CHM 004 (Dr.

ADESINA ADEDOTUN)

SEPARATION AND PURIFICATION TECHNIQUES

1 Simple Distillation
Simple distillation is a process of heating and cooling liquids to separate and purify
them. As the liquid being distilled is heated, the vapours that form are richest in the
component of the mixture that boils at the lowest temperature. Purified component boils,
and thus turns into vapours, over a relatively small temperature range (2 or 3 °C). A
careful observation of the temperature in the distillation flask helps to carry out a good
separation. As distillation progresses, the concentration of the lowest boiling component
steadily decreases. Eventually, the temperatures within the apparatus begin to change
and a pure compound is no longer being distilled. As the temperature continues to
increase the boiling point of the next-lowest-boiling compound is approached. When the
temperature again stabilizes, another pure fraction of the distillate can be collected. This
fraction of distillate is primarily the compound that boils at the second-lowest
temperature. This process can be repeated until all the fractions of the original mixture
are separated. For simple distillation to perform, the two liquids’ boiling points must have
a difference of at least 25 °C (or about 77 °F).

2. Liquid extraction
This is based on the fact that some organic compounds are more soluble in one
solvent than others. For example, caffeine in tea leaves is more soluble in
trichloromethane (chloroform) than in hot water. We can separate the caffeine from other
substances in the tea by first boiling the tea leaves and then shake the cooled solution with
chloroform in a separating funnel. The caffeine passes from the water (aqueous layer) into

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the chloroform (or the organic) layer. Thus, the pure solute caffeine is obtained by
carefully evaporating off the chloroform.

3. Sublimation
This method is used to separate a mixture of two solids in which one sublimes. The
mixture is heated in a dish with an inverted funnel. The solid that sublimes is cooled and
forms a sublimate at the cold surface of the funnel which can be scrapped off.

4. Recrystallization
This is a technique that’s used in the purification of solid compounds. Solids tend
to be more soluble in hot liquids than in cold liquids. During recrystallization, an impure
solid compound is dissolved in hot liquid or solvent until the solution is saturated and
then the liquid is allowed to cool. The compound then forms relatively pure crystals. Any
impurities that are present will remain in the solution and will not be incorporated into
the growing crystals. The crystals can then be removed from solution by filtration.

5. Melting Point determination

An organic compound must be in its pure state, if its properties are to be studied
and understood. Melting point and boiling point can provide useful information which

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can help in the identification of a sample or to establish its purity. The temperature at
which a solid melt and becomes a liquid is the melting point.

In the capillary method of determining melting points and boiling points, a small amount
of the substance to be tested is placed in a capillary tube, or small test tube, which is in
turn tied to a thermometer and placed in heating bath. For our samples, the heating bath
will be Mineral Oil in a Thiele Tube.

The Oil in the Thiele Tube’s arm is heated with a Bunsen Burner. Convection causes the
hot Oil to circulate, heating the thermometer and the sample simultaneously. The sample
is then observed to note when melting occur. With this method, it is important to heat the
sample slowly, so thermal equilibrium between the Oil-Sample and Thermometer can be
established and accurate melting points can be obtained.

Equipment Needs

Thermometer
Thiele Tube
1-Holed Split Stopper Rubber Bands Eyedropper Microburner

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Very Small Test Tubes Ring Stand
Clamp for Thiele Tube

Mineral Oil
Melting Point Capillaries
Sample to be tested

Procedure

All your measurements must be made to the correct number of significant

figures and must have the unit of measurement indicated.

1. Obtain a melting point capillary tube and the solid sample. Make observations
concerning the sample. Pack the capillary tube with the sample. It is very
important that the sample be packed properly and that it be of minimal amount.
2. The capillary is attached to the thermometer using a small rubber band. You want
the band placed high enough so that it will not be in the Oil when placed in the
Thiele Tube. The sample must be positioned so that it is at the same height as the
bulb of the thermometer.
3. Fill a Thiele Tube with Mineral Oil until the level of the Oil is just above the top of
the side-arm neck.
4. Place the thermometer into a split one-holed stopper that will fit into the top of
the Thiele Tube. Place this into the Thiele Tube and position the thermometer
bulb so that it is slightly below the bottom of the side-arm neck of the Tube.
5. Use a microburner to gently heat the Oil in the arm of the tube. The rate of
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heating should be about 1 C/min near the melting range. Greater rates can be
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used until 10-15 C below the melting range.
6. Heat your sample until melting is observed. Typically, samples will melt over a 1-
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2 C range in temperature. Greater ranges in melting temperatures generally
indicate the sample is impure. Note the point at which your sample begins to melt
and the point at which it is completely melted. For your first sample it is okay to
come up on the melting point fairly rapidly. This crude determination will then
be used to fine tune a second determination.
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7. Once the first sample has melted, allow the Oil to cool to about 20 C below the
melting point. Place another sample in the Tube and allow it to equilibrate for at
least 10 minutes. Then begin heating the Oil slowly and make an accurate
determination of the melting point for the substance.

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6. Chromatography
Chromatography is a method of separation in which the components to be separated are
distributed between the two different phases, one is the stationary phase and the other
one is a mobile phase that moves in a definite direction. There are different types of
chromatographic techniques such as column chromatography, paper chromatography,
partition chromatography, thin-layer chromatography, gas-liquid chromatography, and
ion-exchange chromatography.

Thin Layer Chromatography (TLC)

Thin-layer chromatography is the simple, fast, easiest, and least expensive of all the
chromatographic techniques which are used in quantitative and qualitative analysis of
separation of organic compounds, and to test the purity of compounds.

