CHROMATOGRAPHY
By :
Dr. Mrs. P. S. Chaudhari
Associate Professor
Rasiklal M. Dhariwal Instittue of Pharmaceutical Education & Research, Pune
• Definition & Principle in chromatography
• It is defined as the process of
separation of the individual
components of a mixture based on
their relative affinities towards
stationary and mobile phases.
• Principle: The samples are subjected to flow by mobile
liquid onto or through the stable stationary phase. The
sample components are separated into fractions based
on their relative affinity towards the two phases during
their travel.
• The fraction with greater affinity to stationary layer
travels slower and shorter distance while that with less
affinity travels faster and longer.
Chromatography
• "Chromato" "graphy" derives its name from two
words as chromo= color and graphy= writing. I.e
color bands are formed in the procedure which
are measured or analysed. These color bands are
due to separation of individual compounds at
different lengths on the column as seen in
column chromatography and on paper in paper
chromatography.
• But in the modern methods like HPLC, Gas
chromatography [Link] color bands can be seen.
Types of Chromatography.
1. Adsorption based:
Here the stationary layer is a solid while the
mobile phase is liquid. The compounds travel on
the solid surface under the influence of mobile
liquid. The separation depends on the extent of
physical adsorption to the solid surface.
2. Partition based:
In this mode both the stationary and mobile
phase are liquids. So the compounds have
affinity based on their partition coefficients into
the individual liquid layer. The one with greater
partition to mobile liquid has higher affinity to it
so travels faster and vice versa.
COLUMN CHROMATOGRAPHY
Column chromatography (CC) is an extremely valuable
technique for purification of synthetic or natural
products.
Compounds are separated by CC through the same
mechanism as TLC;
through differential intermolecular forces between
the components of the mixture with the mobile
phase, and between the components with the
stationary phase.
A variety of adsorbents can be used as the stationary
phase;
silica gel (which is very polar) is most commonly
used in organic chemistry.
The principle of column chromatography is
based on differential adsorption of substance by
the adsorbent.
The usual adsorbents employed in cc are silica,
alumina, calcium carbonate, calcium phosphate,
magnesia, starch, etc.,
selection of solvent is based on the nature of both the
solvent and the adsorbent.
The rate at which the components of a mixture are
separated depends on the activity of the adsorbent
and polarity of the solvent.
If the activity of the adsorbent is very high and polarity
of the solvent is very low, then the separation is very
slow but gives a good separation.
On the other hand, if the activity of adsorbent is low
and polarity of the solvent is high the separation is
rapid but gives only a poor separation, i.e., the
components separated are not 100% pure.
The column
• Column chromatography works on a much larger
scale by packing the adsorbent materials into a
vertical glass column.
• Various sizes of chromatography columns are
used
Procedure of separation of component
• Suppose you wanted to separate a mixture of
two colored compounds - one yellow, one
blue. The mixture looks green.
What is happening?
• The blue compound is obviously more polar
than the yellow one - it perhaps even has the
ability to hydrogen bond.
• blue compound doesn't travel through the
column very quickly. That means that it must
adsorb more strongly to the silica gel or
alumina than the yellow one.
• The less polar yellow one spends more of its
time in the solvent and therefore washes
through the column much faster.
• It is going to take ages to wash the blue
compound through at the rate it is travelling
at the moment.
• Suppose you replace the solvent you have
been using by a more polar solvent once the
yellow has all been collected.
• That will have two effects,
• both of which will speed the blue compound
through the column.
• The polar solvent will compete for space on the silica
gel or alumina with the blue compound. Any space
temporarily occupied by solvent molecules on the
surface of the stationary phase isn't available for blue
molecules to stick to and this will tend to keep them
moving along in the solvent.
• There will be a greater attraction between the polar
solvent molecules and the polar blue molecules. This
will tend to attract any blue molecules sticking to the
stationary phase back into solution.
• The net effect is that with a more polar solvent, the
blue compound spends more time in solution, and so
moves faster.
What if everything in your mixture
is colourless?
COLUMN CHROMATOGRAPHY
Polar components (b) adsorb more strongly to the polar silica and elute after
the less polar components (a), which move more quickly with the non-polar
(relative to silica) solvent.
COLUMN CHROMATOGRAPHY
Skoog and Leary: Principals of Instrumental Analysis, 4th ed. Suanders, 1992
The process of washing a compound through
a column using a solvent is known
as elution. The solvent is sometimes known
as the eluent.
