2. ANALYTICAL TOOL –PART 3
CHROMATOGRAPHY
Dr.K.J.KARTHIKA
DEPARTMENT OF RASASHASTRA AND BHAISHAJYA KALPANA
GAVC,TPRA

3. Contents…
•What is chromatography?
•History
•Technical terms in chromatography.
•Biochemistry of separations.
•Selected chromatography techniques.
•Advanced chromatography techniques.
•Application of chromatography in Ayurveda.

4. What is chromatography???
Chromatography is a physical method of separation
that distributes components to separate between two
phases, one stationary (stationary phase), the other
(the mobile phase) moving in a definite direction.
Basic principle- components in a mixture have different
tendencies to adsorb onto a surface or dissolve in a solvent.

5. History
•Chromatography was first employed by the Russian scientist Mikhail
Tsvet in 1900 for the separation of plant pigments such
as chlorophyll, carotenes, and xanthophylls.
•Archer John Porter Martin and Richard Laurence Millington
Synge during the 1940s and 1950s established the principles and
basic techniques of partition chromatography, and their work
encouraged the rapid development of several chromatographic
methods.

6. Technical terminology
Analyte
It is the substance to be separated during chromatography. It is
also normally what is needed from the mixture.
Analytical chromatography
Chromatography used to determine the existence and possibly
also the concentration of analyte(s) in a sample.
Chromatogram
The visual output of the chromatograph. In the case of an
optimal separation, different peaks or patterns on the
chromatogram correspond to different components of the
separated mixture.

7. Eluate
The mobile phase leaving the column.
Eluent
The solvent that carries the analyte.
Mobile phase
The phase that moves in a definite direction.
It may be
- Liquid - (LC and Capillary Electrochromatography
(CEC)),
- Gas - (GC)
- supercritical fluid (supercritical-fluid
chromatography, SFC). with the stationary
phase and is separated.

8. Immobilized phase
A stationary phase that is immobilized on the support particles, or on
the inner wall of the column tubing.
Retention time
The characteristic time it takes for a particular analyte to pass
through the system (from the column inlet to the detector)
Solute
Refers to the sample components in partition chromatography.

9. Solvent
Any substance capable of solubilizing another substance, and
especially the liquid mobile phase in liquid chromatography.
Stationary phase
Substance fixed in place for the chromatography procedure.
Examples include the silica layer in thin layer chromatography
Detector
Refers to the instrument used for qualitative and quantitative
detection of analytes after separation.

10. Various modes of separation
•Adsorption
•Partition
•Ion exchange
•Molecular exclusion

11. Adsorption
•It Has solid stationary phase and liquid or gaseous mobile
phase
•The best adsorbed solute travel slow.
Eg: column chromatography

12. Partition
•Stationary phase is a non volatile liquid held as a thin
layer on the surface of an inert solid and a mobile phase
which is gas or liquid.
•The solute distribute themselves between the mobile
and stationary phase.
Eg paper chromatography

13. Ion exchange
•Similar to partition chromatography.
•It has a resin coated stationary phase and the mobile
phase is always liquid.
•When mobile phase is eluted through the resin the
electrostaticaly bound ions are released and other ions
are bonded preferentially. Eg, Domestic water softners.

14. Molecular exclusion
•Different from all other chromatography.
•Separation according to particle size.
•Mixture passes as a gas or liquid through a porous gel.
•Pore size allow large particles to pass through and small
particles permeate the gel.

15. Planar
chromatography
Column
chromatography
According to the
shape of
chromatographic
bed
Classification
According to the
physical state of
mobile phase
Liquid
chromatography
Gas
chromatography

16. According to the
direction of flow
of mobile phase .
Ascending
Descending
According to the
purpose
Analytical
Preparative

17. Selected chromatographic techniques
•Planar chromatography- Thin Layer Chromatography(TLC)
High Performance Thin Layer
Chromatography(HPTLC)
•Liquid chromatography- Column Chromatography(CC)
Flash Column Chromatography
High pressure liquid
chromatography(HPLC)
•Gas chromatography

18. THIN LAYER CHROMATOGRAPHY /TLC
•Process of separation – Adsorption
•Stationary phase- thin layer of solid as alumina or silica(5-50
micrometre) supported on an inert base such as glass, aluminum
foil or insoluble plastic(20*20cm/20*10cm).
•Mobile phase- mixture of organic solvents chosen by trial and error
method.
•Driving force for mobile phase- capillary action .

