Final year Bpharm 7th semester
Vikrant Ashok Shirke
Gsmcop, Wagholi, Pune
BP701T- INSTRUMENTAL METHODS OF ANALYSIS
UNIT-III [05-21 MARKS]
Syallabus:-
Introduction to chromatography:-
Adsorption and partition column chromatography:-
Methodology, advantages, disadvantages and applications.
Paper chromatography:-
Introduction, methodology, development techniques, advantages, disadvantages and applications.
Thin layer chromatography:-
Introduction, principle, methodology, Rf values, advantages, disadvantages, and applications.
HPTLC:-
Introduction, instrumentation, and applications.
Q.1. Attempt the following:- (3 Marks Each)
1) Explain the principle and methodology of thin layer chromatography?
★ Principle of Thin Layer Chromatography (TLC):-
➢ TLC is based on the principle of adsorption chromatography. The components of a
mixture are separated due to differences in their affinity (adsorption strength) towards the
stationary phase and solubility in the mobile phase.
● Stationary Phase: A thin layer of adsorbent material (usually silica gel, alumina, or cellulose)
coated on a glass, plastic, or aluminum plate.
● Mobile Phase: A suitable solvent or solvent mixture that moves up the plate by capillary
action.
➢ Mechanism:
1) Components that interact strongly with the stationary phase move slowly.
2) Components that interact weakly or are more soluble in the mobile phase move quickly.
3) This leads to differential migration, resulting in separation of the components.
★ Methodology of TLC
1) Preparation of TLC Plate
Use a pre-coated TLC plate (e.g, silica gel on glass).
Handle with gloves or forceps to avoid contamination.
2) Sample Application
Use a capillary tube to spot a small amount of sample near the bottom edge (~1 cm from the
base), Mark the spot lightly with a pencil.
3) Development Chamber Preparation
Use a developing chamber (e.g, a beaker with a lid).
Add a small amount of mobile phase (solvent) to cover the bottom (~0.5 cm depth).
Line the chamber with filter paper to saturate it with solvent vapors (improves separation).
4) Development of the Plate
,Place the plate vertically in the chamber so that the sample spot is above the solvent level.
Allow the solvent to travel up the plate by capillary action.
Remove the plate before the solvent front reaches the top.
Mark the solvent front immediately with a pencil.
5) Drying and Visualization
Air dry the plate, Visualize the separated spots using UV light (for UV-active compounds), Iodine
chamber, or Spraying with chemical reagents (e.g, ninhydrin for amino acids).
6) Rf Value Calculation
Measure the distance travelled by the compound and solvent front.
● Calculate the Rf value:
Rf = [Distance travelled by compound/Distance travelled by solvent front]
2) Describe various development techniques used in paper chromatography? (3/5Marks)
★ Various Development Techniques in Paper Chromatography
A) Ascending Development
● Setup: 1) The solvent is placed in a chamber, and the lower edge of the paper (with sample
spot) is dipped in the solvent.
2) The solvent moves upward by capillary action.
3) Commonly used due to its simplicity and ease of operation.
● Advantages: Simple to perform, Good separation for most compounds.
B) Descending Development
● Setup: 1) The paper is hung from the top of the chamber, and the solvent is supplied from a
trough at the top.
2) The solvent moves downward, aided by gravity and capillary action.
● Advantages: 1) Faster development due to gravity,
2) Better separation for components with similar Rf values.
C) Radial or Circular (Centrifugal) Development
● Setup: 1) The sample is spotted at the center of a circular paper. The solvent moves
radially outward from the center toward the edges.
2) A wick connects the center of the paper with a solvent reservoir.
● Advantages: 1) Quick and effective for rapid screening.
2) Useful for multiple samples applied at different angles.
D) Two-Dimensional (2D) Development
● Used for complex mixtures.
● Step 1: Spot the sample and develop the chromatogram in one solvent (1st direction).
● Step 2: Dry the paper, rotate it 90°, and develop in a second solvent (orthogonal direction).
● Advantages: 1) Excellent resolution of complex mixtures.
2) Separates compounds with similar properties in one direction but different in another.
E) Horizontal or Edgewise Development
● The paper strip is kept horizontally, and solvent is allowed to migrate across the paper
from one end.
● Less commonly used in standard analytical labs.
● Advantage: Can be adapted for specific experimental needs.
3) Write the advantages of TLC over paper chromatography?
, ★ Advantages of Thin Layer Chromatography (TLC) over Paper Chromatography:
1) Faster Separation:- TLC usually takes less time than paper chromatography due to
faster solvent movement.
2) Better Resolution:- TLC provides sharper and better-separated spots, making
identification more accurate.
3) Choice of Adsorbent:- In TLC, various adsorbents like silica gel, alumina, or modified
phases can be used, Paper chromatography is limited to cellulose as the stationary phase.
4) Wider Range of Solvents:- TLC is compatible with a broader range of solvents, including
organic and non-aqueous solvents, Paper is sensitive to many solvents (especially strong
acids/bases), limiting the choice.
5) Greater Sensitivity:- TLC can detect smaller quantities of substances due to strong
adsorbent interactions and better visualization techniques.
6) Reproducibility:- TLC plates provide uniform coating and thickness, leading to more
reproducible results compared to hand-cut paper strips.
7) Visualization Flexibility:- TLC plates often have a fluorescent indicator, making it easier
to visualize compounds under UV light, Paper has limited options for direct visualization.
8) Quantitative Analysis:- TLC is more suitable for semi-quantitative or quantitative
analysis using densitometry or scanning methods.
9) Chemical Resistance:- TLC plates (glass or aluminum-backed) are chemically more
stable and can tolerate corrosive solvents better than paper.
10) Temperature Tolerance:- TLC plates can withstand higher temperatures, enabling
techniques like derivatization or heating for development.
4) Differentiate between normal phase and reverse phase chromatography?
Feature Normal Phase Chromatography Reverse Phase
(NPC) Chromatography (RPC)
Stationary Phase Polar (e.g., silica gel, alumina) Non-polar (e.g., C18, C8
hydrocarbon chains bonded to
silica)
Mobile Phase Non-polar or less polar solvents Polar solvents (e.g., water,
(e.g., hexane, chloroform) methanol, acetonitrile)
Polarity of Polar compounds are retained Non-polar compounds are
Compounds longer retained longer
Separation Based on adsorption Based on partitioning
Mechanism (hydrophobic interactions)
Elution Order Non-polar compounds elute first Polar compounds elute first
Common Applications Separation of isomers, lipids, and Widely used in
natural products pharmaceuticals, peptides,
and proteins
Water Compatibility Poor – water not usually used Good – water and aqueous
buffers often used
Use in HPLC Less common in modern HPLC Most widely used mode in
HPLC
Vikrant Ashok Shirke
Gsmcop, Wagholi, Pune
BP701T- INSTRUMENTAL METHODS OF ANALYSIS
UNIT-III [05-21 MARKS]
Syallabus:-
Introduction to chromatography:-
Adsorption and partition column chromatography:-
Methodology, advantages, disadvantages and applications.
Paper chromatography:-
Introduction, methodology, development techniques, advantages, disadvantages and applications.
Thin layer chromatography:-
Introduction, principle, methodology, Rf values, advantages, disadvantages, and applications.
HPTLC:-
Introduction, instrumentation, and applications.
Q.1. Attempt the following:- (3 Marks Each)
1) Explain the principle and methodology of thin layer chromatography?
★ Principle of Thin Layer Chromatography (TLC):-
➢ TLC is based on the principle of adsorption chromatography. The components of a
mixture are separated due to differences in their affinity (adsorption strength) towards the
stationary phase and solubility in the mobile phase.
● Stationary Phase: A thin layer of adsorbent material (usually silica gel, alumina, or cellulose)
coated on a glass, plastic, or aluminum plate.
● Mobile Phase: A suitable solvent or solvent mixture that moves up the plate by capillary
action.
➢ Mechanism:
1) Components that interact strongly with the stationary phase move slowly.
2) Components that interact weakly or are more soluble in the mobile phase move quickly.
3) This leads to differential migration, resulting in separation of the components.
★ Methodology of TLC
1) Preparation of TLC Plate
Use a pre-coated TLC plate (e.g, silica gel on glass).
Handle with gloves or forceps to avoid contamination.
2) Sample Application
Use a capillary tube to spot a small amount of sample near the bottom edge (~1 cm from the
base), Mark the spot lightly with a pencil.
3) Development Chamber Preparation
Use a developing chamber (e.g, a beaker with a lid).
Add a small amount of mobile phase (solvent) to cover the bottom (~0.5 cm depth).
Line the chamber with filter paper to saturate it with solvent vapors (improves separation).
4) Development of the Plate
,Place the plate vertically in the chamber so that the sample spot is above the solvent level.
Allow the solvent to travel up the plate by capillary action.
Remove the plate before the solvent front reaches the top.
Mark the solvent front immediately with a pencil.
