Flash chromatography is a medium-pressure liquid chromatography technique that uses slightly smaller silica gel particles and pressurized gas to drive solvents through the column more rapidly than gravity-fed chromatography. It allows for efficient separation and purification of compounds. Chiral chromatography separates chiral compounds like enantiomers using a chiral stationary phase or chiral mobile phase additive. These techniques have various applications in natural product purification, organic synthesis, and the pharmaceutical industry.

1. FLASH CHROMATOGRAPHY
BY: Gauthami. K.B
Roll no: 256213886009
M.pharmacy 1st yr (pharmaceutics)
Pharmaceutical Analytical Techniques
Under the guidance of
UTTAM sir
AND
CHIRAL CHROMATOGRAPHY

2. FLASH CHROMATOGRAPHY

3. INTRODUCTION
• Flash chromatography, also known as medium pressure chromatography
was introduced by CLARK
• It is alternative to slow and often inefficient gravity-fed chromatography.
• Flash chromatography differs from the conventional technique in two ways:
1. first, slightly smaller silica gel particles (250- 400 mesh)are used
2. second, due to restricted flow of solvent caused by the small gel particles,
pressurized gas (ca.10-15 psi) is used to drive the solvent through the
column of stationary phase.
• The net result is a rapid(“over in a flash”) and high resolution
chromatography.

4. STEPS INVOLVED IN FLASH CHROMATOGRAPHY
• Selecting a Solvent System
• Determining the Quantity of Silica Gel Required
• Packing the Column
• Applying the Sample
• Eluting the Sample

5. • Selecting a Solvent System:
• The compound of interest should have a TLC Rf of ≈0.15 to 0.20
in the solvent system, choose binary (two component) solvent
systems with one solvent having a higher polarity than the other
are usually best, since they allow for easy adjustment of the
average polarity of the eluent.
• The ratio of solvents determines the polarity of the solvent
system, and hence the rates of elution of the compounds to be
separated.
• Higher polarity of solvent increases rate of elution for ALL
compounds.
• Common binary solvent systems in order of increasing polarity
are dichloromethane/hexane, ether/hexane, hexane/ethyl
acetate, and dichloromethane/methanol.

6. CONTD…
• Some solvents list according to their increasing eluting
power 1) cyclohexane 2)pet. ether 3)Pentane 4)
Dichloromethane 5)Ethyl ether 6)Ethyl acetate
7)Ethanol 7)Water 8)Acetone 9)Acetic acid
10)methanol.
• If Rf is ≈0.2, you will need a volume of solvent ≈5X the
volume of the dry silica gel in order to run your column.

7. • Determining the Quantity of Silica Gel
Required
• The amount of silica gel depends on the Rf
difference of the compounds to be separated,
and on the amount of sample.
• For ngrams of sample, you should use 30 to
100 ngrams of silica gel. For easier
separations, ratios closer to 30 : 1 are
effective, for difficult separations, more silica
gel is often required.
• However, by using more silica gel, the length
of time required for the chromatography is
extended.

8. • Selecting the column and column diameter Plastic
column :
• Recommended length 46cm, Diameter - depends on
sample size and the difference in Rf value.
• Packing the Column:
• Obtain a glass column and make sure that it has either a
glass frit or a plug of cotton wool directly above the
stopcock to prevent the silica gel from escaping from the
column through the stopcock.

9. • Next, put a ~1/2 in. layer of clean sand above the plug of glass
wool. Make sure the surface is flat. Then pour in the silica gel
using a funnel.
• Two methods for packing the column are:
1)wet packing
2)dry packing

10. • Applying the Sample:
• Loading the sample is important .
• Application of sample can be made by the small Pasteur pipette.
• Allow the solvent which remains above the silica to drain down
until it is flush with the surface of the silica.
• If the top surface of the silica gel is not flat, gently tap the side
of the column until it is.
• Dissolve sample into the minimum volume of the elution
solvent. Apply this to the top of the column, being careful not to
disturb the top of the silica.
• Add a small amount of sand to protect the top surface of the
silica when you add more solvent.

11. • Eluting the Sample:
• Add elution solvent to the column.
• Apply pressure to force the solvent through the column.
• The pressure should be the minimum necessary to keep a
steady stream coming out of the column.
• Begin collecting the eluted solvent into separate test tubes
(fractions).
• To maximize the efficiency of chromatography, the fractions
collected should not be more than about one tenth of the
column volume.
• Prepacked column Online uv-vis detection Automatic
Collection Sample Solvent reservoirs can be used.

12. • Locating the Sample:
• Use TLC to determine
which fractions contain
your compound.
• Combine the fractions that
contain your sample
together in a flask, then
concentrate the sample.

13. • Detection:
• Detection is usually done by UV-Vis detectors.
• Fully automated method with user-friendly software Online UV –
Vis detectors are used.
• Modern online FC systems have improved the method by
incorporating reusable plastic cartridges prepacked with the
sorbent, ultraviolet (UV) detection, computer software control,
mobile phase gradients, and automated fraction collection.
• Normal phase (NP) FC of polar compounds on silica gel
columns is still the most widely used mode, but reversed phase
(RP), ion exchange, and other types of sorbents are becoming
used more frequently.

