Column chromatography is a separation technique that uses a column packed with a stationary phase to separate mixtures based on how compounds partition between the stationary and mobile phases. Martin and Synge introduced partition column chromatography in 1941 using differences in how compounds partition between two liquid phases. Column chromatography can use a solid stationary phase for adsorption chromatography or a liquid stationary phase for partition chromatography. The technique works by selectively retaining compounds based on their interaction with and attraction to the stationary phase.
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•Martin and Synge introduced partition column
chromatography in 1941.
•CC may be defined as a separation process involving
the uniform percolation of a liquid solute through a
column packed with finely divided material.
•If solid is used as stationary phase then it is
Adsorption CC (solid – liquid chromatography)
•If liquid is used as stationary phase then it is Partition
CC (liquid – liquid chromatography)
INTRODUCTION
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Principle: Adsorption
• The rate of absorption varies with a given adsorbent
for different materials. This principle of selective
adsorption is used in column chromatography.
• A compound attracted more strongly by the mobile
phase will move rapidly through the column, and
elute from, or come off, the column dissolved in the
eluent.
• In contrast, a compound more strongly attracted to
the stationary phase will move slowly through the
column. Thus the compounds are separated.
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• The various bands present in the column become
more defined. The banded column of the adsorbent is
termed as chromatogram.
• The portion of a column which is occupied by a
particular substance is called as zone.
• The narrower the zones, the longer the number of
substances which can be separated in a column of a
definite length and the more concentrated are the
elutes.
• This type of interaction between the stationary phase
(adsorbent) and the solute is reversible in nature.
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2. PARTITION
• In partition column chromatography it is based on the
differences in partition coefficient of the individual
components of a mixture.
• The technique is similar to column adsorption
chromatography except the stationary phase is liquid.
• Stationary phase: liquid is supported in the column by means
of a solid material.
Eg. silica gel and cellulose powder are frequently used
as solid supports.
• Mobile phase: liquid
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Experimental aspects of Column
chromatography
1. COLUMN CHARACTERISTICS
• The main function of all the columns is to support the
stationary phase.
• The material of the column is mostly good quality
neutral glass since, it shouldn’t be affected by
solvents.
• An ordinary burette can also be used as column for
separation.
• Column dimensions - length & diameter ratio
(10:1,30:1 or 100:1)
10. Column efficiency
• It is expressed by the number of theoretical plates
• It is determined by the formula
• The number of theoretical plates is a measure of the
• “goodness” of the column
• If the retention time is high and peak width is narrow
then it
• shows excellent chromatograms
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11. 2. ADSORBENT
IDEAL PROPERTIES:
Their particle should be spherical in shape and
uniform in size.
Their mechanical stability must be great enough to
prevent the formation of fine dust which might be
deposited in the channels of the packing.
They should not react chemically, either with the
eluting solvents or with the sample components.
They should contain as small amount of soluble
component as possible.
They should be categorically inactive and as a rule,
have neutral surface. Exceptions are ion exchangers.16
12. It should be colorless to facilitate of zones and
recovery of components.
It should allow free flow of mobile phase.
Useful for separating wide variety of compounds.
The adsorbent is generally an active solid with a large
surface area.
Three types:
1. Weak – talc, sucrose, starch, inulin etc.,
MgCO3,
2.Intermediate – magnesia, CaCO3,
Na2SO4, etc.,
3. Strong – alumina, charcoal, bauxite, etc.,
Silica gel (acidic) may strongly retain basic
compounds.
Alumina (basic) should not be used for base sensitive
compounds
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3) Mobile Phase
They act as solvent, developer & eluent. The function
of a mobile phase are:
As developing agent
To introduce the mixture into the column – as solvent
To developing agent
To remove pure components out of the column – as
eluent
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PREPARATION OF COLUMN
• The bottom portion of the column is packed with
cotton wool or glass wool or asbestos pad, above
which the column of adsorbent is packed.
• A Whatman filter paper disc can also be used.
• After packing the column with the adsorbent, a
similar paper disc is kept on the top to avoid the
disturbance of adsorbent layer during the introduction
of sample or mobile phase.
• Disturbance in the layer of adsorbent will lead to
irregular bands of separation.
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• There are two types of preparing the column, which
are called as packing techniques. They are:
1. Dry packing technique
2. Wet packing technique
• Dry packing is the method of choice for a microscale
column.
• There are two versions of dry packing;
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The disadvantages of this technique are:
Air bubbles are entrapped between the solvent
& stationary phase.
Column is not packed uniformly.
Cracks appear in the adsorbent present in the
column.
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Wet packing method (slurry
method)
• The slurry method is often used for macro scale
separations.
• Combine the solid stationary phase with a small
amount of nonpolar solvent in a beaker.
• Thoroughly mix the two until a consistent paste is
formed, but is still capable of flowing.
• Never allow your column to run dry.
• It is allowed to settle under gravity until a column of
the desired height is obtained.
20. INTRODUCTION OF THE SAMPLE
1. Wet application
• Dissolve the sample in the initial mobile phase and
apply by pipette to the top of the column.
• This is very good method but in most of cases the
samples are not soluble in the initial mobile phase.
2. Dry loading
• Dissolve sample in any volatile solvent.
