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3
Chemical Methods of Separation and Purification

These methods involve carrying out appropriate chemical reactions for preparing
selected derivatives of all or particular constituents of the given mixture of which all
the components are, of course, present originally in one phase. Each such selected
derivative is produced in a form which may either (i) constitute a new phase by itself,
(ii) pass into a second phase that is immiscible with, and hence easily separable from,
the first phase that orginally contained all the components of the given mixture, or
(iii) remain in the original phase but is separable from it by such a physical process
as fractional distillation, fractional crystallisation or dissolution. Physical separation
of the phases is carried out as the next step, followed by suitable processing of these
isolated phases to obtain the original components as separated individuals. The
following examples illustrate the versatile applicability of chemical methods for
separation of mixtures into their respective components in the fields of Organic
Chemistry, Biochemistry and Inorganic Chemistry.

3.1 Separations in Organic Chemistry

3.1.1 Separation of a Mixture of an Alkane, an Alkene and an Alkyne into its

constituents
A mixture of ethane, ethylene and acetylene can be resolved into its constituents
by first passing the gaseous mixture through an ammoniacal solution of cuprous
chloride which retains acetylene as a precipitate of cuprous acetylide.
The escaping gaseous mixture consisting of ethane and ethylene is next bubbled
through cold concentrated sulphuric acid which dissolves out ethylene by converting
the latter into ethl hydrogen sulphate; ethane remains unaffected and can be collected
separately as gas.
 The precipitated cuprous acetylide is collected by filtration and acetylene
recovered from it by treatment with dilute acid.

C. Cu CH
+ 2H + + 2Cu +
C . Cu CH

Ethylene is recovered by heating the solution of ethyl hydrogen sulphate in

concentrated sulphuric acid to 160º–170ºC.

CH 3 CH 2
160° – 170 ° C
+ H SO 2 4
CH . HSO 2 4 CH 2

3.1.2 Separation of Carbonyl from Non-Carbonyl Compounds A carbonyl

compound (an aldehyde or a ketone) can be separated from its admixture with non-
carbonyl compounds by shaking the mixture with a saturated solution of sodium
bisulphite. This treatment yields a crystalline addition bisulphite derivative of the
carbonyl compound:
OH
C = O + NaHSO 3 C SO Na 3
This solid derivative is separated out by filtration and the original carbonyl
compound regenerated from it by heating with dilute acid or sodium carbonate
solution.
OH
C OH + +
+ H C = O + Na + HO + SO 2 2
SO Na 3

2C + Na CO 2 3 C = O + 2Na SO + CO + HO 24 2 2
SO Na 3

3.1.3 Separation of Neutral and Basic Components of Mixture The constituents of a

homogeneous mixture of aniline and toluene can be separated from each other by
shaking the mixture with dilute hydrochloric acid in a separating funnel. The reaction
mixture is allowed to stand for some time, and the aqueous layer of aniline
hydrochloride separating at the bottom of the funnel is withdrawn, leaving the
toluene layer in the funnel. This aqueous solution is transferred to another separating
funnel and rendered alkaline with sodium hydroxide solution whereby aniline is
renererated as an oily layer. 3.1.4 In the Hofmann's method of separating a mixture
of primary, secondary and tertiary amines into its components, the mixture is treated
with diethyl oxalate
Chemical Methods of Separation and Purification 59

whereby the primary amine yields a solid oxamide, the secondary amine gives a
liquid oxamic ester and the tertiary amine is left unaffected.

2 RNH + 2 R NH + 2 HC OOC 5 2 HC OOC 5 2RH N . OC RH N . OC

oxam ide HCO . OC 5 2 + 2 CH OH 2 5 + CH OH 2 5
(solid) oxamic ester
(liquid)
HC OOC 5 2

HC OOC 5 2 RN . OC 2

The solid oxamide is separated out by filtration and the solid yields the original
primary amine on its being heated with caustic potash:
amine
The liquid oxamic ester is separated from its admixture with the unchanged
tertiary amine by fractional distillation. On decomposition by heating with caustic
potash, the oxamic ester regenerates the original secondary amine:
R NH + am ine
CO . NR2 CO. OC H2 5 + 2KOH 2 secondary + CH OH 2 5
COOK COOK