Procedure:
• Adsorbents
• Preparation of the Column
• Solvents used
• Adding the Sample to the Column
• Developing the Chromatogram:
• Recovering the Constituents
Step 1: Preparation of the Column
tamp it down on the bench top to pack the
silica gel
When properly packed, the silica gel fills the column to just below the indent on
the pipet. This leaves a space of 4-5 cm on top of the adsorbent for the addition
of solvent. Clamp the filled column securely to a ring stand using a small three-
pronged clamp.
Step 2: Pre-elute the column
• Add hexanes (or other solvent,
as specified by the procedure)
to the top of the silica gel. The
solvent flows slowly down the
column; on the column above,
it has flowed down to the point
marked by the arrow.
• Monitor the solvent level, both
as it flows through the silica gel
and the level at the top. If you
are not in a hurry, you can let
the top level drop by gravity,
but make sure it does not go
below the top of the silica
• You can speed up the process by using a pipet bulb to force the solvent through
the silica gel. Place the pipet bulb on top of the column, squeeze the bulb, and
then remove the bulb while it is still squeezed. You must be careful not to allow
the pipet bulb to expand before you remove it from the column, or you will
draw solvent and silica gel into the bulb.
• When the bottom solvent level is at the bottom of
the column, the pre-elution process is completed
and the column is ready to load.
keep the top solvent level above the top of the
silica
Step 3: Load the sample onto the silica gel
column
• Two different methods are used to load the column:
wet and dry
Once the sample is in the column, fresh eluting solvent is
added to the top and you are ready to begin the elution
process.
• For the dry method, first dissolve the sample to be
analyzed in the minimum amount of solvent and add
about 100 mg of silica gel.
Swirl the mixture until the solvent evaporates and
only a dry powder remains.
Place the dry powder on a folded piece of weighing paper...
... and transfer it to the top of the prepared column.
Add fresh eluting solvent to the top. Now you are ready to
begin the elution process.
Step 4: Elute the column
Force the solvent through the column by pressing on the top of the Pasteur pipet
with a pipet bulb. Only force the solvent to the very top of the silica: do not let the
silica go dry. Add fresh solvent as necessary.
The colored bands will travel down the column as the
compound is eluted.
As soon as the colored compound begins to elute, the collection
beaker is changed. The process is complicated if the compound is not
colored. In such experiments, equal sized fractions are collected
sequentially and carefully labeled for later analysis.
• Step 5 (Optional): Elute the column with the
second elution solvent
• Step 6: Analyze the fractions
• If the fractions are colored, you can 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.
Capacity Factor:
• The factor that control the distribution of any material
between the two competitive phases is called
“Distribution coefficient” or “Capacity factor” or
“Mass distribution ratio” K’
(n)s
K’ =
(n)m
(n)s : Is the total number of moles of the compound in the
stationary phase.
(n)m : Is the total number of moles of the compound in the
mobile phase.
tr – t0
K’ = t0
tr : Time required for the sample to cross the
column (retention time).
t0 : Time required for the solvent molecules to cross
the column.
• Bigger K’ means that the material retained more
time on the column i.e move slowly. Smaller K’
means faster movement.
2- Movement of materials through the chromatographic system in the
form of “zones” or “bands”:
• It was assumed that the chromatographic system
composed of number of “distribution systems” or
“equilibrations” called “Theoretical Plates”. Each
theoretical plat is composed of stationary phase and
mobile phase. The height of each plate is called “Height
equivalent to Theoretical Plate” (HETP).
• The number of theoretical plates “N” is important for
separation. Increasing “N” resulted in narrower bands and
better separation.
Basics of Chromatography
• Column Efficiency
L tR 2
N = 5.54( )
N= W1
H 2
tR 2
N =( )
W
L
N = HETP
L: Column length
“N” can be increased by:
1- Increase the length of the column (impractical).
2- Decrease the HETP.
How to decrease HETP:
➢ Decrease the particle size of the stationary phase.
➢ Proper selection of good mobile phase.
Materials move through the column as bands or zones and
velocity is controlled by K'.
Material with K' = 1 (64 molecules)
32 16 8 4 2
32 16 8 4 2
16 16 12 8
16 16 12 8
8 12 12
8 12 12
4 8
4 8
2
2
The material will be present in the middle of the system in the
form of band. If we increase “N” the material will from
narrower. Narrow bands allow better separation of mixtures.
2
2
8
8
12
12
8
8
2
2
N= 5 N= 25 N= 150
A A
A
B B
B
N= 5 N= 25 N= 150
• Multiple Flow Paths
• Tortuosity
Basics of Chromatography
• The Van Deemter Equation
H = A + B/u + Cu
• Components of
the
VanDeemter
Resolution
Basics of Chromatography
• Resolution
(tR ) B − (tR ) A
RS =
W
The Effect of Mobile Phase on Retention
More on Resolution
Isocratic & Gradient
Isocratic
Gradient
