FLASH COLUMN CHROMATOGRAPHY

This presentation includes basic principle, intrumentation and theory of flash chromatography.

1. GURU GHASIDAS
VISHWAVIDYALAYA,BILASPUR
SESSION-2023
TOPIC-FLASH COLUMN CHROMATOGRAPHY
GUIDED BY PRESENTED BY
DR. VINOD D. RANGARI SIR VINAY BISEN
M.PHARM 1
ST
SEM

2. INTRODUCTION
Flash chromatography is an air pressure driven hybrid of medium
pressure and shorter column chromatography which has been
optimized for rapid separation of organic compounds.
Flash chromatography is also known as medium pressure
chromatography(10-15psi)
This chromatography was popularized several years ago by
Clark still
It is a technique used to separate mixtures of molecule into their
individual constituents, frequently used in drug discovery process
Flash chromatography is a rapid form of preparative column
chromatography based on an optimized pre-pakedcolumn
through which is pumped solvent at a high flow rate
It uses plastic columns or cartridges with high flow rates and
column packed with silica gel (200-400mesh particle size) is
widely used to purify synthetic comounds.

3. Principle
•The principle is that the eluent which is a liquid, under
gas pressure (normally nitrogen or compressed air )
rapidly pushed through a short glass column
•Applied pressure to column reservoir to speed up mobile
phase
•The column is packed with an adsorbent of defined
particle size with large inner diameter
•The most used stationary phase is silica gel 40-63 µm
•Particles smaller than 25µm should only be used with very
low viscosity mobile phases, because otherwise the flow
rate would be very low
•Efficiency of column will be attained by optimum flow
rate of mobile phase
•Normally gel beds are about 15cm high with working
pressures of 1.5-2.0 bars.
•The net result is a rapid over in a flash and high resolution
chromatography

5. THEORY
•The basic theory involved in the flash chromatography is similar to
column chromatography except the involvement of pressure
•In column chromatography the flow of mobile phase is due to the
gravitational force
•In flash chromatography the flow of mobile phase is under the
influence of pressure(10-15psi)
•Pressure is applied through a pump to the column by using
air/nitrogen.
•The mobile phase is pushed rapidly through the stationary phase
packed in a short column with large inner diameter
•The analytesin the mixture are separated according to their
distribution between adsorbent and eluent and reaches the
bottom of the column
•The solvent polarity decides the separation of the analytes. A
mobile phase combination of solvents starting with non polar to
higher polar is passed through the column and various fraction are
collected

6. Phases used in flash chromatography
a)Mobile phases
This chromatography is usually carried out with a mixture of two
solvents, with a polar and a nonpolarcomponent. Occasionally,
just one solvent can be used. The only appropriate one-
component solvent systems (listed from the least polar to the
most polar):
Hydrocarbons:pentane, petroleum ether, hexanes
Ether and dichloromethane:(very similar polarity)
Ethyl acetate
b)Stationary phase
most commonly used stationary phases are silica gel (sio2) and
alumina (Al
2O
3)

7. INTRUMENTATION

8. Flash chromatography generally consist of
follwingparts:-
1.Pump systems –It consist of a pump controller and vacuum
pump/peristalicpump
-A pressure range up to either 10 bar or 50bar gives optimum
separation results for a broad range of application
2. Sample injection systems –samples are introduced into the column
using sample injection valves
-the valves are of specific volumes. Around 3-5 ml volume of
injection valves are used
3.Column cartridges –Pre packed plastic cartridges
•The cartridges are of different sizes , capable of loading from 50
up to 700g of silica
•Solvent is pumped through the cartridge , which is much quicker
and more reproducible
•The introduction of gradient pumps means quicker separation ,
less solvent usage and greater flexibility.

9. 4. Precolumns-pre-column are placed before the main
column and used to minimize dead volumes and
enhance the life time of the main column by trapping
contaminants.
5.Fraction collector –fraction collector consist of a panel
that has provision to keep test tubes for collecting
fractions
6.Detectors and recorders/software
-For most application UV/visdetectors are commonly
used
-In the absence of adequate UV/Vis absorption, likely for
sugar or polymer, a differential refractometer(RI
Detector) in combination with a UV/visdetector is used.
7. Computing system –a computer with software is used to
monitor the separation process
-The peaks are displayed when separation occurs.

