Adsorption and Partition

Chromatography

By- Gauri Pillai

Neola Tauro
Meher Biju

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Adsorption Chromatography

- In 1906, Russian botanist Tswett originally developed the technique during the
course of an investigation into the nature of leaf pigments
- He found that leaf pigments extracted with light petroleum were adsorbed on
the top of a column of calcium carbonate supported in a glass tube
- As more solvent was allowed to percolate through the column, the region of
pigmentation became broader and finally separated into distinct and
differently coloured bands
- Prolonged washing with solvent caused complete separation of the bands

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- The principle underlying separation of compounds is adsorption at the solid-liquid
interface.
- Compounds of a mixture must show different degrees of affinity for the solid support
(adsorbent).
- Interaction between adsorbent and component must be reversible.
- Washing of the adsorbent with fresh solvent will allow the various components to move
down the column until they are arranged in order of their affinity for the adsorbent.

- Least affinity-move faster-eluted first

- The technique in which the individual components of a mixture are separated by eluting
the column with fresh solvent is elution analysis.

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Frontal Analysis
- When the frontal technique is used, the eluent containing several components is continuously
fed to the column.
- An adsorbent which is already saturated with respect to one substance may take up a small
quantity of a second.
- Least adsorbed component elutes out first and is the only component which is obtained in the
pure form.
- For instance, if the eluent contains three substances B, C and D with the sorption ability order
B < C < D, then separation in the column and on the chromatogram can be represented as
shown below

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Displacement Analysis
- A small volume of the mixture is added to the column which is developed by a
solution of a substance which is capable of displacing all the components of the
mixture.
- Although the division between components is sharp there are no intervening fractions
of solvent only, and some mixing is bound to occur between pairs unless more
displacing agents are used to separate A from B, B from C, and C from D.

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Adsorbent Requirements

- Insoluble in solvents
- Chemically inert
- active (not so active that no movement of components occurs)
- Colourless to facilitate observation of zones
- Allow suitable flow of mobile phase
- Reproducible properties from batch to batch

*Not always possible for adsorbents to comply as the mobile phases

and particle size also play a part

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Adsorption Isotherms
- The amount of a substance adsorbed from solution by an adsorbent can be determined
by shaking a known weight of adsorbent with a known volume of solution at a fixed
temperature until equilibrium is attained
- Filtering the adsorbent, the concentration of the substance in the filtrate is determined.
- The procedure is carried out with solutions of different concentrations and
graphs are plotted called ‘adsorption isotherms’

amount of substance
adsorbed per gram of
adsorbent is plotted
against the
concentration of
substance remaining
in solution

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Linear Adsorption Isotherms

- Obtained when the amount of substance adsorbed per

gram of adsorbent is proportional to the
concentration of solution.

- The adsorption coefficient (m/Cs) is a constant value

independent of the initial amount of the substance shaken
with the adsorbent

- When a substance moves as a band through a column of

adsorbent there is no tendency for any portion of the band
to be adsorbed more strongly than another.

- Therefore, a symmetrical peak is obtained as the eluate

from the column is examined.

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Convex Adsorption Isotherms

- These are obtained when adsorption from weak solutions is

greater than from strong solutions.

- Therefore, even if the pattern of the band of substance is

initially symmetrical, the substance, in low concentration, at
the front of the band is held more strongly by adsorption
than is the centre of the band as the band of substance
moves down the column.

- The centre of the band 'catches up' with the front and a
sharp leading edge to the band is obtained.

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Concave Adsorption Isotherms

- Obtained when adsorption from strong solutions is greater than from weak solutions

- This type of isotherm does not occur often

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Partition Chromatography:

Separations based upon the differences in

partition characteristics (partition
coefficients) of the individual components
of a mixture between a liquid stationary
phase supported on a solid and a
gaseous or liquid mobile phase.

Principle : partitioning behaviour of

substances between two immiscible
liquids.

Solute equilibrates between the mobile

phase and stationary liquid.
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● Substances with large differences in their partition coefficients may be
completely separated by simple solvent extraction techniques involving few
(one to three) extractions.
● During the chromatographic process the two liquid phases are in intimate
contact and this allows the partitioning of the components of the mixture to
occur rapidly.
● The components whose partition coefficients favour the moving liquid travel
down the column faster than those whose coefficients favour the adsorbed
stationary phase. The components thus emerge from the column in the order of
their partition coefficients.
● Tailing (seen in Adsorption Chromatography) is rarely seen in partition
chromatography because the partition coefficients of substances do not
vary with concentration unless adsorption effects are also present.

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Practical Pharmaceutical Chemistry by A.H.
Beckett and J.B. Stenlake

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Plate theory of Chromatography :
Martin and Synge in 1941 developed the concept .

The column is considered as being made up of a large number

of parallel layers or theoretical plates

The rate of movement of mobile phase is assumed to be such

that equilibrium is established within each components .

This equilibrium is dynamic - moves down the column depending on the rate of
mobile phase movement.

Height Equivalent to a theoretical plate (HETP):Thickness of the layer in which one

partition is considered to occur

The efficiency of a chromatography column is measured by its number of theoretical

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plates.
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Supports and liquid phases:

● Stationary Phase - Solid support coated with a thin layer of liquid phase which
is ordinarily polar in character, e.g. water or aqueous buffer solution.In GLC-
non polar solvents are also used
● Cellulose powder is also frequently used.
● Reversed Phase Chromatography: organic liquids (generally octanol) are
used as stationary phase with kieselguhr commonly used as support.

