CHROMATOGRAPHY BRIEF DESCRIPTION IS GIVEN.

1. Presentation on
Chromatography
Submitted by Sukanta Debnath
M.Pharm 1st Semester
Submitted to Mr. Rajat Gosh
Tripura University
Modern Pharmaceutical Analytical
Techniques

2. Contents
• Introductions
• Principle
• Classification
• TLC
• Gas Chromatography
• HPTLC
• Column Chromatography
• Ion Exchange Chromatography
• Gel Filtration Chromatography
• High performance Liquid Chromatography
• Affinity Chromatography
• References

3. Introduction
• Chromatography is a physical process where the components of sample mixture are
separated as a results of there differential distribution between stationary phase and
mobile phase.

4. Principle
•Chromatography is usually based on principle of partition of solute between two
phases. It usually consists of a Mobile Phase and a Stationary Phase.
•The Mobile Phase usually refers to the mixture of the substances to be separated
dissolved in a liquid or a gas
•The Stationary Phase is a porous solid matrix through which the sample contained
in the mobile phase percolates.

5. Classification
1. Thin Layer Chromatography(TLC)
2. High Performance Thin Layer Chromatography(HPTLC)
3. Gel Chromatography
4. Column Chromatography
5. Ion Exchange Chromatography
6. Gel Filtration Chromatography
7. High performance Liquid Chromatography
8. Affinity Chromatography

6. Thin Layer Chromatography
Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar to
paper chromatography However, instead of using a stationary phase of paper, it involves a
stationary phase of a thin layer of adsorbent like
silica gel, alumina, or cellulose Compared to paper, it has the advantage of faster runs, better
separations, and the choice between different adsorbents.

7. Cover plates
Solvent
mixture
Thin
Plate

8. Application
• It is widely used in separating multicomponent pharmaceutical formulations.
• It is used to purify of any sample and direct comparison is done between the sample
and the authentic sample.
• It is used in the food industry, to separate and identify colors, sweetening agent, and
preservatives
• It is used in the cosmetic industry.

9. • Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography,
(GLC), is a separation technique in which the mobile phase is a gas.lt is the method of
choice for the separation of volatile substances or the volatile derivatives of certain non-
volatile substances
• Stationary phase is an inert solid material impregnated with a non-volatile liquid
Gas Chromatography

10. • The principle of separation in GC is "partition."
• The mixture of component to be separated is converted to vapour and mixed with gaseous
mobile phase
• The component which is more soluble in stationary phase travel slower and eluted later. The
component which is less soluble in stationary phase travels faster and eluted out first
• No two components has same partition coefficient conditions. So the components are separated
according to their partition coefficient.
• Partition coefficient is "the ratio of solubility of a substance distributed between two immiscible
liquids at a constant temperature.’’
Principle

11. He,
N,
Hydrogen

12. • The cylinder/ gas tank is fitted with a pressure controller to control the pressure of
gas, a pressure gauge that indicates the pressure, a molecular sieve to transfer
filtered dry gas and a flow regulator to ensure a constant rate of flow of mobile
phase to the column.
• It should meet the following criteria….
• -- Should be chemically inert
• --Should be cheap and readily available
• --Should be of high quality and not cause any fire
accidents
• --Hydrogen has low density and batter thermal
conductivity. Nitrogen is inexpensive and gives reduced
sensitivity.

13. Retention Of GC
For example-
Take a compound
mixture of X,Y,Z
And calculate and
determined the major
product .
Formula :- Area of peak
Total area X 100

14. Applications
• GC-MS is used in research and development, production, and quality
control.
• It is used in identification of impurities in active pharmaceutical
ingredients.
• In medicinal chemistry, GC-MS is used in the synthesis and
characterization of compounds and in pharmaceutical biotechnology.

15. High Performance Thin Layer
Chromatography
• HPTLC (High performance thin layer chromatography) is the automated, sophisticated
form and improved method of TLC.
• It is a powerful analytical method equally suitable for qualitative and quantitative
analytical tasks.
• It is also known as planer or flat bed chromatography.

16. Principle
• Same theoretical principle of TLC (Adsorption chromatography )i.e. the principle of
separation is adsorption.
• Mobile phase flow by capillary action effect. And component move according to their
affinities towards the adsorbent.
• The component with higher affinity toward adsorbent travels slowly. And the component
with lesser affinity towards the stationary phase travels faster.
• Thus the components are separated on a chromatographic plate according to their affinity
and separation also based on their solubility in mobile phase.

17. Sample preparation
Application of Sample
Chromatography Development
Detection of Spots
Screening and Documentation
Selection Of Chromatographic
Layer
Pre washing
Pre conditioning
Steps Involve in HPTLC

18. • HPTLC is suitable for both the qualitative and quantitative analysis of herbal extracts and
formulations.
• Due to integrated software, all the analysis parameters and results can be stored digitally.
• It enables rapid analysis of herbal extracts with a minimum mobile phase.
• In HPTLC, the application of sample, scanning, and data analysis can be performed
individually.
Applications of HPTLC

19. • Column chromatography separates substances based on differential adsorption of
compounds to the adsorbent as the compounds move through the column at different
rates which allow them to get separated in fractions.
• This technique can be used on a small scale as well as large scale to purify materials that
can be used in future experiments.
Column Chromatography

20. Principle
• When the mobile phase along with the mixture that needs to be separated is introduced
from the top of the column, the movement of the individual components of the mixture is
at different rates. The components with lower adsorption and affinity to stationary phase
travel faster when compared to the greater adsorption and affinity with the stationary
phase. The components that move fast are removed first whereas the components that
move slowly are eluted out last.
• The adsorption of solute molecules to the column occurs in a reversible manner. The rate
of the movement of the components is expressed as:
• Rf = the distance travelled by solute/ the distance travelled by the solvent
• Rf is the retardation factor.

