Flash chromatography, developed by Mr. Sanket P. Shinde, is a rapid preparative column chromatography technique that operates under pressure, facilitating the separation of analytes based on their distribution between an adsorbent and eluent. This method utilizes a pre-packed column with silica gel and employs both dry and slurry packing techniques for optimal efficiency. Modern advancements have led to automated systems with pre-packed cartridges, enhancing reproducibility and reducing solvent use in various applications, including the purification of synthetic compounds and isolation of natural products.

1. Flash Chromatography
Mr. Sanket P. Shinde
Assistant Professor
Pune-Maharashtra.

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Mr. S. P. Shinde
Flash chromatography is also known as medium pressure chromatography.
It is widely used for laboratory scale fractionation of mixtures from organic
synthesis or for analysis.
Flash Chromatography is a rapid form of preparative column chromatography
based on an optimized pre-packed column through which is pumped solvent
at a high flow rate.
It uses glass or plastic columns with high flow rates and column packed with
silica gel is widely used to purify synthetic compounds in many labs.
It is simple and easy to use, but it is time consuming, irreproducible and
difficult to pack a good column.

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Mr. S. P. Shinde
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.
The mobile phase is pushed rapidly through the stationary phase packed in a
short column with large inner diameter.
The analytes in 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 highly polar is passed
through the column and various fractions are collected.
According to the solubility of the analytes in particular solvent combinations
separation occurs.

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Mr. S. P. Shinde

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Mr. S. P. Shinde
The conventional flash chromatography apparatus consists of a glass column
of a suitable length containing a small glass-wool plug and a layer of acid-
washed sand or glass frit at its base.
This column is partially filled with sorbent using either dry-packing or slurry-
packing technique.
Sample is placed on the top of the sorbent layer. Above the sample a layer of
glass wool sand is placed to prevent disturbance of the column bed by
solvent added for elution.
Above this, a solvent reservoir may be placed or there will be sufficient space
for solvent of volume equal to the volume of fraction to be collected.
The level of the solvent is maintained throughout the chromatographic
procedure.
On the top of the column an air inlet adaptor is provided and which in turn
connected to air or gas supplier through rubber tubing.
Continue…

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Mr. S. P. Shinde
Pressure of the gas or air is regulated by regulation valves.
The typical pressures employed are less than 1-2 atm, with the various parts
of the apparatus held in place by springs, clamps or screw thread
connectors.
In this type of apparatus the apparatus should be disassembled and
reassembled while changing the solvent.
In further development of the apparatus, a solvent reservoir with tap at the
reservoir-to-column inlet is attached to the side of the column.
The inlet for the reservoir is situated above the height of the sorbent bed.
A second tap at the top of the column allows air pressures to be equalized
for rapid solvent addition to the column.
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Mr. S. P. Shinde
1. Packing Technique
a. Dry Packing
The sorbent is added into the column in small increment followed by
tapping of the column with a hard object.
The addition of sorbent in small increments is better for dry packing than
filling of the column in bulk with the sorbent.
After packing, a weak solvent is forced through the column to remove any
air present between the sorbent the column.
Several column volumes are passed through the sorbent thus the sorbent
bed is made stable.
b. Slurry Packing
The column is initially partially filled with a weak solvent such as hexane.
The sorbent is powdered well in a mortar to remove any clumps and then
mixed with the same weak solvent to prepare slurry.
This slurry is poured into the column in small increments. The excess
solvent in the column is intermittently drained away.
Generally tapping the column in this method is not followed. Periodic
pressurization of the sorbent bed is used to aid consolidation.

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Mr. S. P. Shinde
2. Phases used in Flash Chromatography
a) Mobile Phases
Mobile phase in flash chromatography is organic solvents.
A fixed combination of organic solvents is used for isocratic elution
whereas a different composition of solvents usually starting from non polar
to polar range is used for method optimization.
b) Stationary Phases
Generally silica or alumina is the choice of stationary phases.
However there are many sorbents are used as stationary phases.
Hence depending upon the nature of the samples to be separated choice of
columns can be done.

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Mr. S. P. Shinde
3. Columns
a) Glass Columns
A wide range of columns offer maximum flexibility for every situation.
Depending on the nature and the quantity of the sample offers a series of
column types which vary in form, size and performance.
b) Plastic and Glass Column
Plastic and Glass-coated Glass Columns are available for larger sample
amounts and higher pressure applications on a high safety level.
The columns are designed for sample amounts from 1- 100 g and pressures
up to 50 bar during preparative separations.
An integrated cooling jacket allows separations under constant conditions.
c) Precolumns
Precolumn are minimizing dead volumes and enhance the life time of the
main column by trapping contaminants.
The small Precolumn, fits to Glass Columns.