14. • Application :
These systems are applied In
1. sample cleanup,
2. Natural products purification,
3. Organic synthesis,
4. Combinatorial chemistry,
5. Drug discovery,
6. Pharmaceutical intermediate purification, and many
other areas.

15. CHIRAL CHROMATOGRAPHY

16. INTRODUCTION
• Chiral Chromatography is a branch of chromatography that is
oriented towards the exclusive separation of chiral
substances.
• Certain stereoisomers that differ only in the spatial
arrangement of their atoms and in their capacity for rotating
the plane of polarized light are termed optically active or
chiral and the individual isomers are called enantiomers.
• Enantiomeric separations are achieved in chiral
chromatography by the judicious use of chiral phases.
• The mobile phase can be a gas or liquid giving rise to chiral
gas chromatography and chiral liquid chromatography.

18. PRINCIPLE OF ENANTIOMER SEPARATION
• On transfer of a pair of enantiomers into asymmetric
environment, two diastereomeric species are formed
with distinct physicochemical property profile. On the
basis of which physical separation into individual
enantiomers may be achieved.

19. • Chiral selector (SO):-
• Capable of undergoing covalent or non-covalent
interaction with the individual enantiomer (Selectand
SAs)
Depending on the nature of the interaction stabilizing
the respective diastereomer. SA-SO species.

20. • Enantiomer separation strategies:-
• Indirect enantiomer separation
• Direct enantiomer separation
• Indirect enantiomer separation:- Chiral Derivatization
agent (CDAs):- Transformation of the SAs of interest into
covalent diastereomer by conversion with suitably
reactive SOs. Followed by separation of diastereomeric
product with achiral chromatographic techniques.
• Applicable only to enantiomer presenting a single or more
but selectively addressable functional group suitable for
derivatization.

21. • Direct enantiomer separation:- 1) Chiral mobile phase
additive 2) chiral stationary phase mode
• Chiral mobile phase additive (CMPA): A combination of
an chiral stationary and a chiral mobile phase is
employed.
• On introduction of a mixture of enantiomers into this
system, the individual of enantiomers form diastereomeric
complex with the chiral mobile phase additive.
• This diastereomeric complex may exhibit distinct
association / dissociation rate, thermodynamics stability,
and physiochemical property therefore may be separated
on an appropriate a chiral stationary phase..

22. • chiral stationary phase mode: Consists of an inert
chromatographic support matrix incorporating
chemically or physically immobilized SO species.
• CSPs may be created by a variety of SO immobilization
technique.
1. Covalent attachment on to the surface of suitably
prefunctionalized carrier materials.
2. Physical fixation employing coating technique.
3. Incorporation into polymeric network by
copolymerization or combination of this procedures

23. • CSPs provide several operational advantages over
CMAs based enantiomers separation:
1. stability of CSPs more and flexibility with respect to
method optimization parameter.
2. Used for wide range of mobile phase solvent and
modifiers.
3. also used for gradient elution and variable temp.
protocol

24. • Classification of Chiral Stationary Phase:
• Type-1 Organic Polymer -Pure -Polymer coating on
inorganic support -Grafted polymer.
• Type-2 Carrier material modified with Chiral moieties -
inorganic material mainly silica gel modified on the
surface. -organic polymer network grafted with chiral
molecules.
• Type-3 Imprinted material -imprinted polymer -inorganic
material imprinted on the polymer.

27. DETECTORS
• Commonly detectors used in chiral
chromatography:
• Polarimeter
• Optical rotatory dispersion detector
• Circular dichroism detector.

28. • Applications:-
• Quinine and Quinidine
• Atropine and Hyoscyamine
• Cetrizine and Levo-cetrizine
• Omeprazole and Esomeprazole(more effective in
GERD)
• Dopamine and levodopamine
• D-Amphetamine and Amphetamine
• Dextromethorphan and Levo-methorphan

29. CONCLUSION
• Modern flash chromatography is applicable to a wide
range of compound types.
• Saves time and solvents.
• HPLC linear gradients can be transformed into step
gradients in flash chromatography.
• Flash chromatography can be a reliable and cost-effective
alternative to preparative HPLC.

30. CONTD..
• Chiral chromatography has become a preferred method
for rapidly separation enantiopure compounds in the
pharmaceutical industry, largely owing to the speed
with which a chromatographic method can be
developed and executed as well as the comparatively
small labour requirements of the chromatographic
approach.
• The use of chiral chromatography within the field of
organic synthesis can be expected to increase as the
technique becomes more familiar to synthetic chemists.

31. REFERENCES
• Peter Atkins & Julio de Paula, Aitkin's Physical Chemistry 7 the Ed., Oxford, New York
(2002) Chapter 22 AR Genaro
• Remington: The Science and Practice of Pharmacy 20 the Ed.
• Lippincott Williams & Wilkins (2000) Part 4 DG Peters, JM Hayes, GM Hefted, Chemical
Separations and Measurements, Saunders, Philadelphia(1974) Chapter 17
• http://www.chromatography.amershambiosciences.com.
• Chiral chromatography by T.E.Beesely

32. THANK YOU