3.1.5 Separation of a Racemic Mixture into its Constituents (a) The most
commonly used method of separation of the racemic mixture into its constituent
enantiomers is as follows:
A chemical reaction is carried out between the racemate and an optically active
form (either laevo-or dextro-) of a substance capable of reacting with the racemate.
This other optically active compound is usually derived from a natural source. To
resolve the racemates of amines (or other bases) and alcohols, for example, use may
be made of the naturally occurring d-tartaric acid (from wine tartar). The reaction
with amines gives salts and esters are formed with alcohols. For the resolution of
racemates of acids, use is frequently made of alkaloids such as quinine or strychnine
extracted from plants in which each of these alkaloids is present in an optically active
form. The racemate mixture forms two diastereoisomers (compounds that are
stereoisomers of each other, but are not enantiomers) of a derivative, with the
optically active reagent used. If the
60 Separation Chemistry

enantiomers constituting the racemic mixture are denoted by letters L and R, and the
optically active reagent by L', then the two compounds (salts, esters etc.) obtained
will be represented as:
LR
L' and L'
These two diastereoisomers posses different properties, say, different solubilities,
so that the two can be separated by one of the ordinary methods, say, by fractional
crystallisation. After separation, the optically active reagent is removed from the
molecule (if the diastereoisomer is a salt, it is treated with acid or alkali and if it is an
ester, hydrolysis is carried out) and pure forms of enantiomers are isolated.
Resolution of dl-tartaric acid is the classical example of the application of this
method.
With modification, the aforesaid method can sometimes be applied to the
 resolution of a neutral compound. Such a resolution is accomplished by first
converting the neutral compound into a derivative which can form a salt. Resolution
of dl-octanol-2 is an example of the application of this modification. The racemic
mixture of the alcohol is converted by treatment with phthalic anhydride into the acid
phthalate. In the next step the acid phthalate is reacted with the naturally occurring
laevorotatory base, brucine. Fractional crystallisation of the resulting mixture of the
brucine salts yields the separated salts. Decomposition of the separated salts with
hydrochloric acid removes brucine, and the two resulting acid phthalates are then
hydrolysed with alkali to get the d- and l-forms of octanol-2.

CH CHOH 6 13 CH 3 + OC COOCHC H6 13 COOH

O
OC phthalic CH 3brucine dl-acid
dl-octanol-2
phthalate COOCHC H6 13
anhydride

CH 3 tw o salts (se parated) (rem oval of brucine d- and l- acid phthalates (rem oval of phthalic acid)
HCl hy drochloride)
NaOH d- and l octanol-2
COO (brucine)

(b) Enzymes are highly specific biological catalysts capable of discriminating

between molecules having similar structures. One compound may be chemically
changed in the presence of an enzyme, while another, otherwise inseparable from the
first, is not affected. This selectivity has been used as a basis of separation. The
method is not frequently used but may be resorted to when two very closely related
Chemical Methods of Separation and Purification 61

molecules cannot be separated by other means. Separation of dl-alanine, for example,

has been accomplished by such an enzymic method. For this separation, dl-alanine,
obtained by synthesis, is acetylated, and the acetyl dl-alanine is treated with a
solution containing an enzyme which can bring about deacetylation. The solution
containing the required enzyme is prepared from fresh pork kidneys. The action on
the acetyl dl-alanins is complete in about four hours. The enzyme acts preferentially
on the acetyl derivative of the natural amino acid viz. l-alanine and the result is a
mixture of l-alanine and acetyl d-alanine.
 The resulting mixture is easily separated into its constituents because free
alanine is insoluble in ethanol and the acetyl derivative is soluble.