19. Procedure
•Activation of plate – heat at 110 degree centigrade for 30
minutes.
•Spot crude extract using micropipette.
•Keep undisturbed in the pre saturated developing
chamber containing appropriate solvent.
•When the solvent front approaches the top of the plate
remove it and record it before drying.
•Calculate the Rf / retention factor.

20. Highly polar molecules interact strongly with the Si-O
bonds of the adsorbent. Thus the weakly polar
molecules move through the adsorbent rapidly .
PRECAUTION- the technique is done in closed vessel to
ensure that the atmosphere is saturated with solvent
vapor.

21. Developing the plates.
•Spraying visualizing agents e.g., iodine in petroleum ether for
aromatic compounds, especially with electron donating groups .
Ninhydrin for amino acids.
•Specially prepared plates that fluoresce in UV light can be used.

22. Detection
•Developed Plates / plates with out spraying are observed in UV
chamber (254nm short UV,365nm long UV).
•In Specially prepared plates the solutes mask the fluorescence so a
dark spot will be observed.
•Compounds with their own fluorescence can directly be identified.
• Known solutes identified with Rf factor.
•Radioactive solutes identified by passing plate under Geiger
counter.
•The plates can be scanned in UV/visible absorption along the lane
which mixture has travelled the reflected light from the lane is then
measured relative to the radiation outside the lane

23. High performance thin layer chromatography
(HPTLC)
Similar to TLC but require shorter time and better resolution.
Stationary phase- Pre coated plates available .
whatmann HPTLC plates are produced from 4-5
micrometre silica gel coated uniformly
over aluminium sheet.
Advantage
•The use of smaller particle sized adsorbent helps increase resolution
and sensitivity.
•About 50-70mm solvent front enough for efficient separation which is
achieved in about 4 minutes.
•Very highly concentrated sample is taken for HPTLC thus it need to be
applied in less amount.

24. Column Chromatography
•Process of separation – Adsorption
•Stationary phase- Inert solids such as silica gel (basic compounds),
alumina (acidic compounds),cellulose, magnesia(unsaturated
organic compounds), dextran(compounds like purines) supported in
a glass column.
•Mobile phase- A wide range of solvents from hydrocarbons to
esters used. Mostly mixture of organic solvents chosen. Optimum
solvent is chosen by running experiments on a small scale using TLC
Plates
•Driving force for mobile phase- Gravitational force .

25. Procedure
•In the glass column initially place a glass wool plug to prevent the
stationary phase from clogging the tap.
•The silica/ appropriate stationary phase is mixed with solvent to
form a slurry and the stationary phase is filled in the column with
out any bubbles.
•The column saturated with solvent is then topped with the solvent
and the solvent is allowed to flow through.
•The solvent is then brought to the exact level of the stationary
phase.

26. •A porous flow adapter like sand is added above stationary phase so
that the solute cannot mix with the solvent above them.
• The sample is then loaded in the column.
•Periodic saturation of column with solvent is needed.
•All fractions of eluate is collected continuously in test tubes and
analyzed using TLC.
•Extract from liquid collected by evaporating the solvent.

27. Loading of the sample
1- wet loading
2- dry loading
Precaution: The stationary phase should be saturated with
solvent or else it may crack and lose its integrity.
Bubbles hinder the smooth flow of solute in the column.

