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1. Dnyanasadhana College, Thane.
Department of Chemistry
M.Sc. Analytical Chemistry
Sem-I Introduction to Separation
Methods
in
Analytical Chemistry
Dr.G.R.Bhgaure
1

2. Introduction to separation methods
In
analytical chemistry:
Precipitation, filtration, distillation, extraction
and chromatography, modes
Of separation in chromatography: adsorption,
partition, ion exchange, size exclusion
And electro chromatography.

3. PRECIPITATION,
• Precipitation is an ionic reaction carried out
• Solution in which positive ion from one
substance combines with negative ion of
another substance to form sparingly soluble
salt is called as precipitation

4.  Other types of reactions can be used to
chemically separate an analyte and
interferent, including precipitation,
 Two important examples of the application of
precipitation are the pH-dependent solubility
of metal oxides and hydroxides, and the
solubility of metal sulfides.
• Separation of cations in to groups (Semi-
Micro qualitative Inorganic analysis )

5. Filtration
• Physical method of separation technique

6. Distillation
 SEPARATIONS BASED ON A CHANGE OF STATE
 Since an analyte and interferent are usually in
the same phase, a separation often can be
effected by inducing a change in one of their
physical or chemical states.
 Changes in physical state that have been
exploited for the purpose of a separation
include liquid-to-gas and solid-to-gas phase
transitions. Changes in chemical state involve
one or more chemical reactions.

7.  Changes in Physical State When the analyte
and interferent (matrix) are miscible liquids,
a separation based on distillation may be
possible if their boiling points are significantly
different.
 Changes in Chemical State Distillation,
sublimation, and recrystallization use a
change in physical state as a means of
separation.

9. Solvent Extraction
 Separations Based on a Partitioning Between
Phases
 The most important class of separation
techniques is based on the selective partitioning
 of the analyte or interferent between two
immiscible phases.
 When a phase containing a solute, S, is brought
into contact with a second phase, the solute
partitions itself between the two phases.

10. Solute
(phase-1)
Solute
(phase -2)

11. Partition coefficient:
 An equilibrium constant describing the
distribution of a solute between two phases; only
one form of the solute is used in defining the
partition coefficient (KD).
[solute]phase-2
 KD= --------------------
[solute]phase-1
 If KD is sufficiently large, then the solute will
move from phase 1 to phase 2. The solute will
remain in phase 1, however, if the partition
coefficient is sufficiently small.

12. Liquid -Liquid Extractions
• A liquid–liquid extraction is one of the most important separation
techniques used in environmental, clinical, and industrial laboratories.
• Two examples from environmental analysis serve to illustrate its
importance.
• Public drinking water supplies are routinely monitored for
trihalomethanes (CHCl3, CHBrCl2, CHBr2Cl, and CHBr3) because of their
known or suspected carcinogeneity. Before their analysis by gas
chromatography, trihalomethanes are separated from their aqueous
matrix by a liquid–liquid extraction using pentane.
• A liquid–liquid extraction is also used in screening orange juice for the
presence of organophosphorous pesticides. A sample of orange juice is
mixed with acetonitrite and filtered. Any organophosphorous pesticides
that might be present in the filtrate are extracted with petroleum ether
before a gas chromatographic analysis.

13. Chromatography
• Chromatography,
• Modes Of Separation in chromatography:
adsorption, partition, ion exchange, size
exclusion And electro chromatography.

14. Chromatography
Defination
No.of Phases involved Involved
Chromatography
Classification of Chromatography
Adsorption Chromatography
Partition Chromatography

15. Classification
of
Chromatography
Adsorption
Chromatography
S+L OR S/G
Partition
Chromatography
L+L OR L+G

16. Adsorption
Chromatography
S+L OR S/G
Thin Layer
S+L
Column
Chromatography
S+L
Ion Exchange
S+L Gas Solid
Chromatogra
phy
S+G
Gel
Chromatography

17. Partition Chromatography
L+L OR L+G
Paper
Chromatography
L+L
HPLC
L+L
Gas Liquid
L+G

18. Size exclusion Chromatography
• Separation of molecule takes place according
to molecular size of an analyte.
• Stationary phase : Small, approximately 10-
mm, porous particles of cross-linked dextrin
or polyacrylamide.
• Mobile Phase: Liquid

19.  Size-exclusion chromatography, which also is called gel
permeation or molecular exclusion chromatography, in
which separation takes place according to molecular size
based on size.
 In this technique a column is packed with small,
approximately 10-mm, porous particles of cross-linked
dextrin or polyacrylamide. The pore size of the particles is
controlled by the degree of cross-linking, with greater
cross-linking resulting in smaller pore sizes. The sample to
be separated is placed into a stream of solvent that is
pumped through the column at a fixed flow rate. Particles
too large to enter the pores are not retained and pass
through the column at the same rate as the solvent.
 Those particles capable of entering into the pore structure
take longer to pass through the column. Smaller particles,
which penetrate more deeply into the pore structure, take
the longest time to pass through the column.
 Size-exclusion chromatography is widely used in the
analysis of polymers and in biochemistry, where it is used
for the separation of proteins.

20. Electro chromatography.
• Electrophoresis is another class of separation techniques
in which analyte's are separated based on their ability to
move through a conductive medium, usually an aqueous
buffer, in response to an applied electric field.
• In the absence of other effects, cations migrate toward the
electric field’s negatively charged cathode, and anions
migrate toward the positively charged anode.
• More highly charged ions and ions of smaller size, which
means they have a higher charge-to-size ratio,
migrate at a faster rate than larger ions, or ions of lower
charge. Neutral species do not experience the electric field
and remain stationary. As we will see shortly, under normal
conditions even neutral species and anions migrate toward
the cathode.
• In either case, differences in their rate of migration allow
for the separation of complex mixtures of analyte's.

21. Gel Electrophoresis
• Electrophoresis is the movement of
molecules by an electric current.
• Nucleic acid moves from a negative to a
positive pole.

22. Capillary electro chromatography :A form of capillary
electrophoresis in which a stationary phase is included
within the capillary column.
• Capillary Electro-chromatography Another
approach to separating neutral species is
capillary electrochromatography (CEC). In this
technique the capillary tubing is packed with
1.5–3-mm silica particles coated with a bonded,
nonpolar stationary phase.
• Neutral species separate based on their ability to
partition between the stationary phase and the
buffer solution (which, due to electroosmotic
flow, is the mobile phase). Separations are
similar to the analogous HPLC separation, but
without the need for high-pressure pumps.
Furthermore, efficiency in CEC is better than in
HPLC, with shorter analysis times.

23. Electrophoresis Buffers
• Carry current and protect samples during
electrophoresis.
• Tris Borate EDTA (TBE), Tris Acetate EDTA (TAE), Tris
Phosphate EDTA (TPE) used most often for DNA.
• 10 mM sodium phosphate or MOPS buffer used for
RNA.
• Buffer additives modify sample molecules.
– Formamide, urea (denaturing agents)

24. Capillary Electrophoresis (CE)
• Separates solutes by charge/mass ratio.
• Capillary gel electrophoresis is used to separate nucleic
acids.
=
+ + +
+
+
+
-
-
=

25. Capillary Electrophoresis
Battery
+ + - -
+ --

26. Electrophoresis Equipment
Horizontal or submarine gel

27. Applications
• Electrophoresis is used to separate molecules by
size and/or charge.
• Nucleic acid fragments can be resolved on agarose
of polyacrylamide gels.
• PFGE is used to resolve very large DNA fragments.
• CGE is more rapid and automated than slab gel
electrophoresis.
• The choice of electrophoresis method depends on
the type and size of sample.