3.2 Separations in Biochemistry

3.2.1 Isolation of Proteins from their naturally occurring Mixtures Many

polyvalant metals form salts with organic acids which are markedly less soluble
than either the free acids or their sodium and potassium salts. Organic bases,
similarly, react with these salts to form complexes which are difficultly soluble.
Although these effects are of general occurrence, they find an important
application in the field of Biochemistry in the purification of proteins, and for
this purpose the polyvalent metals may be classified into the following groups
on the basis of their interactions with the different functional groups present in
2+ 2+ 2+ 2+ 2+ 2+
proteins. The first group which includes Mn , Fe , Co , Ni , Cu , Zn dan
Cd2+ is bound strongly both to carboxylic acids and to nitrogenous substances
such as amines and heterocylic compounds. The second group which includes
2+ 2+ 2+ 2+
Ca , Ba , Mg and Pb is bound strongly to carboxylic acids, but shows
negligible affinity to nitrogenous ligends. Zn2+ and Ba2+ may be taken as
representatives of the two classes, and their interactions with proteins have been
studied extensively. A third
62 Separation Chemistry

group of metals have a particularly strong affinity for sulphydryl groups. These
include Hg2+, Ag+ and Pb2+.
These metals can react with the carboxyl, amino, imidazole, or sulphydrl
groups of proteins, and the particular type of complex formed will depend on
the nature of the metal and of the protein and on the extent of ionization of the
ligand groups.
An important property of the protein metal complexes is that their solubilities
are particularly influenced by the dielectric constant of the medium. Thus, a
moderate reduction of the dielectric constant of an aqueous solution will
2+ 2+
precipitate the Ba or Zn salts of many proteins which are readily soluble in
the aqueous solutions. This property has been used for the fractionation of the
plasma proteins. The use of barium and zinc salts not only gave superior
fractionation, but also allowed the use of lower ethanol concentrations with
diminished risk of denaturation.
The recovery of proteins from their complexes with metals has been greatly
facilitated by the introduction of ion-exchange desalting, and of the
complexones such as ethylene diamine-tetra-acetic acid (EDTA), which remove
the metals as non-ionised complexes. The use of specific precipitants for
effecting separations covers a large field in Biochemistry, and some examples
from protein chemistry are given below to illustrate the possibilities of this
method.
Electrophoretically well characterized α1-and γ-globulins may be prepared
directly from serum by precipitation with 0.5 per cent uranyl acetate solution.
The γ-globulins were precipitated at pH 4.3 essentially free from other serum
proteins, while the α1-globulins could be precipitated at pH 3.2.
Another method for the isolation of a serum protein fraction uses
amylopectin sulphate which is particularly effective for the precipitation of β1-
lipoproteins and this observation forms the basis of its potential use for the
estimation of these proteins in plasma.
The acridine dye, Rivanol (2-ethoxy-6:9-diaminoacridine lactate), has been
shown to be useful for the preparation of γ-globulins. A 0.4 per cent solution of
the dye added to plasma at pH 7.6–7.8 will precipitate all the plasma proteins
except the γ-globulins. The excess dye can then be removed by adsorption on
Norit charcoal, and the γ-globulins recovered by precipitation with 25 per cent
ethanol at –5ºC.

3.3 Separations in Inorganic Chemistry

3.3.1 Refining of Metals

One method of refining of metals, called the van Arkel process, involves the
formation of a volatile halide by the direct action between the metal and a
halogen followed by purification of the volatile halide (by fractional distillation,
for example) and its subsequent decomposition to the metal and halogen at a
higher temperature. The sequence of reactions involved in the van Arkel process
is
Chemical Methods of Separation and Purification 63

exemplified below for the refining of Zr:

Zr + 2I2 ZrI4
filam ent
ZrI4 Zr + 2I2
800 °C

Besides Zr, the elements Ti, Hf, V, W, Si and Be have been purified by this
method.
Purification via the volatile carbonyl compounds is similar to the van Arkel
process, though used widely only for nickel:
50°C
N i + 4CO N i (CO ) 4
180° C
N i(CO) 4 N i + 4CO

