study of chromatography

1. Chromatography
presented by:
ramza rahat hashmi
Department of pharmacy
IIMT COLLEGE OF PHARMACY
Greater noida

2. Introduction:
• It was first invented by M.tswett.
• In 1906 he was successful in doing the separation of
chlorophyll, xanthophyll.
• Chromatography defined as separating a mixture of
components in to individual components through
equilibrium distribution between two phases.

3. Classification of chromatographic
technique:
• Partition chromatography
• Adsorption chromatography

5. Paper chromatography:
Principle:
• Chromatography that uses paper sheet or strips
as the adsorbent being the stationary phase
through which a solution is made to pass is called
paper chromatography.
• The principle involved can be partition or
adsorption chromatography
• Partition chromatography because the substance
are partitioned or distributed between liquid
phases.

6. • Adsorption chromatography between solid and liquid
phases, where the solid surface of the paper is the
stationary phase and the liquid phase is the mobile
phase.
• In paper chromatography a drop of test solution is
applied as small spot on filter paper and the spot is
dried
• Then paper is kept in closed chamber.
• Edge of paper is dipped in to solvent called
developing solvent.
• Substances are moved by solvent system.

7. Paper chromatography:

8. Paper chromatography procedure:
• Selecting a suitable type of development.
• Selecting a suitable filter paper.
• Prepare the sample.
• Spot the sample on the paper.
• Chromatogram development.
• Paper drying and compound detection.

9. Advantage of paper chromatography:
• It requires very less quantitative material.
• Both unknown inorganic and organic compounds can
be identified by paper chromatography.
• More efficient for polar and water soluble
compounds.
• A wide range of stationary phase available.

10. Disadvantages:
• In quantitaive analysis paper chromatography is
not effective.
• Complex mixture can not be separated.
Application:
• To check the purity of pharmaceuticals.
• To detect adulterants.
• To detect contaminants in drink and foods.
• To determine dopes and drugs in humans and
animals.

11. Thin layer chromatography:
Principle:
• The separation relies on relative affinity of compounds
towards both the phases.
• The compound in the mobile phase move over the surface
of stationary phase.
• The movement occurs in such a way that the compounds
which have higher affinity to the stationary phase move
slowly while the other compounds travel fast.
• Therefore separation of mixture is attained.
• On completion of the separation process the individual
component from the mixture appear as spot on the plate.

12. Basic operation involved in TLC:
1. Method for production of thin layer plates.
2. Application of sample on chromatoplates.
3. Choice of adsorbent.
4. Choice of solvent.
5. Detecting reagent
6. Developing chamber.

13. Preparation of thin layer
chromatography:
• Pouring
• Dipping
• Spraying
• Spreading
• Percoated plates

15. Advantages:
• Require large preparation scale
• Applicable almost all chemical compounds.
Disadvantages:
• thin layer chromatography plates do not have
longer stationary phase.
• The results generated from tlc are difficult to
reproduce.

16. Ion exchange chromatography:
Principle:
• This chromatography distributes the analyte
molecules as per charge and their affinity towards
the oppositively charged matrix.
• The analyte bound to the matrix are exchanges with
the competitive counter ion to elute.
• The interaction between analyte and matrix is
determined by net charge , ionic strength and Ph of
the buffer.

18. Classification:
• Cation exchange chromatography.
• Anion exchange chromatography.

19. Affinity chromatography:
Principle:
• Inject a sample in to an initially equilibrated affinity
chromatography column.
• Only the substance with affinity for the ligand are
retained in the column.
• Other substance with no affinity for the ligand are
eluted from the column.
• The substance retained in the column can be eluted
from the column by changing ph or salt or organic
concentration of the eluent.

21. Advantages:
• Easy to perform
• Specificity
• Purification yield
• Easy to perform

22. Methodology:
• Equilibration
• Sample preparation
• Elution
• Column regeneration

23. Gas chromatography:
• Mobile phase: gas
• Stationary phase: liquid, solid
• In gas chromatography two types of column used:
1.Packed column
2.Capillary column
• In gas chromatography sample injector and column is
enclosed in an oven and increases temperature up to
400c.
• Elevated temperature is used for temperature
programming.

24. Instrumentation:

25. Instrumentation
• Carrier gas
• Sample injection system
• Column
• Detectors:
1. thermal conductivity detector
2. flame ionization
3. electron capture detector
4. nitrogen phosphorous detector
5. flame photometric detector

26. Procedure:
• Open valve
• Gas flow forward there will be flow controller
• Through sample inlet sample is introduced
• Sample convert gas then only analyze the sample
• Then gas go with mobile phase and elution takes
place
• Sample inlet and column enclosed in chamber which
is oven.
• Increases temperature of oven .

27. Cont…
• Detector will detect the sample then give signal to
recorder.
• Recorder gives peak of the sample
• After elution sample will go to detector
shapes of column:
• Straight (size small)
• U-shaped ( slightly higher)
• Coiled (very long)

28. Hplc:
• High performance liquid chromatography
• Analytical technique used to separate, identify,
quantify the mixture.
• It gives high performance due to smaller particlr size
of stationary phase.
• High pressure is appiled for rapid separation of
compounds.
• There are two phases: mobile phase,
stationary phase.

29. Types of hplc
• Normal phase chromatography
• Reversed phase
• Size exclusion chromatography
• Ion exchange
• affinity

30. Instrumentation:

31. Instrumentation
• Solvent reservoir
• Degasser
• Vaccum pump
• Solvent mixing valve
• Hplc pump
• Guard column/ pre column
• Sample injector
• Analytical column

32. Detectors:
1. Optical detectors: fixed wavelength
variable wavelength
2. Differential detector
3. Electrochemical detector
4. Mass spectrometer
5. Fluroscence detector

33. Chromatographic parameters:
• Chromatogram
• Retention time
• Retention value
• Baseline width
• Void time
• Void volume

34. Essential chromatographic paramters:
• Efficiency
• Retention factor
• Selectivity
• Resolution
• pressure

35. References: