chapter7

 Samples to be analysed are usually a mixture

of many components in a complex matrix.
 For samples containing unknown compounds,
the components must be separated from each
other so that each individual component can
be identified by other analytical methods.
 Measurement techniques used for chemical
analysis are usually specific for a single
chemical species.
 The presence of a foreign species will
interfere the analytical signal.
 This foreign species is called interferent.
 An Interferent is a chemical species that
courses a systematic error in an analysis by
enhancing or reducing the analytical signal.
 A mixture can be separated using the
differences in physical or chemical properties
of the individual components.
 Example of physical properties that are useful
for separations are density and size.
 Some useful chemical properties by which
compounds can be separated are solubility,
boiling point, and vapor pressure.
 Masking
 Precipitation
 Distillation
 Solvent extraction
 Ion exchange
 Chromatography
 Electrophoresis
 Chromotography is the separation of
components of a mixture based on the
different degrees to which they interact with
two separate material phases
 The two phases are:
1) the stationary phase
2) the mobile phase.
 The stationary phase – a phase that is fixed in
place either in a column or on a planar
surface . The stationary phase is either a
porous solid used alone or coated with a
stationary liquid phase.
 The mobile phase- a phase that moves over
or through the stationary phase, carrying with
it the analyte mixture.
It is also called the eluting fluid. The mobile
phase can be a gas, a liquid, or a supercritical
fluid.
 In Column chromatography- the stationary
phase is held in a narrow tube and the mobile
phase is forced through the tube by gravity.
 In Planar chromatography the stationary
phase is supported on flat plate or in the
pores of paper ( Paper chromatography and
Thin layer chromatography)
 Mechanism by which separation takes place
can be divided into two types:

Partition
Adsorption
 This form of chromatography is based on a
thin film formed on the surface of a solid
support by a liquid stationary phase.
 Solute equilibrates between the mobile
phase and the stationary liquid.
 In partition chromatography the solute
moves to and pro between the stationary
phase and the mobile phase and are
partitioned between them.
 Both stationary and mobile phases are liquid.
 The rate of movement depends on relative
solubility of the solute in the two phases.
 In the adsorption process, the solute
molecules are held on or attached to the
surface of the stationary phase.
 Because of the different degree of
intermolecular attraction, some
components of the mixture will be more
attracted or adsorbed to the stationary
phase.
 The stationary phase is a polar solid and the
solutes are polar molecules.
 The separation of the solutes depends on the
difference in their polarity. The more polar
solutes are more readily adsorbed than the
less polar solutes.
 The equilibration between the mobile and
stationary phase accounts for the separation
of different solutes
 In normal phase chromatography the
stationary phase is POLAR and the mobile
phase is NONPOLAR

 In reverse phase chromatography it makes

use of the NON-POLAR STATIONARY phase
and the mobile phase a POLAR LIQUID
 In this technique, a solution of a sample
mixture to be separated is applied to a strip
of chromatography paper.
 The paper is then hung in a glass tank so that
the end dips into a solvent (the mobile phase)
at the bottom of the tank.

 As the solvent rises up through the paper, it
meets the sample mixture, and the
component bands spread out.
 Filter paper can also be used because the
paper is made of cellulose fibers which
contain trapped water molecules.
 The trapped water acts as the stationery
phase and the filter paper itself is the
support.
 The mobile phase is the liquid solvent that
moves over the paper
 Paper chromatography is of type partition
chromatography, in which the component
to be separated are partitioned between the
stationary phase (water absorbed on paper)
and the mobile phase ( the solvent).

 Paper chromatography is a liquid-liquid

chromatography. If a component is more
soluble in the mobile phase, the component
will be carried further up on the paper and
will be separated from the other
components.
 Thin layer chromatography is of the type
planar chromatography.
 In this technique, a small quantity of
solution of mixture to be analyzed is
deposited as a small spot on a TLC plate
 TLC plates consist of a thin layer of silica
gel or alumina (stationary phase) coated on
a glass plate or plastic sheet.
 Preparing and activating TLC plates
 Spotting the TLC plates with sample
 Developing the TLC plates
 Drying the plates
 Visualizing the components spots
 Identifying the components
Pre coated TLC plates are available
commercially.
The most common TLC plates are coated
with a thin layer of silica (SiO2) or aluminium
oxide (Al2O3) .
These polar solids constitute the stationary
phase.
Other types include cellulose TLC plates.
 TLC plates can also be prepared in the lab.
The steps involved are as follows:
a) The glass plates/plastics sheets are
cleaned with soaps and any trace
of grease/oil on the plates is
removed by using acetone.
b) An aqueous slurry of powder ( SiO2 or
Al2O3) is prepared by mixing with a binder
such as plaster of paris, gypsum, or
poly(viny alcohol) to help it adhere to the
backing material.

c) The slurry is spread on the plate to form

a thin film (0.1–0.3 mm thick) by using a
spreader to assure uniform thickness.
d) The TLC plates are then activated
To activate the plates; the plates are air dried
for ½ hour and then are heated in the oven at
100 oC for another ½ hour .
They are cooled in a desiccators before use.
a) Spotting a TLC plate first involves preparing
a glass spotting capillary. (available
commercially)
b) These capillaries are made using melting
point capillary which has been elongated
and thinned down to a small diameter
capillary tube by heating.
c) To spot a TLC plate, obtain a silica gel TLC
plate (4 cm x 10cm), draw a straight line
parallel to the short dimension of the plate
with a pencil.
d) Load the glass capillary tube with the sample
mixture solution.

e) Hold vertically the capillary tube containing the

sample mixture close to the line drawn on the TLC
plate and slowly let the tube touches the plate.

f) The sample mixture will be deposited

on the plate as a small spot.

g) Allow the solvent to evaporate from the spot.

