Final year Bpharm 7th semester
Vikrant Ashok Shirke
Gsmcop, Wagholi, Pune
BP701T- INSTRUMENTAL METHODS OF ANALYSIS
(UNIT-IV) (21-36 MARKS)
Syallabus:-
Theory of Chromatography:-
Plate theory, Rate theory, System suitability parameters.
Gas Chromatography:-
Introduction, theory, instrumentation, temperatures programming, advantages,
disadvantages, and applications.
High Performance Liquid Chromatography (HPTLC):-
Introduction, theory, advantages and applications.
Q.1. Attempt the following:- (3 Marks Each)
1) Give a detail account on various pumps used in HPLC?
Give a detail account on any two pumps used in HPLC?
★ The various types of pumps used in HPLC:
➢ In High-Performance Liquid Chromatography (HPLC), pumps are critical components
responsible for delivering the mobile phase through the column at a consistent flow rate and
pressure. The performance of the pump directly affects the reproducibility, resolution, and
efficiency of the chromatographic separation.
1) Reciprocating Pump
● Principle: i) Uses a piston that moves back and forth in a chamber to draw in and push out
the mobile phase.
ii) Often combined with check valves to control the direction of flow.
● Types: 1) Single piston pump: Simple design but causes pulsed flow.
2) Dual or triple piston pump: Uses multiple pistons out of phase to reduce or
eliminate pulsation.
● Advantages: 1) Delivers high pressures (up to 6000 psi or more).
2) Suitable for gradient elution.
3) Accurate and reproducible flow rate.
● Disadvantages: 1) May require pulse dampeners to smooth flow.
2) Requires maintenance due to moving parts and seals.
2) Syringe Pump
● Principle: i) Uses a motor-driven syringe to deliver the mobile phase.
ii) Typically used for isocratic runs (constant mobile phase composition).
● Advantages: 1) Delivers pulse-free flow.
2) High accuracy and precision in flow rate.
● Disadvantages: 1) Limited volume capacity, requiring frequent refilling.
2) Not suitable for gradient elution without complex setup.
● Applications: Common in research settings or for low flow rate applications (e.g.,
micro-HPLC).
3) Pneumatic Pump
, ● Principle: Utilizes gas pressure (e.g., compressed air or nitrogen) to push the mobile
phase from a reservoir.
● Advantages: 1) No mechanical parts, hence low maintenance.
2). Produces a smooth, pulse-free flow.
● Disadvantages: 1) Low pressure capability compared to reciprocating pumps.
2) Flow rate depends on gas pressure, so it's less precise.
3) Not ideal for analytical HPLC.
4) Peristaltic Pump (used rarely in analytical HPLC)
● Principle: A set of rollers compress a flexible tube, pushing the mobile phase forward.
● Advantages: 1) Simple design and low cost.
2) No contact between the pump mechanism and the mobile phase (good for corrosive
solvents).
● Disadvantages: 1) Pulsatile flow.
2) Low pressure and limited accuracy.
3) Mostly used in preparative HPLC or auxiliary roles (e.g., waste removal).
2) Discuss the importance of gradient elution over isocratic elution techniques?
★ Importance of Gradient Elution Over Isocratic Elution in HPLC
➢ In High-Performance Liquid Chromatography (HPLC), elution techniques determine
how the composition of the mobile phase changes during the separation process.
● The two primary modes are:
1) Isocratic elution – mobile phase composition remains constant throughout the run.
2) Gradient elution – mobile phase composition changes gradually during the run (e.g.,
increasing organic solvent).
a) Better Separation of Complex Mixtures
Gradient elution is ideal for separating compounds with a wide range of polarities or retention
times.
In isocratic elution, highly retained components may take too long to elute, resulting in poor
resolution or broad peaks.
[ Example: Mixture containing both polar and non-polar compounds—gradient elution adjusts
solvent strength to elute all efficiently. ]
b) Shorter Analysis Time
In isocratic mode, compounds with strong retention require a longer run time to elute.
Gradient elution increases the strength of the mobile phase over time, which speeds up elution of
late eluting compounds.
[ Result: Faster analysis, especially for complex samples. ]
c) Improved Peak Shapes
Gradient elution reduces peak tailing and broadening, especially for late-eluting compounds.
