1. Column chromatography.

1.1. Introduction.
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. In this chromatography process, the molecule mixture is separated depending
on its differentials partitioning between a stationary phase and a mobile phase.

1.2. Application.
A. Separation of mixture of compound
B. Removal of impurities or purification process.
C. Isolation of active constituents.
D. Isolation of metabolites from biological fluids.
E. Estimation of drug in formulation and crude drug extract.
1.3. 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.
When the mobile phase along with the mixture that needs to be separated is introduced
from the top of the column, the movement of the individual components of the
mixture is at different rates. The components with lower adsorption and affinity to
stationary phase travel faster when compared to the greater adsorption and affinity
with the stationary phase. The components that move fast are removed first whereas
the components that move slow are eluted out last.
The adsorption of solute molecules to the column occurs in a reversible manner. The
rate of the movement of the components is expressed as:
thedistance travelled by solute
Rf = 
the distance travelled by solvent

1.4. Types of Column Chromatography.

F. Adsorption column chromatography
Adsorption chromatography is a technique of separation, in which the components of
the mixture are adsorbed on the surface of the adsorbent.
G. Partition column chromatography
The stationary phase, as well as mobile phase, are liquid in partition chromatography.
 H. Gel column chromatography
In this method of chromatography, the separation takes place through a column
packed with gel. The stationary phase is a solvent held in the gap of a solvent.
I. Ion exchange column chromatography
A chromatography technique in which the stationary phase is always ion exchange
resin.

1.5. Factors affecting column chromatography.

There are various factors that affect column efficiency and so when comparing
columns these factors need to be stated:
 Stationary phase (and how thick)
 Temperature
 Carrier gas and flow rate
 Particle diameter (packed column)
 Column pressure
 Column diameter.
 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.

1.6. Column
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 procedure to prepare the column by packing with silica or alumina:
A. 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.
B. Wet method: In wet method firstly a 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.
1.7. Examples.
Ex1: Anion exchange chromatography.
The 30 ml plasmid DNA sample prepared in accordance with Example 1 was divided
in half. To one of the samples was added solid PEG-8000 to a final concentration of
1% (wfv). PEG-8000 was not added to the other 15 ml sample.
A Q-SEPHAROSETM Fast Flow anion exchange column (Pharmacia, Piscataway,
N.J.) (5.0x5.0 cm) was pressure packed using the Pharmacia "BioPilot"TM system.
The col umn was equilibrated with either Buffer A (10 mM Tris HCL, pH. 8.0, 0.7M.
NaCl, 1 mM EDTA) containing 1% PEG-8000 or with Buffer A. Each 15 ml aliquot
was individually loaded into the "BioPilot”TM superloop.
After the PEG-containing sample was loaded, a four bed volume wash with buffer
Acontaining PEG was performed. After the control sample (no PEG) was loaded, a
four bed volume wash with buffer A was performed. The flow-through peak was
collected and retained for analysis on an agarose gel.
The DNA was then eluted from the column with four bed volumes of 100% buffer B
(10 mM Tris-HCl, pH 8.0, 1 mM EDTA) containing 1% PEG-8000 for the PEG-
containing DNA sample or with buffer B (noPEG) for the control DNA sample. 50 ml
fractions were collected.
Both the flow through and the fractions eluting after washing with buffer B were
analyzed by electrophoresis on a 0.8% agarose gel in the presence of ethidium
bromide.
The results indicated that in the presence of 1% PEG 8000, nearly the entire DNA
sample was retained on the column as virtually no DNA was present in the flow
through.
In contrast, in the absence of PEG-8000, significant leakage of plasmid DNA from the
column occurred in which plasmid DNA eluted in the flow-through with various
contaminants, indicating that complete binding of DNA was not obtained.
The presence of PEG-8000 improved the recovery of plasmid DNA from as little as
20% to generally 80%.
 Column: A Q-SEPHAROSETM Fast Flow anion exchange column
(Pharmacia, Piscataway, N.J.) (5.0x5.0 cm) was pressure packed using the
Pharmacia "BioPilot"TM system.
 Buffer A (10 mM Tris HCL, pH. 8.0, 0.7M. NaCl, 1 mM EDTA).
 Buffer B (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
Ex2: Anion exchange chromatography
A Pharmacia BPG 100/500 column was pressure packed using the Pharmacia
"BioPilot"TM system according to the manufacturer's instructions. Final column
dimensions were 10x13 cm resulting in a bed volume of about 1,000 ml.
The column was equilibrated with four volumes of buffer A containing 1% PEG-8000
at a flow rate of 35 ml/min. The partially purified plasmid DNA prepared in
accordance with Example 3 was passed through a 0.45pm sterile "Acrodisc" syringe
filter (Gelman Sciences) and loaded into the "Bio Pilot” Superloop.
After the sample was loaded onto the column, a three bed volume wash with buffer A
was per formed.
The flow-through peak was collected in a 2 1 roller bottle and reserved for gel
analysis. The DNA was then eluted from the column with two bed volumes of 100%
buffer B containing 1% PEG-8000 for an additional two bed volumes. Fractions
(45ml) were collected after the step to 100% buffer B.
Following chromatography, the column was washed with one column volume of 2M
NaCl, followed by two column volumes of 1M NaOH. Fractions and flow through
were analyzed by electrophoresis on a 0.8% agarose gel and fractions containing
plasmid DNA were pooled and precipitated with 0.6 volume cold 2-propanol.
Similar to the results obtained in Example 2, basically no plasmid DNA was present in
the flow-through. Plasmid DNA eluted only after application of the step salt gradient
(buffer B).
The plasmid DNA obtained by anion exchange chroma tography in the presence of
1% PEG-8000 was further purified by gel filtration chromatography as described
below.