FRACTIONATION

Apt. Novitaria Br Sembiring., M.Farm

 FRACTIONATION

Fractionation is a process of separation of plant extracts into various fractions. It further segregates the fractions into
portions comprising a number of compounds. The process continues until pure compound is isolated.When several
solvents are required for the fractionation, they should be added according to the order of increasing polarity.
Fractionation techniques are basically classified into physical or chemical method.

Chemical methods
This extraction method is based on the type of functional groups possessed by a compound in the given mixture.
Separation or purification can be achieved by chemical reactions using appropriate reagents.

Physical methods
Physical methods used in separation of compounds from mixtures include separation funnel method,
chromatographic techniques, fractional distillation, fractional crystallization, fractional liberation, and sublimation.

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PHYSICAL METHODS

Separation funnel method

When four different solvents (n-hexane, chloroform, acetone, and n-butanol) are selected,
fractionation begins by moistening or complete dissolution of crude extract with 250mL of water.
This is followed by transfer into a separating funnel, shaken, and allowed to settle. Furthermore, to
250mL of n-hexane, the least polar solvent was added and shaken. The content can settle, and the
bottom of the separating funnel opened to remove the aqueous layer. The remaining content in the
separating funnel was poured into a clean container to get n-hexane fraction.Equal volume of n-
hexane was added again, shaken, and separated. The addition continued until after adding n-hexane
and shaken no reasonable quantity of extract appeared to move into the n-hexane portion. Similar
cycle was performed for chloroform, acetone, n-butanol to get chloroform, acetone, and n-butanol
fractions. The remaining portion left after the fractionation is termed as residual aqueous fraction
(RAF) as the crude extract was first dissolved in water.

Fractional crystallization
Large numbers of compounds that exist naturally in plant extracts are crystal in nature. Separation
is achieved via formation of crystals during concentration of an extract using heat or refrigeration

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PHYSICAL METHODS

Fractional distillation.
This is a process of separating or purifying compounds from a mixture. It is usually used in separation of
hydrocarbons such as crude oil, citral, and eucalyptol. Purification is achieved based on the differences in
their boiling points. Fractional distillation apparatus is constructed in such a manner that when heat is
applied each compound will evaporate and separates at its boiling point. Consequently, each compound
fractionated will condense and collected as a separate entity through several siphons attached to fractional
distillation apparatus.

Fractional liberation.
This method is suitable for separating compounds that can easily form precipitate from the mixture. The
precipitate is usually formed by changing the compounds into their salt form. Fractional liberation is
commonly applicable in purification cinnamon alkaloids

Sublimation.
This method involves changing from solid to gaseous state without passing through liquid state. Substances
such as camphor and volatile oils when heated get separated and converted directly into gas.

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METHODS USED FOR
ACHIEVING
FRACTIONATION

1.Precipitation

2.Solvent-solvent extraction

3.Distillation

4.Dialysis

5.Chromatographic procedures

6.Electrophoresis

20XX Presentation title 5

 1. PRECIPITATION

Precipitation occurs when the concentration of a substance in a solution exceeds its maximum
solubility. A compound can be precipitated by a variety of methods, each dependent on altering
one of the factors influencing solubility. Precipitation can be used either to remove the substances of
interest or to remove unwanted material and retain that of interest in solution.

A simple method of achieving precipitation is to lower the temperature of the solution of the extract.
The less soluble components will precipitate out and can be separated by centrifugation or filtration.
If one substance is present as a major constituent, it may precipitate out as crystals. This technique is
widely used as a final stage in the isolation of single compounds from aliquots derived from the last
fractionation stages.

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2. SOLVENT-SOLVENT EXTRACTION

a. Immiscible liquids

When a liquid is added to an extract dissolved in another liquid which does not mix with the first,
two layers are formed. Any component of the mixture will have a solubility in each of the two layers
(usually called phases) and after a time an equilibrium of concentrations in he two layers is reached.
The time taken to equilibrium of concentrations in two layers is reached. The time taken for
equilibrium to be achieved is usually shortened by fairly vigorous mixing of the teo phases in a
stoppered separating flask.

