Assignment Title:

Column Chromatography
Submitted by:
Name: Waris Hameed
Roll No: PCH07193057
Program: Master of Philosophy
Submitted to:
Dr Saima Zaheer
Introduction to column chromatography:

Column chromatography is a chromatography technique used to separate mixture

of chemical substances into its individual compounds. Column chromatography is
a widely used method for the purification or separation of chemical compound
mixture in lab.

Principles of column chromatography:

Column Chromatography consists of two phases: one mobile phase and one
contiguous stationery phase. The stationery phase is solid and the mobile phase is
liquid. The compound mixture moves along with the mobile phase through
stationery phase and separates depending on the different degree of adhesion (to
the silica) of each component in the sample or the compound mixture.

Explanation:

The stationery phase:

A glass tube with a circle large inlet and a small outlet with a plug or tap,
named as column is used for this column chromatography. The column is placed
vertically with a stand where the outlet is downward.
A piece of cotton wool is entered into the outlet and placed over the plug if there
are no glass wool present to stop escaping the stationery phase from the column.
There are two procedures to prepare the column by packing with silica or alumina:

Dry method: 
In dry method at first the column is filled with dry powdered silica. Then the
mobile phase, a suitable solvent is flushed through it until all the silica are wet and
settled. From this point till the end always the column need to keep wet with
solvent.

Wet method:
 In wet method firstly slurry of silica and solvent is prepared and then
poured onto the column using a funnel. More solvent must be used until the silica
is settled into it.

Process:
Column chromatography works in few steps:

Step 1:

The mobile phase or eluent is either solvent or solvent mixture. The upper
level of mobile phase should be same as the stationery phase. That means the
stationery phase should be wet with the solvent. On this stage the compound
mixture what need to be separated, are added from the top of the column in such a
way that the top level of it is not disturbed. By turning on the tap below it is
allowed to adsorb on the surface of the silica.
Step 2: 
Then the solvent or a suitable solvent mixture is added at first touching the
side of the glass column slowly and carefully so that the top level of the stationery
phase is not disturbed. The solvent is repeatedly added as many times as needed
throughout the process.

Step 3:
When the tap, is on the compounds in the compound mixture moves along
with the eluent depending on the polarity of the sample molecule. The non polar
components travel faster than the polar component.
 Suppose if any compound mixture contains three compounds blue, red and
green. According to polarity the order of these compounds are blue>red>green.
That means blue is the most polar compound and thus will have fewer tendencies
to move along with the mobile phase.

Step 4:
The green colored compound will travel first as it is less polar that other two.
When it is near end of the column a clean test tube is taken to collect the green
sample. After this the red and at last the most polar blue compound is collected, all
in separate test tubes.

Thus a compound mixture is separated or purified by using column

chromatography.

Summary

1. Column layer chromatography is an chromatography technique used to

separate mixture of chemical substances into its individual compounds.

2. Chromatography consists of two phases: one mobile phase and one

contiguous stationery phase.

3. Column is prepared my mixing the silica with suitable solvent and poured in
into a glass column.

4. A suitable solvent (mobile phase) is moved along with compound mixture

through the column according to the polarity.

Extraction and Purification of Flavonoids:

