Introduction to flash chromatography

1. FLASH
CHROMATOGRAPHY

2. INTRODUCTION
 Developed by W.Clark Stills of Columbia University, New York,
USA, “An air pressure driven hybrid of medium pressure and short
column chromatography optimized for rapid separation."
 An alternative to slow and often inefficient gravity-fed
chromatography.
 It is a rapid form of preparative column chromatography that employs
prepacked columns through which the solvent is pumped at high
pressure.
 The pressure for flash chromatography is about 1-2 bars (1,00,000-
2,00,000 Pa or 10-15 psi) and it is provided by an aquarium pump or
several pumps in parallel.
 Sometimes also called as “Medium Pressure Liquid
Chromatography”. However, the later technique is sometimes
considered separately.

3. FLASH VS. CONVENTIONAL
TECHNIQUES
 Differs from the conventional technique in 2 ways:
 Slightly smaller silica gel particles, i.e.,40-63µm are used,
and
 Due to restricted flow of solvent caused by the small gel
particles, pressurized gas at 1-2 bars (1,00,000-2,00,000 Pa
or10-15 psi) 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.

6. Modern flash chromatography systems are sold as pre-packed plastic
cartridges, and the solvent is pumped through the cartridge.
Systems may also be linked with detectors and fraction collectors
providing automation.
The introduction of gradient pumps resulted in quicker separations
and less solvent usage.
Flash chromatography is not expected to provide the resolution
or reproducibility of HPLC; it is a technique that can quickly improve the
purity of samples to an acceptable level.

7. APPARATUS

8. PACKING MATERIALS
 Silica gel is most commonly used packing material.
 But it is now increasingly being replaced by superior adsorbent
materials such as alumina.
 Major advantage of alumina is that it is amphoteric in nature
and can be used in specifically defined pH ranges.
 Particle size distribution of 40-63µm is favored for more
uniform packing of column, superior resolution and separation.

9. PACKING THE COLUMN
1.Dry Filling Method
 Glass column / plastic cartridges are used.
 Have 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)
 ~1/2 inches layer of clean sand above the plug of glass wool
 Make sure that surface is flat
 Pour in the silica gel using a funnel.

10. 2. Slurry Packing
 Easier to perform and gives better packing.
 Very dilute silica gel-solvent slurry is needed to minimize air
bubble formation.
 Sufficient solvent is placed in the column and the slurry is
poured into it.
 The slurry falls under gravity to give a homogeneous packing
which shouldn’t be disturbed by stirring.
 After the slurry has settled, the stopcock is opened to speed up
sedimentation.
 Pressure can then be applied on the column using a pump.
 Further fractions of slurry are added in the same way until the
desired height of packing material is obtained.

11. SOLVENT SELECTION FOE ELUTION
 Two types of solvent systems are used: ISOCRATIC and
GRADIENT.
 In isocratic (same solvent strength) system, single –strength
mobile phase mixture brings about the desired separation.
 Gradient system is used in case of complex mixtures containing
compounds that differ greatly in column retention times. A non-
polar solvent (hexane) is used to elute a non-polar compound.
Then a more polar compound(ethyl acetate) is added to elute the
more polar compounds.
 Hence rapid and effective separations can be achieved by using
gradient solvent systems.

12. PROCEDURE FOR MICROSCALE
FLASH COLUMN
CHROMATOGRAPHY
 In microscale flash chromatography, the column need
neither a pinch clamp or a stopcock at the bottom of
the column to control the flow, nor does it need air-
pressure connections at the top of the column.
 Instead, the solvent flows very slowly through the
column by gravity until you apply air pressure at the
top of the column with an ordinary Pasteur pipette
bulb.

13. (1) Prepare the column.
1. Plug a Pasteur pipette with a small amount
of cotton; use a wood applicator stick to
tamp it down lightly.
2. Take care that you do not use either too
much cotton or pack it too tightly. You just
need enough to prevent the adsorbent from
leaking out.
Add dry silica gel adsorbent, 40-63µm
usually the jar is labeled "for flash
chromatography." One way to fill the
column is to invert it into the jar of
silica gel and scoop it out . . .

14. . . . then tap it down before scooping
more out
Another way to fill the column is to pour
the gel into the column using a 10 mL
beaker.

15. (2) Pre-elute the column

16. (3) Load the sample onto the silica gel column
Two different methods are used to load the column: the wet method and
the dry method:
Wet loading 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.

17. Dry loading method

18. (4) Elute the column.

19. 5) Elute the column with the second elution solvent.
If separating a mixture of one or more compounds, at this point change
the eluting solvent to a more polar system, Elution would proceed as in
step (4).
(6) Analyze the fractions.
If the fractions are colored, simply combine like-colored fractions,
although TLC before combination is usually advisable.
If the fractions are not colored, they are analyzed by TLC (usually). Once
the composition of each fraction is known, the fractions containing the
desired compound(s) are combined.

20. ADVANTAGES
 Large quantities of the sample can be separated (0.5-2g)
 Fast (10 to 15 minutes)
 Cost efficient
 Much quicker and more reproducible
 If high resolution is required, flash chromatography is carried
out before HPLC to avoid contamination of the expensive plates

21. APPLICATIONS
 Purification of various peptides, antibiotics
 Separation of closely related organic compounds
 Purification of closely related drug intermediates
 High speed fractionation of natural products – tocopherols, alkaloids,
lignans, xanthones, stilbenes, flavonoids
 Drug discovery
 Agrochemistry
 Petrochemistry

22. MEDIUM PRESSURE LIQUID
CHROMATOGRAPHY
 It is a modern version of flash chromatography.
 It appears to be a column chromatographic separation technique
best suited for large laboratory scale isolation of pure
compounds from semi-purified plant extracts.
 The technique is thus complimentary to flash chromatography.
 Favorably it can be stated that MPLC combines a resolution
which approaches that of HPLC with a preparative capacity and
a simplicity in operation similar to that of low pressure liquid
chromatography.
 Pressure required in the range of 5-20 bars (5,00,000-20,00,000
Pa or 70-250 psi) and is provided by a piston pump with a
variable flow rate.

23. MPLC EQUIPMENT
Principle components of a MPLC system

24. FLASH CHROMATOGRAPHYVS.MPLC
FLASH CHROMATOGRAPHY
 Low pressure (1-2bars)
 Sample size is 0.5-2g
 Flexible column length (30-
40cm)
 Simple technology
 Low or moderate resolution
 Low reproducibility of
separation
 Column support cannot be
reused
 Low cost
MPLC
 High pressure( 5-20bars)
 Sample size is much larger 0.5-
50g)
 Fixed column length (23cm)
 Moderate technology
 Moderate or high resolution
 High reproducibility of
separation
 Column support can be reused
 Moderate cost

25. CONCLUSIONS
 Flash chromatographic systems have been developed for the
separation and purification of organic molecules from natural
sources and from reaction mixtures.
 Method development for separations can be optimized with
proper selection of adsorbents, solvent systems and flow rates
of solvents used for the separation.
 Scale-up of flash chromatographic methods can easily be
achieved with minimum optimization.
 HPLC linear gradients can be transformed into step gradients in
flash chromatography.
 Therefore, flash chromatographic methods are considered to be
efficient and cost-effective methods to purify compounds in
little time.

26. THANKS