28. Flash Chromatography
•Alternative technique similar to liquid / column chromatography.
The difference is in the driving force for the solvent.
•The solvent is forced through the column at a faster rate by
applying pressure from nitrogen cylinder.
•A valve is attached at the top of the column and nitrogen is
introduced at a pressure up to 200000N/m sq.
•The solvent is replaced by closing the tap and releasing the
pressure. Before opening the tap after refill, the the valve is replaced
and the gas supply is turned on.

29. High performance/presure liquid chromatography /HPLC
•Process of separation – Adsorption if stationary phase is silica.
Partition if the stationary phase contain bonded non volatile solutes
to allow interaction of solutes with different polarities.
•Stationary phase-uniform porous silica particles of 1 micrometre
diametre and surface pores of nanometre range(high surface area).
Coated particles also used.
•Mobile phase- A wide range of solvents from hydrocarbons to
esters used. Mostly mixture of organic solvents chosen. Optimum
solvent is chosen by running experiments on a small scale using TLC
Plates
•Driving force for mobile phase- High pressure of 10 mega
Newton/sq m or 100 atmosphere from twin cylinder reciprocal
pumps.

31. Type of HPLC Stationary
phase
Mobile phase relevance
Normal phase
chromatography
Polar Non-polar Non-polar
compounds
elude first
Reverse phase
chromatography
Non-polar Polar Polar compound
elude first.
Used for
separating plant
constituents
which are polar
Types of HPLC

32. •High pressure means that the instrumentation has to be strong. The
pump and the piping is constructed out of stainless steel. It will also
be inert to the solvent and solutes.
•Flow rate of HPLC column is slow 0.5-5 cc/min.
•A small sample volume of 5- 20 cc is injected through the injection
valve.

33. Detection of compound after HPLC
Most compound separated by HPLC absorb UV radiation, the
elute is passed through a small cell so that UV rays can be passed
through the solute

34. Other methods used in conjunction with HPLC are:-
1. Mass spectrometry
2. Infra red spectroscopy
3. Visible spectroscopy
4. UV spectroscopy
5. Fluorescence spectroscopy
6. Conductivity measurement
7. Refractive index measurement

35. GAS CHROMATOGRAPHY
•Process of separation – Partition b/w mobile gas and the stationary
liquid phase.
•Stationary phase- Solid or non volatile liquid coated in a narrow
column in an oven .
•According to the stationary phase it is classified to solid-gas or
liquid-gas chromatography.
•Mobile phase- gas .eg, Helium.
• The sample must be capable of being volatilized without being
decomposed.

36. Procedure
•The sample is injected through a self sealing disc into a small
heated chamber where it is vaporized.
•The injector oven is 50-100 degree hotter than the start of the
column.
•Sample is taken through the column by an inert gas /carrier eg,
helium, nitrogen.
•The column is coiled to fit into the thermostatically controlled oven.
•The nature of the stationary phase and the temperature is adjusted
according to the polarity and volatility of the substance to be
separated.
•The solute pass to the end of the column to a detector.

37. •The carrier must be dry to avoid interference from water.
•The carrier is dried by passing through anhydrous copper(II)
Sulphate or self indicating silica and unwanted solvent vapors are
dried by passing through activated charcoal.

38. Detectors
•Thermal conductivity detectors
•Respond to the change in the thermal conductivity of gas leaving
the column.
•A hot tungsten film is kept in an oven set at a given temperature .
•Only hydrogen has thermal conductivity more than helium so any
solute with helium will increase the temperature of the film.

39. Flame ionization detector.
•Particularly used for detecting organic solvents.
•The gas from the column is mixed with hydrogen and air and it is
burned.
•CH. Radicles formed on combustion are oxidised to CHO+ ions
which allow a current to transmit via cathode.
•This is then converted to signal.

41. Some advanced techniques of
chromatography

42. Affinity chromatography
based on selective non-covalent interaction between an analyte and
specific molecules. It is very specific, but not very robust. It is often used
in biochemistry in the purification of proteins bound to tags.
Supercritical fluid chromatography
Supercritical fluid chromatography is a separation technique in which
the mobile phase is a fluid above and relatively close to its critical
temperature and pressure
Size-exclusion chromatography
Size-exclusion chromatography (SEC) is also known as gel permeation
chromatography (GPC) or gel filtration chromatography and separates
molecules according to their size.