Leads to sharper and more symmetrical peaks, enhancing resolution and quantification.
d) Enhanced Sensitivity
Due to better peak shape and reduced dilution of analytes, detection sensitivity increases in
gradient elution.
Particularly important in trace analysis or when using detectors like UV or MS.
e) Increased Column Lifespan
In gradient mode, strong solvents are introduced gradually, preventing sudden stress on the
stationary phase.
Reduces chances of column degradation or bleeding, thereby extending column life.
f) Flexibility in Method Development
,Gradient elution offers more control and flexibility in optimizing separations.
Parameters like gradient slope, initial and final composition, and hold times can be adjusted for
fine-tuning.
3) What is activation of plates? Write it's importance?
★ Activation of Plates in Chromatography
1) Activation of plates refers to the process of removing moisture or volatile contaminants
from the adsorbent layer (usually silica gel or alumina) coated on chromatographic plates,
especially TLC (Thin Layer Chromatography) plates, to restore or enhance their adsorptive
activity.
2) Commercial TLC plates often absorb moisture from the air during storage.
3) Moisture or impurities can interfere with adsorption between the sample and the stationary
phase.
4) Activation involves heating the plate to drive off adsorbed water or volatile materials and
ensure the stationary phase is in a highly adsorptive, dry state.
5) Typically, plates are heated in an oven at 110–120°C for 30 minutes to 1 hour.
6) The plates should then be cooled in a desiccator before use to prevent reabsorption of
moisture.
★ Importance of Activation of Plates
1) Ensures Optimal Adsorption
Moisture acts as a competing substance on the stationary phase.
Activation removes water, allowing better interaction between the analyte and adsorbent.
2) Improves Separation Efficiency
Activated plates provide sharper and more defined spots or bands.
Enhances resolution between closely migrating components.
3) Reduces Tailing and Smearing
Unactivated (moist) plates often result in tailing or diffused spots.
Activated plates ensure compact and symmetrical spot formation.
4) Improves Reproducibility
Consistent activation helps achieve reliable and reproducible chromatograms.
Essential for quantitative TLC or when comparing multiple samples.
5) Essential for Sensitive or Low-Concentration Samples
For trace analysis, any interference from moisture can significantly impact detection sensitivity.
➢ Conclusion:- Activation of TLC plates is a crucial preparative step to ensure reliable,
efficient, and reproducible chromatographic separation. By removing moisture and
contaminants, activation maximizes the adsorptive power of the stationary phase,
resulting in better spot definition, resolution, and accuracy.
, 4) Differentiate between guard column and analytical column?
Feature Guard Column Analytical Column
Purpose Protects the analytical column Performs actual separation of
from contaminants analytes
Location Placed before the analytical Placed after the guard column
column
Size Short (typically 1–5 cm in length) Longer (typically 10–25 cm or
more)
Internal Diameter (ID) Same or slightly larger than Standard (e.g., 4.6 mm, 3.0
analytical column mm)
Packed With Same stationary phase as the Optimized stationary phase
analytical column for separation
Cost Relatively low Expensive component
Role in Separation Does not participate in Main column for separation
separation; acts as a filter and resolution of components
Replaceability Frequently replaced to protect Replaced less often due to
main column higher cost
Maintenance Role Traps particulates, strongly Used for retention, resolution,
retained impurities, or matrix and quantification
components
Functions of a Guard Extends the life of the analytical Determines retention time,
Column column. peak shape, and resolution.
Functions of an Prevents column fouling or Crucial for qualitative and
Analytical Column clogging. quantitative analysis.
5) Write a note on system suitability of HPLC?
★ System Suitability in HPLC
● System suitability testing (SST) is a critical quality control procedure in High-Performance
Liquid Chromatography (HPLC) used to ensure the system is performing adequately
before or during analysis. It confirms that the chromatographic system meets the criteria
required for reliable and accurate data.
● System suitability refers to a set of tests performed to verify that the HPLC system,
column, mobile phase, detector, and method are functioning correctly before sample
analysis.
➢ Purpose of System Suitability Testing
1) To ensure precision, accuracy, and consistency of chromatographic results.
