Lesson 3
METHODS OF PURIFICATION AND SEPARATION TECHNIQUES
( CHAPTER 4 )
Today
In this Chapter ……
Chromatography
Types of Chromatography
Uses of Chromatography
What is Chromatography ?
It is a separation technique that is used to separate components in the given mixture .
The mixture is dissolved in a fluid ( solvent, water, ...) called the mobile phase,
which carries it through a system (a column, a capillary tube, a plate, or a sheet) on
which is fixed a material called the stationary phase.
Types of Chromatography
► Ascending Paper Chromatography
► Descending Paper Chromatography
► Radial Paper Chromatography
► PROCEDURE
► In all these methods , Base line is drawn with a lead pencil above the solvent level .
► A small spot of mixture is introduced on the base line .
► The chromatogram is suspended in the given solvent system contained in a glass
equipment ( gas jar in case of ascending and descending paper chromatography while
petri dish in case of Radial Paper Chromatography )
Ascending Paper Chromatography vs
Descending Paper Chromatography
► In ascending chromatography, for the separation of components, the solvent
moves upward by capillary action on the stationary phase. In descending
chromatography, the mobile phase travels along with the gravity by which the
separation takes place.
Ascending Paper Chromatography
Descending Paper Chromatography
Radial Paper Chromatography
Uses of Chromatography
► Used to check the legality of the sample .
► Used to identify blood and urine samples
► Used for separation of components of the mixture
Some Important Questions ?
► 1. Why do you think the Base line is drawn with a lead pencil and above the
solvent level in chromatography ? ( 2 marks )
► Ans ; The Base Line is drawn with a lead pencil as the ink smudges . It is drawn
above the solvent level because at the solvent kevel the mixture spots may smudge
in the start of the experiment and ruin the results .
► 2. Which piece of equipment is used for spotting in Chromatography ? ( 1
mark )
► Ans : Capillary tube
Questions !
► Q3. What is Solvent Front ? ( 2 marks )
► It is the maximum distance travelled by solvent on the chromatogram .
► Q4. Why do you think the mixture spots should be small ? ( 2 marks )
► The bigger mixture spots smudge and ruin the results . The separation does not take place
properly .
► Q5. what is Rf?
► It stands for Retention Factor or Rate of flow of substance on the chromatogram. It is a
ratio so has no units . For calculation of Rf all distances are to be measured from the
BaSe Line .
Solvent System used for Chromatography
Chromatography is based upon the principle of solubility .
More soluble component will travel more distance on the chromatogram .
The Solvent Systems used are :
1. Ethanol
2. Water
3. Mixture of Ethanol and Water
Rf Value
Locating Agent
► Locating Agent is used to locate the colorless mixture components in
chromatography .
► E.g Ninhydrin Reagent is used to locate mixture of Amino Acids
Thank You !
► That is it from today and from the chapter !
► Keep Revising