43. Expanded Bed Adsorption (EBA) Chromatographic Separation
Expanded Bed Adsorption (EBA) Chromatographic Separation captures a
target protein from a crude feed stream when it passes through a
chromatography column system containing adsorbent beads. With this
technique the crude feedstock can be treated directly in the
chromatographic column, avoiding the traditional clarification and pre-
treatment steps.
Simulated moving-bed chromatography
The simulated moving bed (SMB) technique is a variant of high
performance liquid chromatography; it is used to separate particles
and/or chemical compounds that would be difficult or impossible to
resolve otherwise. This increased separation is brought about by a
valve-and-column arrangement that is used to lengthen the stationary
phase indefinitely. In the moving bed technique of preparative
chromatography .In the simulated moving bed technique instead of
moving the bed, the sample inlet and the analyte exit positions are
moved continuously, giving the impression of a moving bed

44. Pyrolysis gas chromatography
Pyrolysis gas chromatography mass spectrometry is a method of
chemical analysis in which the sample is heated to decomposition to
produce smaller molecules that are separated by gas chromatography
and detected using mass spectrometry.
Fast protein liquid chromatography
Fast protein liquid chromatography (FPLC), is a form of liquid
chromatography that is often used to analyze or purify mixtures of
proteins.
Countercurrent chromatography
Countercurrent chromatography (CCC) is a type of liquid-liquid
chromatography, where both the stationary and mobile phases are
liquids.
Chiral chromatography
Chiral chromatography involves the separation of stereoisomers. In the
case of enantiomers, these have no chemical or physical differences
apart from being three-dimensional mirror images.

45. CHROMATO GRAPHY IN AYURVEDA
From recent researches
Standardisation of Kutajarista by HPLC analysis based upon
the presence of the biomarker conessine in the formulation.
The Gas chromatography was set up for the quantitative
estimation of alcohol. The method was developed by
changing various parameters of gas chromatography. In
ayurvedic preparation arishtha, the estimated alcohol (as self
generated alcohol) was found to be 8.1% (khadirarishta),
10.2% (drakshakumari), 7.8% (saraswatarishta) and obeyed
the label claim

46. It was observed that all commercial samples of Talishadi churna, and
standard are similar in their organoleptic and qualitative chemical
analysis but physical characteristic, fluorescence analysis and High
Performance Thin Layer Chromatography (HPTLC) chromatogram of
various formulations are not matching with each other, and it may be
due to the raw material collection time, geographical variation, etc.
HPTLC and HPLC fingerprinting analysis using a marker compound
Bebeerine isolated from roots of Cissampelos pareira. The roots
of Cissampelos pareira can be distinguished from other two plants by
presence of high concentration of alkaloids especially the presence of
high concentration of pharmacologically active alkaloid bebeerine,
which was found to be present in very low concentration in Stephania
japonica and absent in roots of Cyclea peltata.

47. Thirty Ayurvedic herbs and formulations have been screened for the
presence of gallic acid by the use of silica gel thin layer chromatography.
The samples showing presence of gallic acid in screening by the silica gel
thin layer chromatography were quantitatively analyzed for gallic acid by
ferric reducing antioxidant power (FRAP) assay.

48. CONCLUSION
1. Drug identification
2. Quantitative and qualitative standardization of crude drugs.
3. To Assess various stages of ongoing reaction like fermentation.
4. To develop Fingerprints for single and poly herbal formulation
5. Extraction of bio active substances for neo -formulations.
6. Discovery of new chemical compounds.

49. Ayurveda is a science that has its own rich
fundamental principles and innate style of
drug standardization thus it is always
better to think twice before blindly
implementing contemporary technical
advancements.