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CONT’

b. Multiple extraction
Further fractionation of a substance into only one phase can be achieved by successive extraction of
the original phase in question with the opposite phase. It is better to use several successive elutions of
relatively small volume than one amount of the total volume.
This is exemplified by the following: an aqueous extract (100 ml) contains a compound, A, with partition
coefficient κ ( chloroform : water) 4:1. The extract can be extracted either with four successive 25 ml
aliquots of chloroform or by one 100 ml aliquot

𝐴 𝐶𝐻𝐿𝑂𝑅𝑂𝐹𝑅𝑂𝑀
Partition coefficient κ = =4
𝐴 𝑊𝐴𝑇𝐸𝑅

i.e. 50% of A is removed from water phase into chloroform.

So, after four extractions, 50% of original + 50% of 50%
(25%) + 50% of 25% (12,5%) + 50% of 12,5% (6,25%) is
removed = 93,75% removed after four extractions.
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CON’T…
Extraction with one 100 ml aliquot
[A] in chloroform = 4 [A] in water (since both are 100 ml) = 80,0%
removed into chloroform, i.e. the extraction is not efficient as
using 4x25 ml aliquots of chloroform.Successive liquid
extractions can be carried out with immiscible liquids with a
range of polarities. Thus a water extract can be extracted
successively with hexane, dichloromethane, ethyl acetate
(or dichloromethane : ethanol 3:1 ) and butanol. The extracting
solvents of this series are of gradually increasing polarity.
Conversely, a petrol or dichloromethane extract may be
extracted with a solvent series of decreasing polarity, e.g., water :
ethanol 3:1, water:ethanol 2:1, water : ethanol 1:1. It is usual
extract each phase several times with the other one and to
combine the like phases.

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CONT’

c. Salting out

The extraction of moderately polar compounds from aqueous extracts by some what immiscible
solvent such as butanol or methyl ethyl ketone can be enhanced by ‘salting out’. The solubility of many
non-ionic compounds in water is considerably reduced when a large amount of a very soluble
inorganic salt is added to the solution. The non-ionic compound may precipitate out if no other solvent
is present but, in the presence of another moderately polar solvent, it will usually dissolve into that one.
Common salt, sodium chloride, is often used for this purpose because it is cheap and freely available,
but compounds such as ammonium sulphate are also widely used. The addition of 1g/ml sodium
chloride to water will render polar solvents such as methanol and acetonitrile immiscible and allow
them to be used for solvent-solvent extraction.

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d. partitions using changes in ph

Acidic and basic fractions can be obtained and purified from extracts
by exploiting the pH –dependent changes in water solubility of
relatively non-polar acidic and basic substances. As noted above, the
ionic forms of these are soluble in an aqueous media whereas the
non-ionized from is soluble in less polar solvents. Ex: Alkaloid

A similar process is used to separate acidic and phenolic substances

as a fraction from a general aqueous extract. Acidification of the
water layer in this case causes the compounds in question to dissolve
preferentially in the organic phase when they can be separated from
non-acidic, non-polar material by extraction with aqueous base.

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e. Removing traces of water from organic solvents

It should be realized that the extraction solvent will also extract small amounts of the other solvent. When
aqueous and non-polar, sometimes called ‘organic’ solvents from the two phases, the resulting organic
layer often contains small amounts of water separation from the aqueous phase. The organic layer
usually has a faintly cloudy appearance and can be dried by the addition of anhydrous sodium sulphate.
When the salt is added, with good mixing by agitation or swirling, it can be seen to appear more
crystalline and the organic solvent often appears much more transparent. The solid residue can then be
filtered off and traces of water removed by the use of filter paper treated with silicone ( which does not
permit passage of water) or by filtration through absorbent cotton (cotton wool) when the cellulose of
the cotton fibres absorbs large amounts of water.