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Mr. S. P. Shinde
Modern Flash Chromatography
In the modern flash chromatography system the glass columns are replaced
with pre-packed plastic cartridges which are much safer and also more
reproducible.
Solvent is pumped through the cartridge, which is much quicker and more
reproducible.
Systems may also be linked with detectors and fraction collectors providing
automation.
The introduction of gradient pumps means quicker separations, less solvent
usage and greater flexibility.
Modern Flash chromatography generally consist of following parts
A. Pump Systems
B. Sample Injection Systems
C. Columns- cartridges, Filling Sets and Column Valves
D. Precolumns
E. Fraction Collector
F. Detectors and Chart Recorders
G. Computing system

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Mr. S. P. Shinde
A. Pump Systems
It consists of a pump Controller and vacuum Pump/peristaltic Pump.
A pressure range up to either 10 bar or 50 bar gives optimum separation
results for a broad range of applications.
The pump modules can be controlled by various units.
These units are specially designed for isocratic or gradient or for both.
B. Sample Injection Systems
Unlike the conventional flash chromatography, in modern instruments
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.

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Mr. S. P. Shinde
C. Columns Cartridges
i. Pre-packed plastic cartridges
The cartridges are of different sizes, capable of loading from 50 up to 700 g
of silica.
They may be disposable, bidirectional and stackable.
Solvent is pumped through the cartridge, which is much quicker and more
reproducible.
The introduction of gradient pumps means quicker separations, less solvent
usage and greater flexibility.
Column Characteristics
1. Plastic cartridges are disposable with different sizes. This allow easy scale-up
and reproducible results.
2. Solid sample module and injection valve helps in easy sample loading.
3. The pressure can be applied up to 100 psi thus leads to rapid separation.
4. Low back pressure and higher efficiency can be achieved through narrow
particle distribution provides

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Mr. S. P. Shinde
ii. Filling Sets for Glass Columns
a) Dry Filling Set
The Dry Filling Set is employed for filling glass columns with silica gel
using compressed gas.
Silica gel in the size range of 25 - 200 μm can be packed with this method.
b) Slurry Filling Set
The Slurry Filling Set is used for wet filling and conditioning of glass
columns with silica gel particles smaller than 25 μm.

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Mr. S. P. Shinde
D. 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.
The small Pre-columns are usually made up of glass columns with inner
diameter of 15, 26, 36 and 49mm whereas the large Pre-column, have inner
diameter of about 70 and 100 mm.
E. Fraction Collector
Fraction collector consists of a panel that has provision to keep test tubes for
collecting fractions.
A tube connects the outlet of detector and the fraction collector.
This setup is automated and can be controlled by software so that the
required fractions will be collected in the collection tubes automatically.

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Mr. S. P. Shinde
F. Detectors and Recorders/Software
For most applications UV/Vis detectors are commonly used.
These detectors detect the analytes and generate signals as peaks.
Compounds that have absorption in the UV-Vis region can be conveniently
analysed using these detectors.
In the absence of adequate UV/Vis absorption, likely for sugars or polymers,
a Differential Refractometer (RI Detector) in combination with a UV/Vis
detector is used.
G. Computing system:
A computer with software is used to monitor the separation process.
The peaks are displayed while separation occurs.
The desired peaks can be collected using fraction collector.

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Mr. S. P. Shinde
Sample Loading
Samples are loaded in two ways
1. Sample is mixed with a weak solvent and then this mixture is poured into
the column on the top of the sorbent.
2. When Sample is not soluble in weak solvent it is dissolved in strong solvent
and mixed with small amount of sorbent. After stirring well with spatula or
glass rod, the solvent is allowed to evaporate either under the influence of
nitrogen or air or high vacuum. This process leaves a dry powder. This is
then applied at the top of the sorbent layer in the column. The amount of
silica and amount of sample to be used are depending on the column
diameter.

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Mr. S. P. Shinde
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 Rf value.
For optimization of flash method just obtaining a separation on TLC is not
enough. We need to get the targeted compound in the proper Rf range.
Ratios of the solvents are optimized using the chosen solvent (or solvents)
from the selectivity study, to get an Rf value of between 0.1 and 0.4, for the
targeted compound.
Compound retention and mass-transfer kinetics are related to separation
efficiency and, therefore, loading capacity.
In flash chromatography, retention is expressed in column volumes (CV).
Method Optimization

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Mr. S. P. Shinde
A column volume is the space in a column or cartridge not occupied by the
stationary phase.
This volume includes both the interstitial volume which is the volume
outside of the particles and the media's own internal pore volume.
These two volumes constitute 70% to 80% of the packed cartridge's
volume, of course this means that the media only occupies 20% to 30% of
the space in the cartridge.
Knowing a cartridge's column volume helps in determining purification run
length, gradient profile, and solvent consumption which becomes more
critical if a purification method requires scaling up.
Column Volume

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Mr. S. P. Shinde
Flash chromatography use for the purification of synthetic compounds during
synthetic process. It is useful in separation of impurities present along with
the product.
Non-aqueous reversed-phase chromatography that can be very effective at
separation and purifying very lipophilic compounds and other hydrocarbons
with minimal polar functionality.
Flash chromatography has been used for the isolation of chemical
constituents from crude extracts of plants.
It has been used for the separation of synthetic peptides, to isolate pure
peptide.
Purification of Fatty Acid Methyl Esters (FAME), Mixture of Glycerides,
Mono-, Di Tristearin and purification of sterols.
It is useful in bile acid purification, in impurity isolation during drug
purification.
Applications

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Mr. S. P. Shinde