5) Drying and Visualization
Air dry the plate, Visualize the separated spots using UV light (for UV-active compounds), Iodine
chamber, or Spraying with chemical reagents (e.g, ninhydrin for amino acids).
6) Rf Value Calculation
Measure the distance travelled by the compound and solvent front.
● Calculate the Rf value:
Rf = [Distance travelled by compound/Distance travelled by solvent front]
2) Describe various development techniques used in paper chromatography? (3/5Marks)
★ Various Development Techniques in Paper Chromatography
A) Ascending Development
● Setup: 1) The solvent is placed in a chamber, and the lower edge of the paper (with sample
spot) is dipped in the solvent.
2) The solvent moves upward by capillary action.
3) Commonly used due to its simplicity and ease of operation.
● Advantages: Simple to perform, Good separation for most compounds.
B) Descending Development
● Setup: 1) The paper is hung from the top of the chamber, and the solvent is supplied from a
trough at the top.
2) The solvent moves downward, aided by gravity and capillary action.
● Advantages: 1) Faster development due to gravity,
2) Better separation for components with similar Rf values.
C) Radial or Circular (Centrifugal) Development
● Setup: 1) The sample is spotted at the center of a circular paper. The solvent moves
radially outward from the center toward the edges.
2) A wick connects the center of the paper with a solvent reservoir.
● Advantages: 1) Quick and effective for rapid screening.
2) Useful for multiple samples applied at different angles.
D) Two-Dimensional (2D) Development
● Used for complex mixtures.
● Step 1: Spot the sample and develop the chromatogram in one solvent (1st direction).
● Step 2: Dry the paper, rotate it 90°, and develop in a second solvent (orthogonal direction).
● Advantages: 1) Excellent resolution of complex mixtures.
2) Separates compounds with similar properties in one direction but different in another.
E) Horizontal or Edgewise Development
● The paper strip is kept horizontally, and solvent is allowed to migrate across the paper
from one end.
● Less commonly used in standard analytical labs.
● Advantage: Can be adapted for specific experimental needs.
3) Write the advantages of TLC over paper chromatography?
, ★ Advantages of Thin Layer Chromatography (TLC) over Paper Chromatography:
1) Faster Separation:- TLC usually takes less time than paper chromatography due to
faster solvent movement.
2) Better Resolution:- TLC provides sharper and better-separated spots, making
identification more accurate.
3) Choice of Adsorbent:- In TLC, various adsorbents like silica gel, alumina, or modified
phases can be used, Paper chromatography is limited to cellulose as the stationary phase.
4) Wider Range of Solvents:- TLC is compatible with a broader range of solvents, including
organic and non-aqueous solvents, Paper is sensitive to many solvents (especially strong
acids/bases), limiting the choice.
5) Greater Sensitivity:- TLC can detect smaller quantities of substances due to strong
adsorbent interactions and better visualization techniques.
6) Reproducibility:- TLC plates provide uniform coating and thickness, leading to more
reproducible results compared to hand-cut paper strips.
7) Visualization Flexibility:- TLC plates often have a fluorescent indicator, making it easier
to visualize compounds under UV light, Paper has limited options for direct visualization.
8) Quantitative Analysis:- TLC is more suitable for semi-quantitative or quantitative
analysis using densitometry or scanning methods.
9) Chemical Resistance:- TLC plates (glass or aluminum-backed) are chemically more
stable and can tolerate corrosive solvents better than paper.
10) Temperature Tolerance:- TLC plates can withstand higher temperatures, enabling
techniques like derivatization or heating for development.
4) Differentiate between normal phase and reverse phase chromatography?
Feature Normal Phase Chromatography Reverse Phase
(NPC) Chromatography (RPC)
Stationary Phase Polar (e.g., silica gel, alumina) Non-polar (e.g., C18, C8
hydrocarbon chains bonded to
silica)
Mobile Phase Non-polar or less polar solvents Polar solvents (e.g., water,
(e.g., hexane, chloroform) methanol, acetonitrile)
Polarity of Polar compounds are retained Non-polar compounds are
Compounds longer retained longer
Separation Based on adsorption Based on partitioning
Mechanism (hydrophobic interactions)
Elution Order Non-polar compounds elute first Polar compounds elute first
Common Applications Separation of isomers, lipids, and Widely used in
natural products pharmaceuticals, peptides,
and proteins
Water Compatibility Poor – water not usually used Good – water and aqueous
buffers often used
Use in HPLC Less common in modern HPLC Most widely used mode in
HPLC