Vikrant Ashok Shirke
Gsmcop, Wagholi, Pune
BP701T- INSTRUMENTAL METHODS OF ANALYSIS
(UNIT-IV) (21-36 MARKS)
Syallabus:-
Theory of Chromatography:-
Plate theory, Rate theory, System suitability parameters.
Gas Chromatography:-
Introduction, theory, instrumentation, temperatures programming, advantages,
disadvantages, and applications.
High Performance Liquid Chromatography (HPTLC):-
Introduction, theory, advantages and applications.
Q.1. Attempt the following:- (3 Marks Each)
1) Give a detail account on various pumps used in HPLC?
Give a detail account on any two pumps used in HPLC?
★ The various types of pumps used in HPLC:
➢ In High-Performance Liquid Chromatography (HPLC), pumps are critical components
responsible for delivering the mobile phase through the column at a consistent flow rate and
pressure. The performance of the pump directly affects the reproducibility, resolution, and
efficiency of the chromatographic separation.
1) Reciprocating Pump
● Principle: i) Uses a piston that moves back and forth in a chamber to draw in and push out
the mobile phase.
ii) Often combined with check valves to control the direction of flow.
● Types: 1) Single piston pump: Simple design but causes pulsed flow.
2) Dual or triple piston pump: Uses multiple pistons out of phase to reduce or
eliminate pulsation.
● Advantages: 1) Delivers high pressures (up to 6000 psi or more).
2) Suitable for gradient elution.
3) Accurate and reproducible flow rate.
● Disadvantages: 1) May require pulse dampeners to smooth flow.
2) Requires maintenance due to moving parts and seals.
2) Syringe Pump
● Principle: i) Uses a motor-driven syringe to deliver the mobile phase.
ii) Typically used for isocratic runs (constant mobile phase composition).
● Advantages: 1) Delivers pulse-free flow.
2) High accuracy and precision in flow rate.
● Disadvantages: 1) Limited volume capacity, requiring frequent refilling.
2) Not suitable for gradient elution without complex setup.
● Applications: Common in research settings or for low flow rate applications (e.g.,
micro-HPLC).
3) Pneumatic Pump
, ● Principle: Utilizes gas pressure (e.g., compressed air or nitrogen) to push the mobile
phase from a reservoir.
● Advantages: 1) No mechanical parts, hence low maintenance.
2). Produces a smooth, pulse-free flow.
● Disadvantages: 1) Low pressure capability compared to reciprocating pumps.
2) Flow rate depends on gas pressure, so it's less precise.
3) Not ideal for analytical HPLC.
4) Peristaltic Pump (used rarely in analytical HPLC)
● Principle: A set of rollers compress a flexible tube, pushing the mobile phase forward.
● Advantages: 1) Simple design and low cost.
2) No contact between the pump mechanism and the mobile phase (good for corrosive
solvents).
● Disadvantages: 1) Pulsatile flow.
2) Low pressure and limited accuracy.
3) Mostly used in preparative HPLC or auxiliary roles (e.g., waste removal).
2) Discuss the importance of gradient elution over isocratic elution techniques?
★ Importance of Gradient Elution Over Isocratic Elution in HPLC
➢ In High-Performance Liquid Chromatography (HPLC), elution techniques determine
how the composition of the mobile phase changes during the separation process.
● The two primary modes are:
1) Isocratic elution – mobile phase composition remains constant throughout the run.
2) Gradient elution – mobile phase composition changes gradually during the run (e.g.,
increasing organic solvent).
a) Better Separation of Complex Mixtures
Gradient elution is ideal for separating compounds with a wide range of polarities or retention
times.
In isocratic elution, highly retained components may take too long to elute, resulting in poor
resolution or broad peaks.
[ Example: Mixture containing both polar and non-polar compounds—gradient elution adjusts
solvent strength to elute all efficiently. ]
b) Shorter Analysis Time
In isocratic mode, compounds with strong retention require a longer run time to elute.
Gradient elution increases the strength of the mobile phase over time, which speeds up elution of
late eluting compounds.
[ Result: Faster analysis, especially for complex samples. ]
c) Improved Peak Shapes
Gradient elution reduces peak tailing and broadening, especially for late-eluting compounds.