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 Advantages and Disadvantages

Advantages Disadvantages

The advantages of partition chromatography are as The disadvantages of partition

follows.
chromatography are as follows.
● easy-to-operate and economical method
● Data cannot be stored long in certain
for separation.
● The partition chromatography technique types of partition chromatography.

can isolate both organic and inorganic ● Loading capacity is low

compounds.
● It saves time because in a short duration it
separates compounds.
● Selectivity is better in partition
chromatography because a mobile phase
can be simply changed.

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Ref- Principles of Instrumental Analysis, 7th edition, Skoog-Holler-Crouch 19
 Partition Chromatography Applications
● Separation of proteins: Proteins can be separated by partition chromatography based on their
differences in size, charge, and hydrophobicity. This is important for the analysis and purification of
proteins for research and biomedical applications.
● Analysis of amino acids in urine: Amino acids in urine can be analyzed by partition chromatography
to diagnose metabolic disorders and other medical conditions.The profile of amino acids in urine can
be used to diagnose a variety of metabolic disorders and other medical conditions. For example, an
elevated level of phenylalanine in urine can be indicative of phenylketonuria (PKU), a metabolic
disorder that can cause intellectual disability if not treated. An elevated level of homocystine in urine
can be indicative of homocystinuria, a metabolic disorder that can increase the risk of blood clots
and heart disease.
● Analysis of drugs in blood: Drugs in blood can be analyzed by partition chromatography to diagnose
drug intoxication and monitor drug levels in patients.
● Analysis of lipids: Lipids, such as fats and cholesterol, can be separated by partition
chromatography based on their polarity and solubility in different solvents. This is important for the
analysis of lipids in food, blood, and other biological samples.
● Purification of natural products: Partition chromatography can be used to purify them from plants and
animals, such as vitamins, hormones, and antibiotics. This is important for the production of
pharmaceuticals and other products from natural sources.
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 Advantages and Disadvantages
Advantages Disadvantages

Cost-effective: Many common adsorbents used in Lack of selectivity: Adsorption chromatography may not
adsorption chromatography, such as silica gel or alumina, provide enough selectivity for complex mixtures, as it mainly
are relatively inexpensive. This makes it a cost-effective relies on non-specific adsorption. This can lead to co-elution
choice for routine analytical work. of compounds with similar adsorption affinities.

Easy to use: Adsorption chromatography is relatively easy Limited reusability: The adsorbent materials used in
to set up and perform, making it accessible to a wide range adsorption chromatography can become deactivated over
of laboratories and researchers. time, limiting their reusability and potentially adding to the
cost.
Separation based on chemical interactions: It allows for
separation based on various chemical interactions, such as Limited scale-up potential: While adsorption
hydrogen bonding, Van der Waals forces, and dipole-dipole chromatography is suitable for analytical purposes, it may
interactions. not be as readily scalable to larger preparative separations
due to limitations in column packing and efficiency.

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 Adsorption Chromatography Applications
● Adsorption chromatography is used for the separation of amino acids: Amino acids are separated by
adsorption chromatography based on their differences in polarity. More polar amino acids will adsorb
more strongly to the stationary phase, while less polar amino acids will elute more quickly.
● Isolation of antibiotics: Antibiotics are isolated by adsorption chromatography based on their
differences in affinity for the stationary phase. Antibiotics that have a higher affinity for the stationary
phase will elute more slowly. Ex: adsorption chromatography is used to isolate penicillin- The
penicillin mixture is dissolved in a mobile phase of water and methanol.The mobile phase is passed
through a column containing silica gel as the stationary phase.The penicillin interacts with the silica
gel to varying degrees, depending on its affinity for the surface.The penicillin elutes from the column
in order of decreasing affinity for the silica gel.The eluted penicillin is collected and purified.
● Identification of carbohydrates: Carbohydrates are identified by adsorption chromatography by
comparing their retention times to the retention times of known carbohydrates
● It can separate and analyze the components of cosmetics and fragrances, including essential oils
and aroma compounds.
Skoog, D. A., Holler, F. J., & Crouch, S. R. (2017). Principles of instrumental analysis (7th ed.). Cengage Learning.
Kellner, R., Merkle, H. P., & Moritz, W. E. (2013). Bioanalytical chemistry: A laboratory guide (6th ed.). Wiley-VCH. 22
Cooper, J. M., & Gunst, R. F. (2018). Quality control in the pharmaceutical industry (5th ed.). Academic Press.
Difference Between Adsorption and Partition Chromatography

• Partition chromatography - separation on the stationary phase occurs by partition due to

differences in partition coefficients.

• Used for liquid-liquid or liquid gas chromatography

• Adsorption Chromatography - relative differences in adsorption of constituents of given

sample. Because of differences in their affinity towards stationary phase, the components
of the mixture adsorb with different rates.

•Used for solid-liquid or solid gas chromatography.

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References

● Practical Pharmaceutical Chemistry by A.H. Beckett and J.B. Stenlake

● Principles of Instrumental Analysis by Skoog, Holler, and Crouch (7th ed.)
● Bioanalytical Chemistry: A Laboratory Guide by Kellner, Merkle, and Moritz (6th
ed.)
● Quality Control in the Pharmaceutical Industry by Cooper and Gunst (4th ed.)
● Analytical Chemistry 7th Edition by G. Christian.
● https://www.semanticscholar.org/paper/Tswett-and-the-Invention-of-
Chromatography-Ettre/056558fccf6d93a88a96fdd258bd3ceda9b12c2a
● https://www.thermopedia.com/cn/content/633/

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