• The sample solution is then adsorbed on small
weight of adsorbent and the solvent is allowed to
evaporate.
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21. • The dry adsorbent loaded with the sample is then
applied to the column.
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Frontal analysis
• It consists of passing the sample solution
continuously through the adsorbent column.
• This makes the strongly adsorbed solutes are
adsorbed at the upper part of the column whereas the
less strongly adsorbed solutes are adsorbed down the
column.
• The solvent comes out first followed by the least
adsorbed solute.
• The other solutes emerge one after another depending
upon their degree of adsorptivity.
24. ELUTION ANALYSIS
ELUATE:
• The eluate, or mobile phase is a
combination of the mobile phase
"carrier" and the analyte material that
emerge from the chromatograph.
• It specifically includes both the
analytes and solutes passing through
the column.
• A small quantity of the sample solution
is introduced at the top of the column
followed by pure solvent which gives
rise to differential migration of solutes
in the mobile phase. 39
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• Depending upon the partition coefficient the solute
comes out from the column.
• For example: A small amount of the mixture having
components A & B at the top of the column and it is
eluted with an eluant C which is having a lesser
affinity for the stationary phase than the sample
components.
• Depending upon their relative affinity for the
stationary phase the components gets eluted in the
order of their affinities.
• The components can be separated completely with a
zone of the mobile phase that is eluant C.
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Elution analysis Types
• The components can be separated by means of elution
analysis by employing 3 different types of methods:
1.STEP-WISE ELUTION:
In this method, a set of eluants with increasing
eluting powers are used to separate components
which are having greater affinity towards the
stationary phase. Solvents of gradually increasing
polarity or increasing elution strength is used.
Example: initially benzene, then chloroform, then
ethyl acetate, then to methanol.
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2.GRADIENT ELUTION:
In this method components are separated by varying the
composition of the eluants.
In gradient elution, a modulator is often used in the
mobile phase to adjust eluent strength for better
results in chromatographic separations.
Compared with isocratic elution, the modulator
concentration in the mobile phase in gradient elution
is increased or decreased continuously with time.
Therefore, gradient elution can be used to separate
components which have a wide range of retentivity
with no loss of resolution.
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Gradient elution is able to produce high peak heights
in a shorter operation cycle compared with isocratic
elution.
For these reasons, gradient elution has been widely
used in high performance liquid chromatography for
analytical purposes.
Increasingly, gradient elution is applied to
preparative- and large-scale chromatography for the
separation of various macromolecules, such as
proteins.
3) Isocratic elution technique: Same solvent
composition or solvent of same polarity is used
throughout the process of separation
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DETECTION OF COMPOUNDS
• If the mixture to be separated contains colored
compounds, then monitoring the column is very
simple.
• The colored bands will move down the column along
with the solvent and as they approach the end of the
column, collect the colors in individual containers.
• However, most organic molecules are colorless.
• To detect the components which are colorless, several
techniques depending on the properties
absorption of radiations,
like
refractive index, TLC,
fluorescence etc are used.
30. • Eluting the sample:
Components a, b and c
separate
progresses.
• Fractions
as column
can be
collected in test tubes,
vials, beakers, or
Erlenmeyer flasks.
Detection of components
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RECOVERY OF COMPOUNDS
• Once you believe all the materials have been removed
from the column, the colours of the materials results
should indicate which fractions contain the
compound(s) you are interested in isolating.
• Combine the like or same fractions and evaporate the
solvent.
• The pure separated compound will be left behind.
• Recrystallization may be used to further purify a solid
product.
• However, on a milligram scale, there is usually not
enough material to do this.
32. Factors affecting column efficiency
• Nature of the solvents – Solvents of low viscosities
are used for high efficiency separations because the
rate of flow is inversely proportional to viscosity.
• Dimension of the column – For better separations,
sample/column packing ratios should range from 1 :
20 to 1 : 100.
• Pore diameter of column packing – polar
adsorbents should possess a pore diameter of ≤ 2nm.
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• Particle size of adsorbent – Column efficiency can
be increased by using the smaller particles (100 to
200 mesh) of adsorbents. It should be non-catalytic
and fine enough to hold the substance and give
relatively sharp bands.
• Pressure – High pressure above the column by
maintaining a column of liquid on the top of the
column. Low pressure below the column by applying
vacuum using vacuum pump.
• Temperature – Speed of elution is increased at
higher temperature, but adsorbent power is decreased.
Hence a room temperature is used.
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APPLICATION
1. In the separation of the mixtures into the pure
individual components.
2. Removal of impurities and in the purification of
compounds.
3. Determination of the homogeneity of chemical
substances.
4. Identification of unknown compounds.
5. Used in the separation of geometrical isomers,
diastereomers, recemates and tautomers.
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6. In the separation and identification of inorganic
anions and cations.
7. The concentrated of substance from dilute
solutions such as those obtained when natural
products are extracted with large volumes of the
solvents from the leaves of plants, trees, roots or
barks.
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Advantages of C.C
» Any type of mixture can be separated
» Any quantity of mixture can be separated
» Wider choice of Mobile Phase
» Automation is possible
Disadvantages of C.C
» Time consuming
» more amount of Mobile Phase are required
» Automation makes the techniques more
complicated & expensive
