College of pharmacy/NUST

Pharmacognosy lab number :4

BY:MSc: Hussein Kamil Hamid

Separation & isolation of the constituents

Different methods may be used in this matter ex:

1. Sublimation: which is sometimes used on the whole drug , as in the isolation of caffeine from
tea .
2.Distillation:fractional distillation has been traditionally used for the separation of the components
of volatile oils.
3.Fractional liberation: some groups of compounds may be separated by fractional liberation from
a mixture ex: when a mixture of alkaloid bases is shaken with NaOH solution the phenolic alkaloids
will be separated as salts.
4. Fractional crystallization: the method exploits the differences in solubility of the components
of a mixture in a particular solvent.
5. Chromatography: the components of the sample mixture are distributed between two phases, a
moving (mobile) phase that is passed over an immobile (stationary) phase. Separation is based on
the characteristic way in which compounds distribute themselves between these two phases.
The stationary phase can be either solid or liquid & the mobile phase can be either a liquid or gas
thus several combinations are possible. The two combinations which are not possible are the gas-
gas & the solid-solid.
Classification of chromatography according to the packing of the stationary phase:
1-Paper chromatography (PC): the stationary phase is a thin film of liquid supported on an inert
support.
2-Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass,
plastic or aluminum plates.
3-Column chromatography (CC): stationary phase is packed in a glass column.
 Thin layer chromatography

TLC is type of adsorption chromatography method of separation , isolation & identification of many
components in specific sample.
1. Stationary phase = a piece of glass, metal, or plastic coated with a thin, uniform layer of a
solid adsorbent.

❖ Usually silica gel (SiO2), alumina (Al2O3), or cellulose

❖ A substance which fluoresces under UV light often incorporated into the stationary
phase like Zinc sulfide
2. Mobile phase = suitable liquid solvent or mixture of solvents.

TLC Procedure

A) Choose a container that is large enough and can be sealed.

B) Add the a few cm of the mobile phase solvent to the chamber.

C) Seal the chamber and allow it to sit overnight if possible in order to let the atmosphere of the
chamber to saturated with the solvent vapors before running samples. Also you may line part
of the inside of your chamber with filter paper to aid in this saturation process and this have
two benefit:
❖Stops the solvent from evaporating as it rises up the stationary phase plate.
❖Allows for better development of the chromatograms.
D) Pointe the base line on TLC plate and activate it by heating in an oven for approximately 10
minutes at 110˚C to remove moisture.
E) If the sample isn’t in solution, dissolve it in an appropriate solvent and by using capillary tube
make a spotting of sample on the base line present on TLC plate and make sure the sample
spot should be dry before continuing the process.
F) When the sample spot has dried, the TLC plate is placed into the chamber containing the
solvent. It is important that the sample spot is above the level of the solvent.
G) Allow the solvent to rise until it almost reaches the top of the plate.
H) Remove the plate from the chamber and mark the position of the solvent front and allow it
to dry.
I) Visualizing: Two ways to make visualization of separated compounds:

A) Use fluorescence: Substance like zinc sulfide which can fluoresce under UV light is
added to stationary phase So, when the TLC plate is exposed to UV light, the entire plate
will glow. On the final chromatogram, the glow will be masked at positions were spots
are located.

B) Use chemical methods: In some cases it may be possible to visualize the spots by reacting
them with something that produces a colored product like Iodine vapors or Dragendorff's
reagent for alkaloids
 Finally :Interpreting the Results

◼ Calculate the Relative mobility (Rf )

◼ Distan ce traveled by the compound

Rf =
Distance traveled by the solvent
Note: The Rf value is a characteristic for any given compound on the same stationary phase using
the same mobile phase for developing the plates. Hence known Rf value can be compared to those
of unknown substance to aid in their identification.

◼ If two substances have the same Rf value they may or may not be the same compound.
◼ The larger the Rf value, the farther the compound traveled up the plate.
◼ An Rf value is a physical property that can be used for identification purposes. But it does
depended on the conditions under which it is measured.
◼ If two substances have different Rf values they are definitely not the same compound.