 Prepare the solvent system/ the mobile
phase.
 Eg. For small scale prepare about 10 mL of
solvent
 n-hexane : dichloromethane ( 9:1)
 pet. Ether : methanol ( 8.5 : 1.5)
 H2O : NH3 ( 8: 2)
 Mix the solvent well and pour the solvent
system into a closed jar
 Place the TLC plate that has been spotted
inside the jar. Make sure the solvent level is
below the starting line ( below the spot)
 The mobile phase (eluent) will travel up,
moving the components of the sample at
various rate because of the different degree
of interaction with the stationary phase and
also solubility in the solvent (mobile phase).
 When the solvent has traveled up the plate
until ~ 1 cm from the top edge , the plate is
taken out and the solvent front is marked
immediately.
 The different components that have been separated
on the plate must be marked immediately.

 Sometimes the components on the plate are

colorless and can not be seen with naked eyes.
The separated spots can be marked or indicated by

i) placing the plate (with fluorescent indicator

added) under UV light.
ii) Spraying the plate with concentrated sulphuric
acid on hot plate.

iii) Exposing the plate to iodine vapour whereby

the iodine vapour will stain the spot)
 The different component in the mixture are
identified by :
a) comparing the positions of spot on
the plate with those of known
pure compound (standard)

b) measuring the Rf value and compare

with the Rf value of known compound.

 Rf stands for Retention factor
Rf = distance moved by centre of solute spot
 distance moved by front of mobile phase.
 Column chromatography is used by organic
chemists to purify liquids (and solids.)

 An impure sample is loaded onto a column

of adsorbant, such as silica gel or alumina.

 An organic solvent or a mixture of solvents

(the eluent) flows down through the
column.
 Components of the sample separate from
each other by partitioning between the
stationary packing material (silica or
alumina) and the mobile eluent.

 Molecules with different polarity partition to

different extents, and therefore move
through the column at different rates. The
eluent is collected in fractions.
 Ion exchange chromatography is based on
the exchange of ions between the mobile
phase containing the analyte and the
stationary ions-exchange resin.

 IEC is a separation technique in which an

ion from a mobile phase is separated by
exchanging position with another ion
localized on a stationary phase.
H+…[solid-resin] + Na+ Na+…[solid-resin] + H+
 In cation-exchange chromatography, the stationary
phase, which consists of a large quantity of acid
groups attached to a polymeric resin, is slurried
with water and applied to a column.

 The mobile phase, which contains the inorganic

salt dissolved in a suitable solvent, is applied to the
column.

 As the mobile phase passes through the column,

exchange between the H+ ions on the polymeric
ion-exchange resin of the stationary phase and the
cations of the salt in the mobile phase occur.

 The solution which is collected at the
bottom of the column contains the acid
form of the inorganic salt.
 The newly formed acidic solution can then
titrated using a standardized base to
determine the number of moles present in
the sample.
 The molecular weight of the substance and
the identity of the unknown can than be
determined based on the number of moles
and the weight of the sample.
 Synthetic ion-exchange resins are high-
molecular weight polymers containing
large numbers of ionic functional group

Functional group Functional group

Of Cation-resin Of Anion-resin

Acidic group Basic group

(-SO3- H+) [-N(CH3)3+OH-]
(-COOH ) [-N(CH3)2OH-]
Cation-exchanger resin- has a replaceable H+ ion that can be
exchanged with metal from the mobile phase.
[cation- resin]SO3-H+ + M+ [cation-resin]SO3M + H+


Cu2+ , Fe2+ H+ , Cu2+

 Anion-exchanger resin has a replaceable
OH- that can be replaced /exchanged with
anion from the mobile phase.

[anion resin]NH3+OH- + A- [anion resin]NH3+A- + OH-

 Water Purification (producing deionised
water)
 Water softening
 Determination of concentration of trace
ionic material.
 For analytical separation.
 The process to produce deionised water from
tap water.
 The tap water is passed through a mixed-bed
ion exchange resin (commercially available)
that contains both cation and anion exchange
resin .
 .
 When the tap water which may contain salts
such as CaCl2 is passed through the IEC, the
cation ca2+ is exchanged for two H+ ions
while the two anions Cl- are exchanged for
two OH- ions.
 The net result is that the salt CaCl2 is
exchanged for H2O
 A 20 mL sample of seawater was passed
through a cation-exchange resin in the
hydrogen –ion form and the column was
rinsed with some distilled water.
The solution collected required 37.30 mL
0.150 M NaOH for titration to the end-point.
Assuming the seawater contains only NaCl
salt, calculate % w/w of salt content in the
seawater whose density is 1.15 g/mL.
First step:
ion -exchange (cation is exchanged with
H+)
Na+(from seawater) + H+…[solid resin]
 Na+…[solid resin] + H+

Second step : titration with NaOH

 H+ + OH-  H2O
 mol of OH- = mol of H+ released
 = mol of Na+ exchanged
= 37.30 x 10-3 L x 0.150 mol/L
= 5.595 x 10-3 mol

 mol of Na+ exchanged = mol of salt in

seawater =5.595 x 10-3 mol
 mass of NaCl =
5.595 x 10-3 mol x 58.5 g/mol = 0.3273 g

mass of seawater = volume x density

20 mL x 1.15 g/mL = 23 g

% NaCl = 0.3273 g x 100

23 g
= 1.42 %
 200 mL of spring water sample was passed
through a cation-exchange resin in the
hydrogen –ion form. The column was then
eluted with distilled water and collected in a
500 mL beaker. The eluted was then titrated
with 0.06 M NaOH solution and needed
25.25 mL to reach the end point. Calculate
the hardness of the water sample.
 ( ans ;ppm CaCO3 = 378.75 )