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13.2 Separation Techniques
13.2 Separation Techniques
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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.
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.
• 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 evaporates and due to reduced vapour pressure, the required organic compound evaporates together with the mixture.
• Settling allows the required liquid to settle at the top and water at the bottom.
Materials:
Immiscible liquids (water and phenylalanine), thermometer, laboratory steam distillation column set up.
Procedure
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.
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.
Electrophoresis
Free Zone
Electrophoresis Electrophoresis
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.
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