Practical Pharmacognosy
nd
2 stage 2 semister
Chromatography
Msc. Arjwan Fouad Hussain
History:
Exp. No.5
Mikhail Tswett, Russian Botanist.
Chromatography
In 1906 Tswett used the chromatography to separate plant pigments
History:
He called the new Tswett,
technique chromatography because the result
Mikhail Russian Botanist (1872-1919).
of the analysisInwas
1906'written
Tswett used the chromatography
in color' to separate
along the length plant pigments
of the adsorbent
He called the new technique chromatography because the result
column.
of the analysis was 'written in color' along the length of the adsorbent
Chroma means “color”
column. Chroma
andmeans “color”
graphein and graphein
means means to “write”.
to “write”.
Importance:
Chromatography has application in every branch of the physical and
biological sciences. 12 Nobel prizes were awarded between 1937 and 1972
Mikhail Tswett separate plant pigment on column of CaCo3 of
solid adsorbent, then pass on solvent (petroleum ether) tried to wash
pigment. Then many rings or spots are separated into many different
colors so the name came from this.
Chromatography is a separation and isolation technique of
organic and inorganic compounds which separate a mixture of
components in to individual components based on physical and chemical
properties.
it is used quantitatively and qualitatively and proved to be more effective
from the other means of separation and identification.
The main uses of chromatography involve: Analytical procedures,
scientific research and Purification.
The separation of a mixture of compounds in chromatography to its
components depend on the action of two forces:
1. Mobile force (driving force) that will try to move the components of
mixture
2. Opposing force (stationary or retardation force) that will try to keep
components in its place depending on many factors:
a. Solubility in mobile phase.
b. Adsorption ability of component to be separated.
c. Ionic forces.
Classification:
There are different types of chromatography:
According to the packing of the stationary phase:
1-Thin layer chromatography (TLC): the stationary phase is a thin
layer supported on glass, plastic or aluminum plates.
2- Paper chromatography (PC): the stationary phase is a thin film of
liquid supported on an inert support.
3- Column chromatography (CC): stationary phase is packed in a glass
column.
According to the mobile phase (modern type of classification).
1. Liquid chromatography.
2. Gas chromatography.
According to the force of separation (classical type).
1. Adsorption chromatography
2. Partition chromatography
3. Ion-exchange chromatography
4. Gel filtration.
5. Affinity chromatography
6. Electrophoresis.
PYPER CHROMATOGRAPHY
Paper chromatography is a method of partition chromatography using
filter paper strips as carrier or inert support.
The factor governing separation of mixtures of solutes on filter paper is
the partition between two immiscible phases.
One is usually water adsorbed on cellulose fibers in the paper
(stationary phase).The second is the organic solvent flows past the
sample on the paper (stationary phase).
Partition occurs between the mobile phase and the stationary aqueous phase
bound by the cellulose.
Techniques of development with various flow directions:
Ascending technique: The paper will be dipped in the solvent mixture
so that the solvent front travels up the paper.
Descending technique: When the trough of solvent may be supported
at the top of the chamber in this case the solvent travels down the
paper.
Radial technique: Circular or horizontal paper chromatography in
another technique used, in which circular filter bearing a wick at the
center of the paper is placed in a petri dish and the solvent system
supplementation is through the central wick.
Two-dimensional technique: When large numbers of substances are
to be separated on a single chromatogram.
https://youtu.be/mz_xcNrTK_U?si=Jv9QhPxgPIqvzMcC
Types of detections:
1. Physical detection: we use the UV light with certain wavelength.
2. Chemical detection: we use a chemical reagent either by spraying or
dipping in both methods the chemical reagent will react with separated
compound and give a color.
3. Biological detection: by using certain microorganisms to detect the
separated compounds.
4. Radioactive detection: if the compound being separated have
radioactivity, such compound can be detected by using special instrument
Retardation factor (Rf-value):
Can be defined as the distance traveled by the compound to the distance
moved by the solvent and it is constant for each compound when
chromatography is carried out using the same technique, mobile phase, and
the same conditions.
Rf = 𝐝𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐭𝐫𝐚𝐯𝐞𝐥𝐞𝐝 𝐛𝐲 𝐬𝐨𝐥𝐮𝐭𝐞 𝐬𝐩𝐨𝐭)
𝐝𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐭𝐫𝐚𝐯𝐞𝐥𝐞𝐝 𝐛𝐲 𝐦𝐨𝐛𝐢𝐥𝐞 𝐩𝐡𝐚𝐬𝐞 ( 𝐬𝐨𝐥𝐯𝐞𝐧𝐭 𝐟𝐫𝐨𝐧𝐭)
Usually the Rf value is used for the identification of the separated compound by
comparison with the Rf value of a standard.
The Rf value is going to change if we:
1. Change the solvent
2. Aging
3. Impurities
4. Temperature
5. Saturation
6. Solvent front must be uniform.
Any Question