10. Sample application/sample loading
Samples are loaded in two ways-
1.Wet loading method -In the wet method, the sample
to be purified (or separated into components) is
dissolved in a small amount of solvent, such as
hexanes, acetone, or other solvent. This solution is
loaded onto the column.
2.Dry loading method -First dissolve the sample to be
analysed in the minimum amount of solvent and add
sufficient amount of silica gel. Swirl the mixture until
the solvent evaporates and only a dry powder
remains. Place the dry powder and transfer it to the
top of the prepared column.
Elution –apply minimum pressure for continuous flow of
the mobile phase
Locating the sample –TLC is used

11. Method optimization
•When optimizing flash purification for component retention ,TLC is
the most efficient tool for evaluating solvent ratios.
•Initially TLC is carried out to get the desired Rfvalue
•For optimization of flash method just obtaining a separation on
TLC is not enough. We need to get the target compound in the
proper Rfrange
•Ratios of the solvent are optimized using the chosen solvent (or
solvents) from the selectivity study,toget an Rfvalue of between
0.1 and 0.4 ,for the target compound .
•Compound retention and mass-transfer kinetic are related to
separation efficiency and therefore ,loading capacity.
•In flash chromatography, retention is expression in column
volumes(CV).
•A column volume is the space in a column or cartridge not
occupied by thstaionaryphase

12. APPLICATION
•Flash chromatography used for the purification of
synthetic compounds during synthetic process. It is usefull
in separation of impurirtiespresent along with product
•Non aqueous reversed phase chromatography that can
be very effective at separation and purifying very
lipophiliccompounds and other hydrocarbon with
minimal polar functionality
•Flash chromatography has been used for the isolation of
chemical constituent from crude extract of plants
•It has been used for the separation of synthetic peptides
to isolate pure peptide .
•Puificationof fatty acid methyl ester (FAME) ,mixture of
glyceroids, mono , di, tristearinand purification of sterols
•It is usefullin bile acid purification, impurity isolation during
drug purification .

13. Natural products/Nutraceuticalsapplication:
1.Separation and Isolation of α-Santaloland β-Santalol
from Sandalwood Extraction
2.Isolation and Purification of Chromophoricand
NonchromophoricCompounds in Giant Knotweed
Rhizome.
3.Isolation and Purification of FlavonoidsfromGinkgo
BilobaLeaves Extract.
4.Isolation and Purification of Ginsenosidesfrom
RedPanaxGinsengExtract.
5.Isolation and Purification of Catechinsfrom Green Tea
Extract .

14. Carbohydrate application
1.Purification of Conjugated Quercetinand Rutinose.
2.Impurity Isolation of ValproicAcid from
CyclodextrinvDuringEncapsulation.
3.Isolation ofAminosugarand Acarbose.
4.FlavanoneGlycoside Purification.
5.Isolation of AminoglycosideAntibiotics.
Lipid application
1.Purification of Fatty Acid Methyl Esters (FAMEs).
2.Purification of a Mixture of Glycerides, Mono-, Di-,
and Tristearin.
3.Purification of Sterols.

15. Pharmaceutical/small molecule
application:
1.Bile Acid Purification During Lead Generation in Drug
Discovery.
2.In Impurity Isolation During Drug Purification.
3.MestranolPurification During Chemical Synthesis.
4 .In Anti-malarial Drug Purification in Drug Discovery.

16. Conclusion
•Purification of drug is an important step in any branch
of research.Preparativechromatography is used to
separate the components of a mixture for more
advanced use and is thus a form of purification.
•Flash Chromatography can be alternative to
preparative HPLC as it saves time and solvent.
Extrapolation of TLC results on preparative scale can
be achieved by Flash chromatography.
•Modern Flash chromatography with disposable
cartridges and advanced detection techniques is
applicable to a wide range of compounds.