Flavonoids are one of the largest groups of secondary metabolites and widely
distributed in leaves, seeds, bark and flowers of plants with more than 4000
different structures which are classified according to their chemical structures as
follows; flavones, flavonols, flavanones, dihydroflavonols, isoflavones,
anthocyanins, catechins and calchones. Flavonoids which are part of human diet
are thought to have positive effects on human health such as reducing risk of
cardiovascular diseases and cancer. Most of the beneficial effects of flavonoids are
attributed to their antioxidant and chelating abilities (Cook & Samman, 1996;
Peterson & Flavonoids are structurally related compounds with a chromane-type
skeleton with a phenyl substituent in the C2 or C3 position (Rijke et al., 2006).
They are consisting of phenylpropane (C6-C3) unit derived from shikimic acid
pathway and C6 unit derived from polyketide pathway biosynthetically.
Flavonoids are present generally as mixtures and it is very rare to find only one
single flavonoid components in plants (Harborne, 1998). They are phenolic
compounds and hydroxyl groups often located at positions 3, 5, 7, 3’, 4’ and/or 5’.
One or more of these hydroxyl groups are frequently methylated, acetylated,
prenylated or sulphated. Flavonoids are present in plants as aglycone or as their
glycosides generally. Extraction of the flavonoids can be performed with solvents
that are chosen according to their polarity. Less polar aglycones such as
isoflavones, flavanones, dihydroflavonols, higly methylated flavones and flavonols
can be extracted with CH2Cl2, CHCl3, ether, EtOAC. However the more polar
aglycones including hydroxylated flavones, flavonols, chalcones and flavonoid
glycosides are generally extracted by polar solvents such as acetone, alcohol, water
and their combinations (Harborne, 1975; 1998). Many different chromatographic
techniques are employed for flavonoids isolation among them column
chromatography remains the most useful technique for large scale isolation
procedure. Conventional opencolumn chromatography is still widely used because
of its simplicity and its value as an initial separation step. Preparative work on
large quantities of flavonoids from crude plant extracts is also possible. Silica gel,
polyamide, sephadex, cellulose are commonly used adsorbents. Silica gel is
recommended mainly for separation of less polar flavonoids (isoflavones,
flavanones, dihydroflavonols and highly methylated/acetylated flavones and
flavonols). However, some flavonoid glycosides also can be purified on silica gel
using more polar solvents as eluants. Cellulose can be considered as a saceld-up
form of paper chromatography. Cellulose is suitable for separation of all class of
flavonoids and their glycosides. However, cellulose has low capacity and limited
resolving power. Sephadex led to the isolation of compounds on the basis of their
molecular size. The hydroxypropylated dextran gel, Sephadex LH-20 is designed
for use of with organic solvents or water/solvent mixtures. For Sephadex gels, as
well as size exclusion, adsorption and partition mechanisms operate in the presence
of organic solvents. Although methanol and ethanol can be used as eluents for
proanthocyanidins, acetone is better for displacing the high molecular weight
polyphenols. Slow flow rates are also recommended. Open-column
chromatography with certain supports (silica gel, polyamide) suffers from a certain
degree of irreversible adsorption of the solute on the column. Modifications of the
method such as dry-column chromatography, vacuum liquid chromatography are
also provide practical usage for the rapid fractionation of plant extracts. VLC with
a polyamide support has been reported for succesive separation of flavonol
glycosides. Medium-pressure liquid chromatography

(MPLC) which is a closed column (generally glass) connected to a compressed air

source or a reciprocating pump covers is also a simple alternative method to open-
column chromatography or flash chromatography, with both higher resolution and
shorter separation times. MPLC columns have a high loading capacity, up to a 1:25
sample-topacking- material ratio, and are ideal for the separation of flavonoids. In
MPLC, the columns are generally filled by the user. Particle sizes of 25 to 200 μm
are usually advocated (15 to 25, 25 to 40, or 43 to 60 μm are the most common
ranges) and both slurry packing and dry packing is possible. When compared a
shorter column of larger internal diameter with a long column of small internal
diameter (with the same amount of stationary phase) resolution is increased.
Choice of solvent systems can be efficiently performed by TLC or by analytical
HPLC.

Conclusion

Remarkable advances have been accomplished in natural products isolation

since the discovery of chromatography. Natural products are present generally as
mixtures which makes hard to separation of them. Different chromatographic
techniques such as paper chromatography (PC), thin layer chromatography (TLC),
gas liquid chromatography (GLC), high performance liquid chromatography and
column chromatography employ for separation and purification of natural
products. Amon them column chromatography which is the oldest
chromatographic technique, is still widely used especially for large scale isolation
procedure. Conventional open column chromatography is preferable because of its
simplicity. Different stationary phases can be available according to the polarity of
the test samples such as silica gel, bonded-phase silica gel, polyamide, sephadex,
cellulose, ionexchange resins. However separation procedure in conventional
column chromatography is time consuming and this method suffers from a certain
degree of irreversible adsorption of the solute on the column. Vacuum liquid
chromatography (VLC) a modified method of conventional column chromatograhy
provides practical usage for the rapid fractionation of extracts. Medium-pressure
liquid chromatography (MPLC) a closed column which connected to a compressed
air source or a reciprocating pump covers is also a simple alternative method to
open-column chromatography or flash chromatography.
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