Chromatography and separation techniques
PHCG 323
PHCG 323.
 The course aims to ensure the students have the necessary knowledge about
quality control from herbal aspects. It also covers modern chromatographic
and separation techniques employed for evaluation of natural products and
their constituents: Adsorption chromatography, partition chromatography,
paper chromatography, thin layer chromatography, gel chromatography and
ion exchange chromatography. Also, the advanced types and their
applications: High performance (pressure) liquid chromatography (HPLC),
Gas chromatography (GC) as well as Liquid chromatography Mass spectrum
(LCMS). Students have laboratory activities for these types of
chromatography.
 It includes an overview on forensic pharmacognosy including plants and their
natural products that constitute health hazards, or intended for criminal uses
to produce abortion, loss of mental control, hallucination, and heart arrest.
Also, it includes Mycotoxins as a series threat to general health and safety of
community, contamination of food materials and poisonous fungi.
Plant constituents
 Phenolic compounds
Examples are flavonoids and their glycosides
 Terpenoids (or isoprenoids)
They are classified according to the number of their isopentene ( or isoprene,
C=5 ) units.
Terpenes
Isoprene
units
Carbon
atoms
1
Monoterpenes
2
10
2
Sesquiterpenes
3
15
3
Diterpenes
4
20
4
Sesterpenes
5
25
5
Triterpenes
6
30
6
Carotenoids
8
40
7
Rubber
> 100
> 500
 SESQUITERPENES
 DITERPENES
Abietic acid
 TRITERPENOIDS
 CAROTENOIDS
 Organic acids, lipids and related compounds
 Examples of simple organic acids are citric, oxalic and ascorbic acids
 Fatty acids occur mainly as esterified forms with glycerol or higher
aliphatic alcohols in the form of oils, fats or waxes.
 Nitrogen containing compounds
Examples are amino acids, peptides and alkaloids
Extraction of the plant material
 Extraction is the process of separating the medicinally active constituents of plant or animal tissues
from the inactive components by using selective solvents.
Preparation of plant sample
1. Careful selection of the plant material to avoid the use of contaminated or infected samples
2. Careful authentication of the selected sample by taxonomic expert.
3. Direct immersion of fresh material in boiling alcohol with few minutes of its collection to stop
enzymatic activity.
4. Drying of plant material as quickly as possible in a good air.
Selection of the solvent
 Non-polar solvents (e.g. hexane) dissolve non-polar compounds ( fats and waxes).
 Polar solvents (e.g. methanol and water) dissolve polar compounds ( sugars and alkaloidal salts).
The solvents in order of increasing ploarity
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hexane (most non polar)
cyclohexane
Carbon tetrachloride
benzene
chloroform
ether
ethyl acetate
acetone
ethanol
water
acids and bases (most polar)
Principal methods of extraction
 Maceration
The ground plant material is immersed in a suitable solvent in a stopper container for at least three
days with frequent agitation.
 Digestion
It is a form of maceration in which gentle heat is used, to increase efficiency of the solvent without
alteration of active principles.
 Infusion
It is maceration for a short time with either cold or hot water as in tea.
 Decoction
This is useful for the harder plant parts such as roots, bark when boiling with water for 15 min. will
extract their active ingredients
 Continuous hot extraction
It is a repeated infusion with hot solvent. It carried in a Soxhlet extractor apparatus.
For thermolabile constituents, a low-boiling point solvent is used. Boiling solvent vapors rise up through
the larger side-arm
Soxhlet extractor
CENTRIFUGE
 CENTRIFUGATION
Centrifugation is used when we want to separate small amounts of suspension. The suspension
of solid in liquid is poured into a centrifuge tube. The spinning motion forces the solid to the bottom
of the tube. Then the liquid can be poured off from the solid.
 Extraction by distillation
 Steam distillation
It is used to isolate volatile oils.
Fractionation of crude extracts
 The crude extract is clarified by filtration and concentrated under reduced pressure in
a rotary evaporator at temperature less than 40 ºC.
 By mixing with a less polar miscible solvent causing the precipitation of the less
soluble plant constituents e.g. precipitation of saponin mixtures by addition of
acetone to the methanolic extract. Also, Precipitation of gum by addition of
alcohol to the aqueous extract.
 By mixing with a more polar solvent e.g. precipitation of resins by addition of water to
the alcoholic extract
Sublimation
It can be applied to the whole drug e.g. isolation of caffeine from tea and
balsamic acids from balsams
Liquid / liquid extraction
 It involves the partition of the solute molecules between two immiscible solvents.
Chromatography
• Chromatography is a technique for the separation of mixture by distributing the
components between a stationary phase and a mobile phase.
• The mixture is first placed on the stationary phase (a solid or a liquid) and then the
mobile phase (a gas or a liquid) is allowed to pass through the system.
Classifications
• TLC Thin layer Chromatography
• CC Column Chromatography
• PC Paper Chromatography
• LC Liquid chromatography
• LSC Liquid solid chromatography
• LLC Liquid Liquid chromatography
• GC Gas Chromatography
• GSC Gas solid Chromatography
• GLC Gas Liquid Chromatography
• HPLC High performance Chromatography
Types of chromatographic techniques
I. According to the method of holding the stationary phase
1. Column chromatography
2. Planar Chromatography
II. According to the physical state of mobile phase
1. .Gas chromatography (GC)
2. .Liquid chromatography (LC)
Packing the Column
Slurry packing
• The adsorbent is slurred with the solvent and added to the column in portions.
Dry packing
• The adsorbent is added in portion with vibration or tamping until the column is
sufficiently filled.
• The column is washed with the first solvent.
Wet packing
The solvent to be used is first added and then the adsorbent is added in portions
Slurry packing
Dry packing
Wet packing
Thin layer chromatography (TLC)
Rate of flow
Distance traveled by substance
Rf = Distance traveled by
solvent