Leads to sharper and more symmetrical peaks, enhancing resolution and quantification.
d) Enhanced Sensitivity
Due to better peak shape and reduced dilution of analytes, detection sensitivity increases in
gradient elution.
Particularly important in trace analysis or when using detectors like UV or MS.
e) Increased Column Lifespan
In gradient mode, strong solvents are introduced gradually, preventing sudden stress on the
stationary phase.
Reduces chances of column degradation or bleeding, thereby extending column life.
f) Flexibility in Method Development
,Gradient elution offers more control and flexibility in optimizing separations.
Parameters like gradient slope, initial and final composition, and hold times can be adjusted for
fine-tuning.
3) What is activation of plates? Write it's importance?
★ Activation of Plates in Chromatography
1) Activation of plates refers to the process of removing moisture or volatile contaminants
from the adsorbent layer (usually silica gel or alumina) coated on chromatographic plates,
especially TLC (Thin Layer Chromatography) plates, to restore or enhance their adsorptive
activity.
2) Commercial TLC plates often absorb moisture from the air during storage.
3) Moisture or impurities can interfere with adsorption between the sample and the stationary
phase.
4) Activation involves heating the plate to drive off adsorbed water or volatile materials and
ensure the stationary phase is in a highly adsorptive, dry state.
5) Typically, plates are heated in an oven at 110–120°C for 30 minutes to 1 hour.
6) The plates should then be cooled in a desiccator before use to prevent reabsorption of
moisture.
★ Importance of Activation of Plates
1) Ensures Optimal Adsorption
Moisture acts as a competing substance on the stationary phase.
Activation removes water, allowing better interaction between the analyte and adsorbent.
2) Improves Separation Efficiency
Activated plates provide sharper and more defined spots or bands.
Enhances resolution between closely migrating components.
3) Reduces Tailing and Smearing
Unactivated (moist) plates often result in tailing or diffused spots.
Activated plates ensure compact and symmetrical spot formation.
4) Improves Reproducibility
Consistent activation helps achieve reliable and reproducible chromatograms.
Essential for quantitative TLC or when comparing multiple samples.
5) Essential for Sensitive or Low-Concentration Samples
For trace analysis, any interference from moisture can significantly impact detection sensitivity.
➢ Conclusion:- Activation of TLC plates is a crucial preparative step to ensure reliable,
efficient, and reproducible chromatographic separation. By removing moisture and
contaminants, activation maximizes the adsorptive power of the stationary phase,
resulting in better spot definition, resolution, and accuracy.
, 4) Differentiate between guard column and analytical column?
Feature Guard Column Analytical Column
Purpose Protects the analytical column Performs actual separation of
from contaminants analytes
Location Placed before the analytical Placed after the guard column
column
Size Short (typically 1–5 cm in length) Longer (typically 10–25 cm or
more)
Internal Diameter (ID) Same or slightly larger than Standard (e.g., 4.6 mm, 3.0
analytical column mm)
Packed With Same stationary phase as the Optimized stationary phase
analytical column for separation
Cost Relatively low Expensive component
Role in Separation Does not participate in Main column for separation
separation; acts as a filter and resolution of components
Replaceability Frequently replaced to protect Replaced less often due to
main column higher cost
Maintenance Role Traps particulates, strongly Used for retention, resolution,
retained impurities, or matrix and quantification
components
Functions of a Guard Extends the life of the analytical Determines retention time,
Column column. peak shape, and resolution.
Functions of an Prevents column fouling or Crucial for qualitative and
Analytical Column clogging. quantitative analysis.
5) Write a note on system suitability of HPLC?
★ System Suitability in HPLC
● System suitability testing (SST) is a critical quality control procedure in High-Performance
Liquid Chromatography (HPLC) used to ensure the system is performing adequately
before or during analysis. It confirms that the chromatographic system meets the criteria
required for reliable and accurate data.
● System suitability refers to a set of tests performed to verify that the HPLC system,
column, mobile phase, detector, and method are functioning correctly before sample
analysis.
➢ Purpose of System Suitability Testing
1) To ensure precision, accuracy, and consistency of chromatographic results.
