Instrumental Methods of Analysis (13PH0701)

Unit 3: Adsorption Column Chromatography

Mr. Harshil Majethiya

Assistant Professor
Department of Pharmaceutical Sciences,
Faculty of Health Sciences,
Marwadi University,
Rajkot, Gujarat, India – 360 003
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Contents

• Chromatographic Terminologies
• Basic Principle of Chromatography
• Column Chromatography
• Types of Column Chromatography
• Introduction to Adsorption Column Chromatography
• Key Components of Adsorption Column Chromatography
• Methodology of Adsorption Column Chromatography
• Factors affecting Adsorption Column Chromatography
• Advantages of Adsorption Column Chromatography
Cont…

• Disadvantages of Adsorption Column Chromatography

• Applications of Adsorption Column Chromatography
Chromatographic Terminologies

Chromatograph: Instrument employed for a

chromatography.
Stationary phase: Phase that stays in place inside the
column. Can be a particular solid or gel-based packing or a
highly viscous liquid coated on the inside of the column.
Mobile phase: Solvent moving through the column, either a
liquid in LC or gas in GC.
Eluent: Fluid entering a column.
Eluate: Fluid exiting the column.
Elution: The process of passing the mobile phase through
the column.
Cont…

Chromatogram: Graph showing detector response as a

function of a time.
Flow rate: How much mobile phase passed / minute
(ml/min).
Linear velocity: Distance passed by mobile phase per 1
min in the column (cm/min).
Retention time: It is a measure of the time taken for a
solute to pass through a chromatography column
Cont…
Basic Principle of
Chromatography
• All chromatographic separations rely on the differences in
interaction between analytes and the two characteristic
phases
• Mobile Phase: Carries/transports the analyte
• Stationary Phase: Interacts with the analytes as they are
moving through it.
• Analytes that strongly interact with the stationary phase are
retained longer, thus elute from the column later than those
interact weakly with the stationary phase.
• Analytes separate into bands.
• Analytes are detected at the exit of the column and their
signals recorded.
Stationary Phase and Mobile phase

Stationary phase:
✓The stationary phase is the substance which is fixed in
place for the chromatography procedure.
✓Hold different drug to a different extent,
✓Mostly solid

Mobile phase:
✓Solvents are continuous moving,
✓Gas, liquid, or super critical fluid.
Column Chromatography

Column chromatography is generally used as a purification

technique: it isolates desired compounds from a mixture.
Types of Column Chromatography

Sr. Types of column Stationary

Mobile phase Sample phase
No. chromatography phase

Adsorption Solid
1 Liquid Solution
chromatography adsorbent

Immiscible
Partition
2 Liquid solvent on Solution
chromatography
solid matrix
Cont…

Types of column Stationary

Sr. No. Mobile phase Sample phase
chromatography phase

Ion exchange Ion exchange

3 Liquid Solution
chromatography resin

Solvent held in
the interstices
4 Gel chromatography Liquid Solution
of a polymetric
solvent
Introduction to Adsorption Column Chromatography

• Adsorption Column Chromatography is a fundamental

technique used in separating and purifying compounds in
analytical and preparative chemistry.
• It is based on the principle of adsorption, where the
components of a mixture interact with a solid adsorbent
material packed in a column.
• This interaction is primarily due to physical forces such as
Van-der Waals forces, Electrostatic Interactions,
Hydrogen Bonding, or Hydrophobic Effects.
Key Components of Adsorption Column Chromatography

1. Adsorbent (Stationary Phase): The solid material

within the column where the adsorption occurs. Common
adsorbents include Silica Gel, Alumina, and Ion-
exchange resins. The surface of the adsorbent is coated
with a thin layer of liquid (the stationary phase) or is itself
the stationary phase.
2. Solvent (Mobile Phase): The liquid that carries the
sample through the column. The choice of the solvent
depends on the nature of the sample and the desired
separation. It can be a single solvent or a mixture of
solvents.
 Cont…

3. Sample: The mixture of compounds that needs to be

separated. The components of the sample will adsorb
onto the stationary phase to varying degrees.