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3. DISTILATION

The separation of a mixture of volatile compounds can be

effected by fractional distillation. This process is used
extensively in the petrochemical industry but has very
limited applications in the fractionation of plant extracts
and can only be used for volatile (sometimes called
‘essential’ oils.

Fractional distillation relies on the temperature geadient

established along a cooling column situated above a
boiling liquid. The least volatile compounds condense
nearest the boiling chamber whereas the most volatile
condense much further away. Withdrawal points are
positioned along the column and the liquid condensate
taken each will differ in composition

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4. DIALYSIS

Dialysis is a method of separating the components of a mixture according to their molecular size.
This process occurs naturally across cell membranes and is very important in many physiological
processes. The essential part of any dialysis procedure is a thin, semipermeable membrane,
consisting of a polymeric material with regular pores, which allows the passage of small molecules (
molecular mass < 1000 daltons). The passage of larger molecules is considerably impeded or not
possible. Under pressure or an osmotic gradient nearly all the small molecules in a mixture pass
through the membrane, leaving behind the larger molecules. Dialysis is often used in the
purification of polysaccharides and proteins in a process sometimes called ‘desalting’. The aqueous
extract containing the large molecules in enclosed in a membrane and immersed in water.

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5. CHROMATOGRAPHIC TECHNIQUES

These are special techniques used in separation of compounds from mixtures based on their size, shape,
and charge. The concept of chromatography involves the use of mobile phase, which is the solvent of
extraction and the stationary phase such as silca gel and sephadex mixed with a calcium sulfate as a
binder. Silica gel is used for parting amino acids, sugars, fatty acids, lipids, and alkaloids. Sephadex is
applicable in isolation of proteins and amino acids. Aluminum is useful in separation of vitamins,
carotenes, phenols, steroids, and alkaloids. Cellulose powder is used in separation of amino acids, food
dyes, and alkaloids. Celite is applicable in separation of organic cations and steroids.Various mechanisms
were involved in separation compounds using chromatographic techniques, namely, adsorption, partition,
affinity, ion exchange, or size exclusion. Chromatographic techniques include TLC, column
chromatography (CC), liquid chromatography (LC), GC, and HPLC.

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MECHANISMS OF SEPARATION IN CHROMATOGRAPHY

Adsorption chromatography
Separation is performed based on the interaction between compounds to be separated and the stationary
phase. In this case, the stationary phase will pull and remove compounds via hydrophobic, non-covalent
Van der Waals forces of attraction. The compound that is loosely bound will first be eluted by the mobile
phase.

Partition chromatography
Compounds are separated by addition of two or more immiscible solvents in to the mixture of an extract.
Each compound will part away from the mixture by dissolving in the portion of solvent where it is soluble.
Subsequently, the immiscible liquids will be separated using separating funnel to obtain the individual
compounds. The partition chromatography is otherwise known as liquid/liquid separation

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MECHANISMS OF SEPARATION IN CHROMATOGRAPHY

Affinity chromatography
The stationary phase is a ligand positioned in a separating column. The mobile phase applied washed
down the compounds that have no affinity for the stationary phase. As such, compounds with high affinity
for stationary phase get attracted and separated.

Ion exchange chromatography

The concept of ion exchange is useful in separation of polar compounds based on the type of charge they
possessed. As such like charges attract, whereas unlike charges repelled. Like-charge substances attracted
to each other and get separated the mixture or extract.

Size exclusion chromatography

This method considers separating compounds based on their molecular size by application of mesh of
different diameters. It is also known as gel filtration or molecular sieving.A smaller size mesh was first
applied followed by medium size, and finally larger pores size mesh.

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REFERENCE

Abubakar, AR & Mainul, H. 2020. Preparation of Medicinal Plants: Basic Extraction and
Fractionation Procedures for Experimental Purposes. J Pharm Bioallied Sci. 12(1): 1-10.

Houghton, PJ & Amala, R. Handbook for the Fractionation of Natural Extracts.

Pharmacognosy Research Laboratories, Department of Pharmacy, King’s College London.

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THANK YOU

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