from a crime scene is compared with a DNA sample from a suspect.
• If the two DNA profiles are a match, then the evidence came from that suspect.
• lf the two DNA profiles do not match, then the evidence cannot have come from the suspect.
• DNA fingerprinting is also used to establish paternity (family relationship), Forensic Science and Health Care
• Apart from identification, paternity and immigration cases, the technique is also used in medical research including cancer and
genetic conditions such as Huntingtons disease.
• The sample of DNA are taken from:
• Blood
• Hair
• Saliva and sweat
• Semen and
• Body tissue cells

• These samples are the same in every cell and retain their distinctiveness throughout a person life.
• Human cells contain 23 chromosomes (packets of DNA) from the father and 23 from the mother.
• Each DNA strand contains a unique sequence or code of genetic information.
• But while most of DNA shows only slight variation from one person to the next, certain areas, called mini satellites (short
sequences of chemical building blocks) show variation in the numbers of repeat units (or stutters) unique to each person.

Process of Genetic Fingerprinting

1. DNA Extraction
• Cells are broken down to release DNA

2. DNA Cutting
• The DNA is cut into fragments using restriction enzymes.
• Each restriction enzyme cuts DNA at a specific base sequence.
• The sections of DNA that are cut out are called restriction fragments.
3. Fragments Separation
• Fragments are separated on the basis of size
using a process called gel electrophoresis.
• DNA fragments are injected into wells and an
electric current is applied along the gel.
• DNA is negatively charged so it is attracted to
the positive end of the gel.
• The shorter DNA fragments move faster than
the longer fragments.
• DNA is separated on basis of size
4. DNA Transfer
• DNA split into single strands using alkaline
solution
• DNA fragments transferred from gel to filter
paper or nylon membrane
• (This is called Southern blotting)
• Gel, with filter paper attached, is removed &
separated.
Fig 13.2.9: DNA analysis
5. Analysis
• The fragments are treated with a radioactive probe which identifies shared design or
patterns.
• These patterns are then captured on X-ray film
• The result will be a pattern of more than 30 stripes, resembling a 'bar code'.
• This involves testing mini satellites one at a time, producing a simpler image.
• The pattern is then revealed.
• Each test will reveal a pattern unique to a particular person, and is therefore suitable for
forensic cases.
• This analysis establishes whether sample X comes from person Y.
ASSESSMENT EXERCISE 13.2 SEPARATION TECHNIQUES
1. A table below shows some properties of certain immiscible liquids under certain conditions.
Substance Boiling points Vapour pressure at
250 C 50𝑜 C 98𝑜 C
X 100𝑜 C 3.2kPa 12.3kPa 94.3kPa
Y 184𝑜 C 1.76kPa 2.36kPa 7.07kPa
Z 68𝑂 C 20k[Pa 53kPa 98kPa

a. What is likely to be substance X? Explain your answer.[2]

b. It was found that a mixture of X and Y or Y and Z can be separated by steam distillation. Calculate the
Total vapour pressure of:
i. X and Y at 250 C,50𝑜 C and 98𝑜 C .[3]
ii. Y and Z at 250 C,50𝑜 C and 98𝑜 C .[3]
c. State which temperature is ideal for steam distillation for a mixture of X and Y. Give a reason for your
answer.[2]

2. State any two application of electrophoresis. [2]

3. Explain what happens if an immiscible liquid is left to settle.[1]
4. Distinguish between paper and thin layer chromatography. [2]