TLC consists of two phases given as follows-

1. A mobile phase (solvent)

2. A stationary phase (glass plate coated with silica gel)

The analysis is done under atmospheric pressure and room temperature.

Principle: thin-layer chromatography is a method of separation, or identification of a

mixture of components by using finely divided adsorbent solid/ liquid over a glass plate,
and liquid as a mobile phase.

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The distinction depends on the relative affinity between the stationary and mobile phases
of the compounds. The compounds move over the surface of the stationary phase under
the influence of the mobile phase (driven by capillary action). The compounds with
greater affinity for the stationary phase travel slowly during this movement while the
others travel faster. The isolation of components in the mixture is thus accomplished. The
individual components are visualized as spots at a different stage of travel on the plate
once separation happens.

Process: the process includes the following steps-

 Adsorption of substances on the stationary phase.

 Separation of adsorbed substances by the mobile phase.

 Separated substances are recovered by the mobile phase through elution.

 Qualitative and quantitative analysis of eluted substances.

In a solid phase, the adsorbent is coated onto a solid support such as a sheet of glass,
aluminium, or plastic as a thin layer of about 0.25mm thick.

The mixture that needs to be separated is dissolved in a solvent to form a solution. Thus,
this solution is applied at the base of the thin-layer plate. The solution is allowed to move
in an upward direction under the influence of capillary action. The solid will absorb some
fraction of each component of the mixture and the remaining will be left in the solution.
Anyone molecule will remain attached to the solid surface while the other components
continue moving up the plate with solvent.

Separation of mixtures on a flat surface by the movement of solvent due to differences in

solubility, migrating rates, size, and charge. Elution is stopped when the solvent reaches
the opposite side of the silica-coated glass.

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Paper Chromatography
Paper chromatography is an analytical chemistry technique for separating and identifying
mixtures that are or can be colored, especially pigments. The way paper chromatography
works is pretty simple. A small concentrated spot of a solution that contains the sample
of the solute is applied to a strip of chromatography paper about two centimeters away
from the base of the plate or paper, usually using a capillary tube for maximum precision
of amount spotted. This sample is absorbed onto the paper and may form an interaction
with it. The paper is then dipped into a suitable solvent, such as ethanol, H2O, or another
aqueous solvent, taking care that spot is above the surface of the solvent. The spotted
chromatogram is then placed in a sealed container. The solvent moves up the paper by
capillary action, which occurs as a result of the attraction of the solvent molecules to the
paper. Differential adsorption of the solute components into the solvent can also occur
and explain the separations observed. As the solvent rises through the paper it meets and
dissolves the sample mixture, which will then travel up the paper with the solvent solute
sample. Different compounds in the sample mixture travel at different rates due to
differences in solubility in the solvent and due to differences in their attraction to the
fibers in the paper. The more soluble the component the further it goes. Paper
chromatography takes anywhere from several minutes to several hours.

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 DETERMINATION OF ELEMENTS IN AN ORGANIC COMPOUND
(QUALITATIVE ANALYSIS)
1. Carbon and hydrogen
These two elements are detected by burning a pure dry sample of the compound in
excess dry oxygen and the resulting gases are tested. Carbon is converted to carbondioxide
which turns lime water milky, while hydrogen is converted to water vapour which turns
anhydrous copper (II)sulphate blue.

2. Sodium fusion test/ Lassaigne’s Test: Nitrogen, sulphur, and

halogens
These elements are determined by sodium fusion test or Lassaigne’s test. A small
portion of the organic compound is heated with a small piece of molten sodium in a test
tube until it is red hot. The mixture is rapidly crushed in a mortar with about 10 cm 3 of
cold water and filtered. The filtrate is then used for the various tests. Note the N, S, and
Xs in the organic compounds are converted to NaCN, Na 2S and NaX respectively.

 Na + C + N → NaCN
 2Na + S → Na2S
 Na + X → NaX ( X= Cl, Br, or I)

Test for Nitrogen: to 2cm3 of the freshly prepared filtrate in the test tube, add
aqueous iron (II)sulphate and heat to boil. A few drops of dilute sulphuric acid are added.
The appearance of a Prussian blue (bluish green) precipitate indicates the presence of
nitrogen in the compound.

The following reactions occur,

1. Fe2+ + 6CN– → [Fe(CN)6]4-

2. Fe2+ + H+→ Fe3+ + e–
3. [Fe(CN)6]4- + 4Fe3+ → Fe4[Fe(CN)6].H2O

Test for Sulphur: to a few drops of the filtrate, acetic acid is added, followed by
a few drops of lead acetate solution. The appearance of a black precipitate shows the
presence of sulphur in the compound.
Or in another portion of the filtrate, add few drops of sodium nitroprusside. An intense
purple colouration indicates presence of sulphur in the original compound.
The following reaction occurs:

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S2- + [Fe(CN)5NO]2- → [Fe(CN)5NOS]4-

Test for Halogens: if nitrogen and or sulphur is present, the filtrate is boiled
with 2cm3 of dilute nitric acid to remove N and S. After cooling, a few drops of AgNO 3
solution are added. The appearance of a white precipitate indicates the presence of
chlorine, a cream or pale-yellow precipitate indicates the presence of bromine, while a
yellow precipitate indicates the presence of iodine.
AgNO3 + NaX → AgX ↓ + NaNO3

EMPIRICAL AND MOLECULAR FORMULA DETERMINATION

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