BGC TRUST UNIVERSITY, BANGLADESH

*TOPIC : COLUMN CHROMATOGRAPHY

 SUBMITTED TO
Prof.Dr.Md.Hazrat Ali Miah
Professor
BGC TRUST UNIVERSITY ,BANGLADESH

 SUBMITTED BY
Srijib Chakraborty
Roll:1824005
Reg. No. : 180824005
Semester :6th
 *COLUMN CHROMATOGHRAPHY & ITS
CLASSIFICATION
Column chromatography in chemistry is a chromatography method used
to isolate a single chemical compound from a mixture. Chromatography is
able to separate substances based on differential adsorption of compounds
to the adsorbent; compounds move through the column at different rates,
allowing them to be separated into fractions. The technique is widely
applicable, as many different adsorbents (normal phase, reversed phase, or
otherwise) can be used with a wide range of solvents. The technique can
be used on scales from micrograms up to kilograms. The main advantage
of column chromatography is the relatively low cost and disposability of
the stationary phase used in the process. The latter prevents cross-
contamination and stationary phase degradation due to recycling. Column
chromatography can be done using gravity to move the solvent, or using
compressed gas to push the solvent through the column.
 CLASSIFICATION
Two types of column (or liquid) chromatography are generally used:
1. Adsorption chromatography: Adsorption chromatography is based on the
interaction between the solute molecules and active sites on the stationary
phase. This attachment or interaction depends on the polarity of solutes. This
technique proves the statement that “polar like polar”. Because if the stationary
phase is more polar than the mobile phase then high polar compounds in the
mixture will tightly bound to the stationary phase whereas less polar compounds
will lightly bound to the stationary phase. Less tightly bound compounds will be
eluted out by the mobile phase earlier than the tightly bonded ones.
2.Ion-exchange chromatography: Ion-exchange chromatography (or ion
chromatography) is a process that allows the separation of ions and polar
molecules based on their charge. It can be used for almost any kind of charged
molecule including large proteins, small nucleotides and amino acids. The
solution to be injected is usually called a sample, and the individually separated
components are called analytes. It is often used in protein purification, water
analysis, and quality control.
 *PACKING OF COLUMN
1. Use a piece of wire to add a plug of cotton to the bottom of the column.
There should be enough cotton that the sand and silica will not fall out
of the column. However, too much cotton or cotton packed too tightly
will prevent the eluent from dripping at an acceptable rate.
2. Clamp the column to a ring stand and add enough sand to fill the curved
portion of the column.
3. Place a pinch clamp on the tubing, then fill the column 1/4 to 1/3 full
with the intial eluent. (The composition of eluent is often changed as
the separation proceeds.)
4. Prepare a slurry of silica in the intial eluent by pouring dry silica into a
beaker of eluent. (Add a volumne of silica gel, such as 20 mL, to
approximately double the volume of eluent, 40 mL.) CAUTION: keep
the dry silica in your hood and be careful not to inhale the lightweight
substance.
5. Quickly but carefully pour the slurry into the column. Stir and pour
immediately to maximize the amount of silica that goes into the column
instead of remaining behind in the beaker. You may find a clean spatula
or glass rod helpful in transfering the silica.
6. Remove the pinch clamp to allow solvent to drip into a clean flask. Tap
on the side of the column with a rubber stopper or tubing to help the
silica settle uniformly.
7. Use a Pasteur pipet to rinse any silica that is sticking to the sides of the
column. Allow the silica to settle while eluent continues to drip into the
flask.
8. Once the silica has settled, carefully add sand to the top of the column.
Sand is heavier than silica. If the silica has not settled, the sand may
sink into the silica instead of forming a layer on top of it. (You may
need to rinse down sand that sticks to the side of the column.
 *ADSORBENT & ITS CLASSIFICATION
Adsorbent

The substance on which adsorbate is adsorbed is called adsorbent. Major types of

adsorbents in use are: activated alumina, silica gel, activated carbon, molecular sieve
carbon, molecular sieve zeolites and polymeric adsorbents.

 CLASSIFICATION
 * METHOD OF SEPERATION IN COLUMN
CHROMATOGRAPHY

Separation in column chromatography relies on differences. Molecules vary in

size, charge, polarity, and solubility. We leverage these differences to distribute
molecules between a stationary phase and a mobile phase. But because
molecules are so different, it’s not possible to have a single method that works
for all.
In my previous article I discussed the basic process of running a
chromatography column. And now, with all the different methods available, I
think it’s important for you to know some of your options and how they work.
The methods shown below take advantage of several molecular properties to
dictate the mode of separation.
Pave the Way
Hydrophobic interaction chromatography separates molecules based on their
hydrophobicity. The stationary matrix has hydrophobic groups that interact with
the hydrophobic regions of molecules, but they have to find each other first.
Remember the “mix oil and water” experiment? Of course you do! Well, they
don’t mix, right? That’s because oil is hydrophobic—it’s not attracted to water.
The same happens with a hydrophobic column and water. Because there are no
interactions between them, a network of hydrogen bonds among water molecules
from the eluent forms instead. This network surrounds hydrophobic groups in the
column, preventing your analyte from binding to the matrix.
To enable binding, break the water shield by treating the column with a chaotropic
salt solution. This introduces ions that interact with water and thus, exposes the
hydrophobic groups. If you use a high-salt solution your molecule will bind
strongly to the matrix. At this point you wash out the junk. After, elute your
molecule with a low-salt solution as it promotes the reformation of the water
shield.

Like Attracts Like

Normal/reversed-phase chromatography separates molecules by polarity. The
stationary phase contains either highly polar or highly non-polar functional groups
that interact with molecules according to their polarity level.
If we go back to the concept of oil and water not mixing, we can also say that
because water is highly polar and oil is highly non-polar, they don’t attract each
other but instead are attracted to their own molecules. In other words, it shows how
polar attracts polar and non-polar attracts non-polar.
In normal-phase chromatography, the stationary phase is more polar than the
mobile phase. So as polar molecules are retained in the column, your elution of
molecules will go from non-polar to polar. For reversed-phase chromatography
things are, well, the reverse. You use a non-polar stationary phase that retains non-
polar compounds and so, you elute first the polar molecules.