3.3.2 Separation of Lanthanides

(a) Complex formation constitutes an important technique of separation in
Inorganic Chemistry. An important example of such an application is provided
by the separation of lanthanides by means of complex formation. Lanthanide
oxalates are insoluble in water but they become soluble on complexation with
EDTA. However, towards acids the stability of each complex formed between
EDTA and a particular member of the lanthanide series is different. Advantage
is taken of this fact for effecting the aforesaid separation. On adding an acid the
least stable complex is destroyed first to yield the precipitate of the
corresponding exalate, whereas the lanthanide which forms the most stable
complex is the last to precipitate out as its oxalate.
(b) Fractional precipitation makes use of the concept of solubility product
and its application can be elaborated by taking the example of separation of
lanthanides. This separation is effected by taking advantage of differences in
solubility products of the hydroxides of the various members of this group of
 elements. On addition of NaOH to a solution of lanthanide nitrates, Li (OH) 3
precipitates out first because of its lowest solubility product, whereas La (OH) 3
precipitates out last of all because of its highest solubility product. By repeating
dissolution and subsequent fractional precipitation of the hydroxides several
times, it becomes possible to achieve complete separation of lanthanides.
(c) Change in oxidation state of an ion brought about by oxidation or
reduction under conditions which do not affect the oxidation state of the other
ions present in the mixture is yet another chemical method of separating
+
lanthanides. The usual oxidation state of members of the lanthanide series is 3
and in this oxidation state they are very close to one another in properties. But
some of these elements exhibit oxidation states of 2 + and 4+ also, and these
changed values of oxidation state cause a marked change in the properties of
such lanthanides.
64 Separation Chemistry

Ce3+ alone is oxidised to the tetrapositive state by alkaline KMnO 4 when it is

present in solution along with other tripositive lanthanide cations. Separation of
cerium from other cations is then effected by carrying out this oxidation
followed by the addition of a small quantity of an alkali. Carium alone is
precipitated out as Ce (OH)4 leaving all other tripositive lanthanides in solution.
Thus 99% pure cerium can be obtained starting with a mixture having a cerium
content of only 40%.
3+
Eu present in a solution along with other tripositive lanthanide ions can be
isolated almost quantitatively through its reduction to Eu 2+. Zn-amalgam
3+
reduces only Eu to the divalent ion leaving the tripositive lons of the other
lanthanides unaffected. Eu2+ is then precipitated out from the aqueous solution
as EuSO4, which is insoluble, leaving behind other lanthanides in solution
because the corresponding sulphates are all soluble in water.

3.3.3 Separations of Isotopes by Exchange Reactions

In 'exchange reactions' chemical reaction gives appreciable exchange of
isotopes between the reactants. These reactions can be used for the separation of
isotopes. Obviously, only partial separations can be achieved through exchange
reactions, because these are equilibrium reactions. Some examples of
application of exchange reactions are given below.
(i) When carbon dioxide is kept in contact with water, the liquid gets richer
in 16O and the gas in 18O:
C16O2 + 2H218OC18O2 + 2H216O
(ii) Isotopic exchange reaction between hydrogen gas and water establishes
the following equilibrium:

HD + H2OH2 + HDO
As a result of this equilibrium, concentration of deuterium in water gets three
to four times as great as in hydrogen.
(iii) Passage of lithuim chloride solution down a column containing sodium
zeolite yields an outgoing solution which is richer in 7Li in comparison
with the starting solution:
Na[AlSiO4] + 6Li+ 6Li [AlSiO4] + Na+
 7 +7 +
Na[AlSiO4] + Li Li [AlSiO4] + Na
6 + 7 +
This happens because Li reacts faster than Li .

3.4 Conclusion
The examples cited in this chapter are only illustrative of the versatile
applicability of chemical methods for carrying out separation and purification in
Chemistry and
Chemical Methods of Separation and Purification 65

Biochemistry and are far from being meant as a comprehensive list of the
numerous chemical reactions that have been used by chemists and biochemists
to obtain substances in the pure state. More examples of chemical reactions used
as aids in separation processes are dispersed throughout the book. Two such
noteworthy examples are: (i) Ion-exchange chromatography, and (ii) electro
refining which is purification by chemical reactions carried out with the help of
an electric current. In the same context one is also reminded of the use of some
chemical reactions for separation of inorganic ions mentioned in the previous
chapter during discussion of Solvent Extraction.