Analytical Technique

1. Submitted by : Pragya Sharma
M.Pharm 1st sem.
B.N. College of Pharmacy
Udaipur(Raj)

2. CONTENT
• Introduction
• History
• Principle
• Types of paper chromatography
• Instrumentation
• Methodology
• Modes of paper chromatography
• Application
• References

3. Chromatography
 Chromatography is an laboratory technique for the
separation of a mixture. The mixture is dissolved in a fluid
called mobile phase , which carries it through a structure
holding another material called stationary phase .
 The various constituent of the mixture travels at the
different rate , causing them to separate.
 The separation is based on the difference in the
partition coefficient of stationary and mobile phase.

4. Paper chromatography
 Paper chromatography is define as a technique in which
the analysis of unknown substance is carried out mainly
by the flow of solvent on specially designed filter paper .

5. History
 Paper chromatography was first introduced by german
scientist christian friedrich (1865).
 Paper chromatography is considered to be a simplest and
most widely used chromatographic technique because of
its applicability to isolation ,identification, and
quantitative determination of organic and inorganic
compound .

6. Types of paper chromatography
 There are two types of paper chromatography
1. Paper absorption chromatography: Paper coated with
silica and alumina act as absorbent (stationary phase) and
solvent as mobile phase
2. Paper partition chromatography : moisture and water
present in the pores of cellulose fibers present in filter paper
act as stationary phase and another mobile phase is used as a
solvent.

7. Principle of sepration
 The principle of sepration is mainly partition
rather than adsorption .
 Cellulose layer in filter paper contain moisture
which act as stationary phase and organic
solvent/buffers are used as mobile phase.

8. Instrumentation
1) Stationary phase and paper used
2) Application of sample
3) Mobile phase
4) Development technique
5) Detection or visualizing agent

9. Stationary phase and paper used
 Whatman filter papers of different grades like no.1,
no.2,no.3,no.4,no.20,no.40,no.42 etc are used.
 In general this paper contains 98 – 99% of α-cellulose ,
0.3- 1% beta cellulose
Factors that governs the choice of paper:
Nature of sample and solvents used.
Based on quantitaive and qualitative analysis
Based on the thickness of paper .

10. Types of papers used in paper
chromatography
 Modified papers – Acid or Base washed filter paper .
 Hydrophilic papers – Papers modified with methanol ,
glycol , glycerol etc.
 Hydrophobic papers – Acetylation of OH group leads to
hydrophobic nature , hence can be used for reversed phase
chromatography .
 Coating of silica , alumna can also be made.

11. Preparation of paper
 Cut the paper into desired shape and size .
 The starting line is marked on the paper with pencil 5cm
from the bottom edge .
 On the starting line marks are made 2cm apart from each
other .

12. Preparation of the solution
 Choice of suitable solvent for making solution is very
important . Pure solution can also be applied direct on the
paper but solids are always dissolved in small quantity of
suitable solvent .
Application of sample
 The sample to be applied is dissolved in mobile phase and
applied as a small spot on the origin line using capillary tube or
micropipette .
 Very low concentration is used to avoid larger zone.
 The spot is dried on the filter paper and placed in developing
chamber .

13. Choice of solvent
 The commonly employed solvents are the polar solvent but the
choice depend on the nature of the substance to be separated .
Mobile phase
 Pure solvents , buffer solutions or mixture of solvent
 Example – Hydrophilic mobile phase
 Isopropanol : Ammonia : Water 9:1:2
 Methanol : Water 4:1
Hydrophobic mobile phase
Dimethyl ether : Cyclohexane
Kerosene : Isopropanol

14. Chromatographic chamber
 This is made up of many materials like glass , plastic ,
stainless steel .
 Glass tanks are preferred most . They are available in
various sizes depending on paper length and development
type.
 The chamber atmosphere should be saturated with solvent
vapor

15. Development technique
 Descending type
 Development of the chromatogram is done by allowing the solvent to travel
down the paper. Here, mobile phase is placed in solvent holder at the top.
The spot is kept at the top and solvent flows down the paper from above.

16. Ascending type
Here the solvent travels up the chromatographic paper. Both descending and
ascending paper chromatography are used for the separation of organic and
inorganic substances. The sample and solvent move upward.

17. Ascending-descending type
This is the hybrid of both of the above techniques. The upper part of ascending
chromatography can be folded over a rod in order to allow the paper to become
descending after crossing the rod.

18. Circular chromatography type (Radial)
A circular filter paper is taken and the sample is deposited at the center of the paper.
After drying the spot, the filter paper is tied horizontally on a Petri dish containing
solvent, so that the wick of the paper is dipped in the solvent. The solvent rises
through the wick and the components are separated into concentric rings.

19. Two-dimensional type
In this technique a square or rectangular paper is used. Here the sample is
applied to one of the corners and development is performed at a right
angle to the direction of the first run.

20. Drying of chromatogram
 After the solvent has moved a certain distance for certain
times the chromatogram is taken out from the tank and
position of solvent front and marked with pencil .
 They are dried by hot and cold air depending on volatility
of solvent .

21. Detecting / visualizing agents
 If the substance are colored they are visually detected
easily .
 For colorless substance physical and chemical methods
are used .
 Physical method –
 Example – Iodine chamber and UV chamber method
 Chemical method –
 Example – Ferric chloride for phenolic compound and
Ninhydrin in acetone for amino acids .

22. Rf value (Retardation factor )
 In paper chromatography the result is represented by Rf
value which represent the movement of solute relative to
the solvent

23. Factors affecting Rf value
i. The temperature .
ii. The purity of solvent used .
iii. The quality of paper , adsorbents and impurities present
in adsorbents .
iv. Chamber saturation techniques , method of drying and
development .
v. The distance travelled by the solute and solvent.
vi. pH of the solution .

24. Applications
 Separation of mixture of drug
 Separation of carbohydrates ,vitamins , proteins etc.
 Identification of drug .
 Identification of impurities.
 Analysis of metabolities of drug in blood or urine.