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The document provides an overview of chromatography, a separation technique developed by Mikhail Tswett in 1906 for isolating plant pigments. It discusses various types of chromatography, including thin layer, paper, and column chromatography, as well as detection methods and the importance of the retardation factor (Rf-value) in identifying compounds. Chromatography is widely used in analytical procedures, scientific research, and purification across various scientific disciplines.

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2 views

Presentation 3

The document provides an overview of chromatography, a separation technique developed by Mikhail Tswett in 1906 for isolating plant pigments. It discusses various types of chromatography, including thin layer, paper, and column chromatography, as well as detection methods and the importance of the retardation factor (Rf-value) in identifying compounds. Chromatography is widely used in analytical procedures, scientific research, and purification across various scientific disciplines.

Uploaded by

moqtadamhmmd
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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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

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