Download Lecture 3

Partition chromatography
 Partition chromatography is carried out on sheets
of filter paper, column or thin layer of powdered
cellulose, moist silica gel or kieselguhr
 Silica acts as support and the water is stationary
phase
 It is known that filter paper consists of numerous
cellulose fibre which accomodate certain
percentage of moisture we can consider that fibre
+ water constitute cells
 Partition of the substance between moisture in
the cell and the solvent flowing over the cells
which bring about separation
 Water of cell is the stationary phase and moving
solvent is the mobile phase
 The extent to which the solute distribute itself
between the two liquid is measured and is known
as partition or distribution coefficient
K=
Conc. of solute in M. Ph
Conc. of solute in St. Ph
 Partition chr. Includes
 A- liquid liquid extraction
 B- column chromatography
 C- paper chromatography
 D- TLC
 E- Gas liquid chromatography
 A- Liquid liquid extraction
 Solute distributes itself between equal volumes
of two immiscible solvents.
 Methods of extraction
 1- discontinuous extraction
 A- single contact
 In separating funnel using
two immiscible liquid
 B- multiple contact
 Several times in separating funnel.
 2- continuous liquid liquid extraction
 1- single stage
 by using liquid liquid extraction app.
 It consists of round bottom Pyrex flask
 Extraction tube connected to the flask by another
glass tube
 Condenser
 2- multiple stage (counter current distribution)
 Several units of extraction app are specially
constructed to transfer the upper phase to the
subsequent tube keeping the lower stationary
phase
 Substance distribute itself between lower
stationary phase and upper according to
distribution coefficient (K)
Counter current extraction app.
Conc. of solute in upper phase
K=
Conc. of solute in lower phase
 The procedure:
 1- the app. Consists of 100 tubes affecting 100
equilibrations
 2- fresh mob. Phase. Is transferred from reservoir
to tube 1
 3- after equilibrium mob. Phase move to fresh
stationary phase, shacked and transferred to a
tube containing stationary phase and so on....
 It depends on
1- number of shacking
 2- time allowed to stand for separation
 Number of equilibrium
B- column (partition chromatography)
 As in column (partition chr.) the difference is the
packing material which is porous material such as
kieselguhr coated with layer of liquid, water, the
stationary phase is the liquid layer and the solid
serves as a support
 More soluble substance travel more slowly down
the column
 Solid support as silica, diatomites, cellulose,
cellulose acetate.
Preparation of the column
 Mixture of support + stationary phase is stirred
with some of mobile phase to be used initially
 The slurry is poured in a small portion into the
tube which contain small portion of the mobile
phase
 Application of the sample
 1- by use of pipette with a bent tip which is
placed against the column wall just above
the adsorbent and the liquid is slowly
drained from the pipette.
 2- use of one or two small disks of filter
paper which perforated with very small
holes of a size which just to fit on the top
of the column
 - Solute is dissolved in volatile solvent
and adsorbed on one of the filter paper
 3- Mixture is dissolved in some of the Mob. Ph.
And mix with some of the stationary phase , the
dry and put the powder on the top of the column.
 Elution
 As in adsorption column
 Identification of the separated substance
 as in adsorption column
Applications of partition column
 1-
fractionation of many alkaloids and
polyphenols
 2- determination of amino end group of proteins
and analysis of hormones
 3- separation of methylated sugars on silica gel
and free sugar on cellulose.
Paper chromatography
 Stationary phase is water hold by adsorption on
cellulose molecules which in turn are kept in a
fixed position by the fibrous structure of the
paper
 Apparatus and method
 Tightly covered jar
 Method
 1- choice of the paper
 2- purification of the sample
 3- application of the sample on the paper
 4- development
 5- detection
 6- identification
 1- Choice of the paper
 Whatman No 1 or 3 for
 analytical
 Whatman No 3 or 3MM for
 preparative
 2- choice of solvent system
 Depend on substances to be separated
 Amino acid..phenol/ water
Butanol / water/ acetic acid
 Sugars...EtOAc/ pyridine/ water
EtOAc/ i-prppanol/ water
 3- sample purification
 By column or liquid liquid extraction
 4- application of the sample to the paper
 Clear solution is prepared
 Take sample by capillary
 Apply the spot on the paper and dry it.
4- Development
A- ascending the flow upward
b- descending downward
C- horizontal
D- radial the flow in the radii of circular paper
 1- ascending development
 Paper is stood vertically and immersed in the
mobile phase
 Level of the spots should be above the liquid by 12 cm
 2- descending development
 Jar is provided by a trough for
the mobile phase in its upper
part
Paper suspended in
trough
wire fixed to side of the tank
on which trough can fest
Serrated edge to allow the
solvent to flow uniformely
off
the paper
 Advantages of descending technique
A- solvent flow faster
B- long distance of development can be achieved 
 3- Horizontal development:
 Shallow tank of glass or metal
 The paper is placed horizontal on glass rods with
one end of the paper is dipped in the mobile
phase
Direction of mobile phase
paper
support
mobile phase
 -sample is spotted on a line on the horizontal
part of the paper 1-2 cm from the edge
 - solvent is flows up by capillary forces
 4- radial development
 The principle of this method is based on
migration of the spot from the centre of the paper
toward the periphery.
.
Detection of the resolved spots
visualization
 1- physical , UV, colour






2- chemical
Aniline phthalate...........reducing sugars
Dragendorff`s..................alkaloids
FeCl3.....................phenolic compounds
Ninhydrin...................amino acid, amino sugars
Anisaldehyde H2SO4......terpene, sterols, sugars
(Not used in paper as it cause charring of the
paper)
 3- biological enzymatic detection of amylase
microbilogical detection of antibiotic
 Uses of paper chromatography
 1- identification of components of mixture
 2- detection of purity of components against
authentic
 3- major use is the separation of flavonoids and
sugars