21. Applications
• Column Chromatography is used to isolate active ingredients.
• It is very helpful in separating compound mixtures.
• It is used to determine drug estimation from drug formulations.
• It is used to remove impurities.
• Used to isolate metabolites from biological fluids.

22. • Ion- exchange chromatography is based on an exchange of ions between a
charged stationary surface and ions of the opposite charge in mobile phase.
• Depending on the conditions, solutes are either cations (positively charged) or
anions (negatively charged). These are also known as ion exchangers.
Ion Exchange Chromatography
Principle

23. • Ion -exchangers are made up of two parts- an insoluble matrix and chemically bonded
charged groups within and on the surface of the matrix.
• It is classified as cationic or anionic based on whether it exchanges cation or anions.
• Cation exchanger -also known as acidic ion exchanger.
• Anion exchanger -also called basic ion exchanger.
• Each type of exchanger is also classified as strong or weak depending on the ionizing
strength of the functional group.

24. • Ion exchange chromatography can be applied for the separation and
purification of many charged or ionizable molecules such as proteins,
peptides, enzymes, nucleotides, DNA, antibiotics, vitamins and etc.
from natural sources or synthetic origin.
• An important use of ion-exchange chromatography is in the routine
analysis of amino acid mixtures
Applications

25. Gel Filtration Chromatography
Principle
• Gel filtration is also known as size-exclusion chromatography or molecular-sieve
chromatography. In this process, separation is based on the differing ability (due
to differing molecular size) of molecules in the sample to enter the pores of the
gel-filtration medium. The stationary phase in this technique consists of beads of
a hydrated, sponge-like material that has pores of molecular dimensions and with
a narrow range of sizes.
• When an aqueous solution, containing molecules of various sizes, is passed
through a column containing such ‘molecular sieves, molecules that are larger
than the pores of the filtration medium move quickly through the column. Smaller
molecules enter the pores of the gel and move slowly through the column.

26. • Gel filtration chromatography, also known as size exclusion chromatography,
is used to separate molecules of different sizes
• Fractionation of molecules and complexes within a predetermined size range
• Size analysis and determination
• Removal of large proteins and complexes
• Buffer exchange
Applications

27. HPLC Chromatography
High Performance Liquid Chromatography (HPLC) is a form of column chromatography that
pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure
through a column with chromatographic packing material (stationary phase). The sample is
carried by a moving carrier gas stream of helium or nitrogen.

29. • The purification takes place in a separation column between a stationary and a mobile phase.
• The stationary phase is a granular material with very small porous particles in a separation column.
• The mobile phase, on the other hand, is a solvent or solvent mixture which is forced at high pressure
through the separation column.
• Via a valve with a connected sample loop, i.e. a small tube or a capillary made of stainless steel, the
sample is injected into the mobile phase flow from the pump to the separation column using a
syringe.
• Subsequently, the individual components of the sample migrate through the column at different rates
because they are retained to a varying degree by interactions with the stationary phase.
• After leaving the column, the individual substances are detected by a suitable detector and passed on
as a signal to the HPLC software on the computer.
• At the end of this operation/run, a chromatogram in the HPLC software on the computer is obtained
The chromatogram allows the identification and quantification of the different substances.
Principle

30. HPLC Chromatogram Interpretation
Interpretation
of Some
Standard &
Pure known
Compounds.
Like :-
Caffeine,
Aspartame,
Benzoic Acid,
Sorbic Acid

31. Base Line Configuration

32. • Analysis of drugs
• Analysis of synthetic polymers
• Analysis of pollutants in environmental analytics
• Determination of drugs in biological matrices
• Isolation of valuable products
Applications

33. • Affinity chromatography is a type of liquid chromatography for the separation,
purification or specific analysis of sample components.
• It utilizes the reversible biological interaction or molecular recognition called affinity
which refers to the attracting forced exerted in different degrees between atoms which
cause them to remain in combination.
Affinity Chromatography

34. • The stationary phase consists of a support medium, on which the substrate (ligand) is bound
covalently, in such a way that the reactive groups that are essential for binding of the target molecule
are exposed.
• As the crude mixture of the substances is passed through the chromatography column, substances
with binding site for the immobilized substrate bind to the stationary phase, while all other substances
is eluted in the void volume of the column.
• Once the other substances are eluted, the bound target molecules can be eluted by methods such as
including a competing ligand in the mobile phase or changing the pH, ionic strength or polarity
conditions.
Principle

35. • Affinity chromatography is one of the most useful methods for the separation and
purification of specific products.
• It is essentially a sample purification technique, used primarily for biological
molecules such as proteins.
• Its major application includes:
• Separation of mixture of compounds.
• Removal of impurities or in purification process.
• In enzyme assays
• Detection of substrates
Applications

36. References
• https://www.thermofisher.com/blog/ask-a-scientist
• https://www.khanacademy.org/science/in-in-methods-of-purification-of-organic-
compounds/a/principles-of-chromatography
• Journal of Chromatography A publishes research papers and critical reviews on all aspects
of fundamental and applied separation science. The scope includes chromatography and related
techniques
• The Journal of Chromatography and Separation Techniques is a peer-reviewed (refereed)
• The journal of Mixed solvents in gas—liquid chromatography : Activity coefficients