VACUUM LIQUID CHROMATOGRAPHY

1. VA C U U M L I Q U I D
C H R O M AT O G R A P H Y
( V L C )

2. INTRODUCTION
• VLC seems to have its origins in Australia.
• The method was first described by Coll and Bowden in 1977 for the
isolation of a cambrenoid diterpene from an Australian soft coral.
• The name was first coined by Targett in 1979.
• It is a preparative layer chromatographic separation run as a column,
the flow of which is activated by vacuum.
• The vacuum is applied to speed up the eluent flow rates.
• It differs from flash chromatography in that the column is allowed to
run dry after each fraction is collected and also that VLC involves step
gradient elution.

3. • It is, hence, similar to preparative TLC because plates can
be dried after a run and then re-eluted. This repetitive
running of plates enhances separation.

4. PRINCIPLE
• The resolving power of a chromatographic column increases with
column length and the number of theoretical plates per unit length.
• As the number of theoretical plates is related to the surface area of the
stationary phase, it means smaller the particle size of the stationary
phase, the better the resolution.
• But smaller the particle size, greater is the resistance to eluent flow.
• Similarly in VLC, TLC grade silica (10-40µm) is used, hence there is
resistance to eluent flow.
Hence vacuum is applied to achieve faster eluent flow rates.
• Thus in VLC, solvent flow is vacuum driven.

5. APPARATUS
A- Sintered glass Buchner filter funnel
with fritted disk (B) (10-20µ) and
adapter with 24/40 joint (C).
D- Three-way stopcock.
E- To vacuum (water aspirator, 15-
25mm Hg).
F- To round bottom flask or
separatory funnel.
G- Rubber tubing.
H- Adsorbent (TLC grade).
I- Substrate, after absorption on
support.
J- Solvent.
Vacuum Liquid Chromatography (VLC) Apparatus

6. • The apparatus consists of a sintered glass buchner funnel
with a fritted disk.
• A ‘T’ 24/40 joint into which a layer of TLC grade
aluminium oxide or silica gel is packed.
• A three-way stopcock is used to control the vacuum
provided by a water aspirator (20-70mm Hg).

7. TECHNIQUE
• The adsorbent is first loaded into the sintered glass funnel and allowed to
settle by gentle tapping under gravity.
• Then vacuum is applied and the adsorbent is compressed to a hard layer
by pressing with a rubber stopper and tapping.
• After uniform and tight packing of the adsorbent, the vacuum is released
and solvent of low polarity is poured quickly onto the surface of the
adsorbent, and then vacuum is reapplied.
• The solvent should pass through the column uniformly. If it does not, then
column should be repacked.
• The column is then sucked dry, and the sample mixture, in the least polar
solvent, is carefully introduced into the surface of the packing (no
vacuum).1

8. • Enough solvent is used to completely cover the top surface of the
adsorbent.
• Then vacuum is applied gently to draw the sample into the packing. A
thin, uniform line of substrate should result at the top of the column.
• The substrate/sample is applied as a solution or sometimes by
preadsorbing on a suitable adsorbent (e.g. Al2O3, SiO2) and the latter is
applied as a uniform layer at the top of the column.
• The column is then developed under gentle vacuum with appropriate
solvent mixtures, pulling the column dry between the collection of
each fraction.

9. • The fractions can be collected in a round bottom flask or in a suitable
separatory funnel. The use of a separatory funnel avoids the problem
of changing the flask for each fraction and also is useful for collecting
the very small fractions without difficulty.
• Then each fraction is collected, an appropriate solvent is added to the
top of the column without vacuum until the surface is well covered.
• Then gentle vacuum is applied and gradient elution is employed.
• Channeling is easily avoided by sucking the column dry each time a
fraction was changed.

10. • Typical solvent ratios used are from 30:1 to 300:1.
• Loss of very volatile solvents was minimized by increasing
the flow rate of the solvent mixture using a larger pore size
of fritted like funnel.

11. ADSORBENTS USED
• Aluminium oxide 60H basic (10µm average particle size
and 60Å mean pore diameter),
• Silica gel 60HR (10-60µm average particle size and 60Å
pore diameter).

12. HEIGHT OF COLUMN BED
• Column bed should be about 5cm in height.
• For larger samples use wider diameter sintered glass funnel.
Hence, with increase in sample size, the diameter of the column is
also increased without increasing the height of the bed.
SAMPLE SIZE COLUMN HEIGHT
<100mg sample 4cm (0.5-0.1cm dia)
0.5-1.0g 2.5*4.0cm
1.0-10.0g 5*5cm
More larger samples 250ml sintered funnel

13. SOLVENTS
• Initially petroleum ether and increasing amount of more
polar solvent (CH2Cl2 , Et2O, EtOAc) are added.
• 10 to 100ml fractions are collected at each polarity step.
• Earlier fractions are collected in the sequence of 1%, 2%,
3% & 4%.
• Later they are collected in the sequence 5%, 10%, 20%,
50% and 100%.
• A more polar solvent like methanol is then added.

14. COLUMN PACKING
• The VLC column is “DRY PACKED", generally with 10-
40µm TLC grade silica, under vacuum to achieve the
maximum packing density.
• The vacuum is then released, low polarity solvent is
poured on to the surface of the adsorbent and the vacuum
is reapplied.
• The column is sucked dry and is then ready for loading.

15. SAMPLE INTRODUCTION
• The sample, in a suitable solvent is applied directly to the
top of the column or onto a layer of preadsorbent
(diatomaceous earth, celite etc.) .
• It is then drawn gently into the packing by applying the
vacuum.

16. ELUTION
• The column is eluted with appropriate solvent mixture , starting with
solvent of low polarity and gradually increasing the polarity, pulling
the column dry between each fraction collected.
Thus, VLC uses reduced pressure to increase the flow rate of mobile
phase. On the other hand, in contrast to other methods which use
pressure applied at top of the column to increase flow rates,
manipulations on the column (solvent changes etc.) are easy because
the head of the column is at atmospheric pressure.

17. APPLICATIONS
• Alkaloids are easily and rapidly separated by the vacuum technique.
e.g. Aconitum columbianum (Ranunculaceae) extract was
chromatographed to isolate talatizamine and cammaconin.
• Diterpenoids from the brown seaweed Dictyoda dentata was separated
from a dichloromethane-methanol (2:1) extract by using TLC grade
silica gel.
The compounds eluted are pachydictoyl A and dictoyl H.
• Antineoplastic diterpene dephnane orthoesters were obtained from
stems and leaves of Pimelia species (Thymelaeaceae) using VLC.
E.g. simpleximacrin isolated from Pimelia simplex using VLC.

18. • VLC is used for separation of 1g of 1:1 mixture of d-
fenchone and d-camphor.
Separation takes place on 50g of silica gel (10-40µm) and
solvent system used is petroleum ether : ethyl acetate.

19. ADVANTAGES
• It is a rapid and efficient chromatographic technique for
the separation of organic compounds.
• Manipulations on the top of column are easy because the
column head is at atmospheric pressure.

20. CONCLUSION
• Thus, VLC is a preparative layer chromatographic
separation run as a column, in which vacuum is used to
speed up the eluent flow rates.
• It is similar to preparative TLC because plates can be dried
after a run and then re-eluted.
• It is successfully being used for the separation of organic
compounds, such as, alkaloids, diterpenoids, antineoplastic
diterpenes, etc.

21. THANKS