2. CHROMATOGRAPHY
• separation technique
• mobile phase carrying a mixture is caused to move in contact
with a selectively absorbent stationary phase.
• STATIONARY PHASE- a solid adsorbent, is placed in a vertical
glass (usually) column.
• MOBILE PHASE- a liquid, is added to the top and flows down
through the column by either gravity or external pressure.
3. COLUMN CHROMATOGRAPHY
• purification technique
• separated into two compositions: STATIONARY PHASE AND MOBILE
PHASE
• separated into two categories: (depending on how the solvents flows
down the column)
• GRAVITY COLUMN CHROMATOGRAPHY- if the solvent is allowed to flow
down the column by gravity, or percolation.
• FLASH CHROMATOGRAPHY- if the solvent is forced down the column by
positive air pressure.
4. PROCEDURE
1. The mixture to be analyzed by column chromatography is placed
inside the top of the column.
2. The liquid solvent (the eluent) is passed through the column by gravity
or by the application of air pressure.
3. An equilibrium is established between the solute adsorbed on the
adsorbent and the eluting solvent flowing down through the column.
4. Due to the different mixture component interaction, with the stationary
and mobile phases, they will be carried along with the mobile phase to
varying degrees and a separation will be achieved.
5. The individual components, or elutants, are collected as the solvent
drips from the bottom of the column.
5. INTERPRETATION
• In a mixture of mobile phase and a sample, the
component with lower affinity and adsorption to
stationary phase move faster and eluted out first while
those with greater adsorption affinity move or travel
slower and get eluted out last.
6. RATE OF MOVEMENT
R= rate of movement of a component
t2= retention time of component 2
t 1= retention time of component 1
W2 and W1 = Corresponding widths at the bases or peaks obtained
by extrapolating the relatively straight sides of the peaks to the
base line.
R = 2 (t2 – t1)
W2 + W1
8. PARTITION CHROMATOGRAPHY
• solute are separated based on their partitioning between a liquid
mobile phase and a liquid stationary phase coated on a solid
support.
• NORMAL: Analyte is non-polar organic;
Stationary phase more polar than mobile phase
• REVERSE: Analyte is polar organic;
Stationary phase less polar than the mobile phase
9. PRINCIPLE
• separation of components of a sample mixture occurs because of partition.
stationary phase is coated with a liquid which is immiscible in mobile phase.
• partition of component of sample between sample and liquid/gas stationary
phase retard some components of sample more as compared to others. this
gives a basis for separation.
• the stationary phase immobilizes the liquid surface layer, which becomes
stationary phase. mobile phase passes over the coated adsorbent and
depending upon relative solubility in the coated liquid, separation occurs. the
components of sample mixture appear separated because of differences in their
partition coefficient.
10. PROCEDURE
1. Clean and dried chromatography jar is taken.
2. A paper impregnated in the mobile phase is applied to the walls to ensure that
atmosphere of the jar is saturated with solvent vapors.
3. Mobile phase is added to the jar at a length of 0.5-1cm from the bottom.
4. Jar is closed.
5. Equilibrium is allowed to be maintained.
6. Base line is marked on adsorbent.
7. Sample is applied on paper with help of capillary tube.
8. Sample spot is air dried.
9. Paper is put in the chromatography jar and lid is closed.
10. The system is allowed to be static until the solvent move to a proper distance from
baseline.
11. Paper is taken out and dried.
11. INTERPRETATION
• based on the determination of peaks due to impurities, expressed
as a percentage of the area due to the drug peak.
• It is preferable, however, to compare impurity peaks to the
chromatogram of a standard at a similar concentration.
• The standard may be the drug itself at a level corresponding to,
for example, 0.5% impurity, or in the case of toxic or signal
impurities, a standard of the impurity itself.
12. INSTRUMENTATION
CHROMATOGRAPHY JAR - jar maintains proper environment that is
required for separation.
CAPILLARY TUBE - used to apply sample mixture
STATIONARY PHASE (LIQUID IMPREGNATED PAPER)
MOBILE PHASE
13. APPARATUS USED IN PARTITION
CHROMATOGRAPHY
• HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