Link for Adsorption Column Chromatography Video: https://www.youtube.com/watch?v=hdj9-w8LAn0&list=PPSV

Methodology
Cont…

1. Selection of Adsorbent and

Solvent:
• Adsorbent: Choose an appropriate
adsorbent material (e.g. Silica Gel,
Alumina) based on the chemical
properties of the compounds to be
separated.
• Solvent: Select a solvent or solvent
mixture (mobile phase) that can
effectively dissolve the sample
components and has the right elution
strength.
Cont…
Cont…
Cont…

2. Column Preparation:
• Column Selection: Choose a column
with an appropriate diameter and
length based on the scale of
separation and the amount of sample.
• Packing of the Column: The
adsorbent is carefully packed into the
column to form a dense and uniform
bed. Air bubbles must be eliminated to
ensure the smooth flow of the solvent.
Cont…
Cont…
Cont…

3. Sample Application:
• Sample Dissolution: Dissolve the
sample in a minimal volume of a
solvent that is compatible with the
elution solvent.
• Application to Column: Apply the
dissolved sample carefully onto the
center of the adsorbent bed without
disturbing the adsorbent.
Cont…

4. Elution Process:
• Initial Elution: Start with a solvent
that has low elution strength to retain
the strongly adsorbed components on
the column.
• Gradient Elution: Gradually increase
the elution strength of the solvent to
desorb and elute the components in
order of their adsorption strength.
• Monitoring: Monitor the elution
process visually (if the components
are colored) or by collecting fractions
for further analysis.
Cont…
Cont…

5. Fraction Collection:
• Collection: Collect fractions of the
eluent at regular intervals or based on
changes in eluent composition or
properties.
• Testing: Test the fractions for the
presence of the desired compounds
using techniques such as TLC, UV-Vis
Spectroscopy, or Mass Spectroscopy.
Cont…

6. Fraction Analysis:
• Identification: Identify the fractions
containing the compounds of interest.
• Purity Check: Assess the purity of
the compounds in the fractions.
Cont…

7. Isolation and Purification:

• Evaporation: Evaporate the solvent
from the fractions containing the pure
compounds.
• Further Purification: If necessary,
subject the compounds to additional
purification steps, such as
recrystallization or further
chromatographic separation.
Cont…

8. Documentation and Analysis:

• Documentation: Record the details
of the chromatographic separation,
including the adsorbent used, solvent
system, flow rate, and fraction
collection times.
• Analysis: Analyse the purified
compounds using appropriate
analytical techniques to confirm their
identity and purity.
Cont…

Troubleshooting and Optimization:

• Resolution Issues: If the separation
is not satisfactory, adjust the solvent
system, adsorbent type, or column
dimensions.
• Flow Rate Issues: Ensure that the
flow rate is consistent and appropriate
for the separation.
• Sample Application Issues: If the
sample spreads too much on the
column, reapply the sample using a
more concentrated solution or a
smaller volume.
Factors Affecting Adsorption Column Chromatography
Cont…

1. Adsorbent Type: Different adsorbents

have different surface properties and
will interact differently with various
compounds.
2. Solvent Polarity: The polarity of the
solvent affects how strongly the
compounds are eluted from the
adsorbent.
3. Flow Rate: The speed at which the
solvent moves through the column can
influence the separation.
4. Column Dimensions: The length and
diameter of the column, as well as the
particle size of the adsorbent, can
affect the resolution of the separation.
Advantages of Adsorption Column Chromatography

1. Versatility: Adsorption Column Chromatography can be

used for a wide range of compounds, from small organic
molecules to large biomolecules like proteins and nucleic
acids.
2. Scalability: The technique can be scaled up from
analytical to preparative scales, allowing for the
purification of small to large quantities of material.
3. High Resolution: With the proper choice of adsorbent
and solvent system, high resolution can be achieved,
leading to the effective separation of complex mixtures.
Cont…

4. Selective Adsorption: Different compounds can be

selectively adsorbed based on their chemical properties,
which allows for targeted purification.
5. Sample Recovery: The technique is non-destructive,
and the compounds can be recovered after separation
with little to no chemical modification.
6. Flexibility in Elution: Gradient elution can be used to
improve separation by gradually changing the solvent
composition.
Disadvantages of Adsorption Column Chromatography

1. Time-Consuming: The process can be time-consuming,

especially for a complex mixture that requires careful
optimization of conditions.
2. Skill Level: Successful chromatography requires
experience and skill in selecting the appropriate
adsorbent, solvent system, and operating conditions.
3. Sample Loss: There can be some loss of sample due to
adsorption to the column or during the transfer of
fractions.
Cont…

4. Equipment Cost: High-performance columns and

associated equipment can be expensive, especially for
preparative scale operations.
5. Solvent Consumption: The technique can consume
large volumes of solvent, which may be costly and
environmentally unfriendly.
6. Requires Monitoring: Continuous monitoring of the
elution process is necessary to identify and collect the
desired fractions, which can be labor-intensive.
Applications of Adsorption Column Chromatography

• Adsorption Column Chromatography is used in various

fields, including biochemistry, organic chemistry, and
environmental analysis.
• It is particularly useful for the purification of proteins,
nucleic acids, and small organic molecules.
• It can also be scaled up for preparative purposes to
isolate larger quantities of pure compounds.