TUMKUR UNIVERSITY

DEPARTMENT OF STUDIES AND RESEARCH IN

CHEMISTRY
University college of Science, Tumkur University , Tumkur
2023 - 2024

Seminar Topic :
HPLC – Principle , instrumentation , Detectors for HPLC – UV detector

Submitted by Under the Guidance of

Soundarya G Dr . Narendra N
I MSC , I Semester DOSR in Chemistry
U n i v e r s i t y c o l l e g e Science University college of Science
Tu m k u r Tu m k u r
CONTENTS
• Introduction
• Principle
• Instrumentation
• HPLC – UV detector
• Application
• Conclusion
• Reference
 INTRODUCTION

• HPLC : High performance liquid chromatography

• HPLC is an analytical technique used to separate , identify and quantify each component in a mixture.

• Csaba Horvath and S R Lipsky built the first HPLC

• HPLC : High pressure liquid chromatography

• Reverse phase HPLC is commonly preferred for separating non polar compounds.
PRINCIPLE

• The mobile phase along with the sample that needs to be separated is introduced into the column , the

movement of individual component of the mixture is at different rates.

• The component with the lower adsorption and lower affinity towards the stationary phase travel faster

when compared to the component with greater adsorption and greater affinity towards the stationary

phase.

• The components that moves fast are removed first whereas the component that move slowly are eluted

out last.

• In column chromatography the solvent being allowed to drip through a column under a gravity , Whereas

in HPLC the solvent is forced through a column under high pressure upto 6000 psi.

• The pressure is necessary for pushing the liquid mobile phase through the chromatographic column.

Without pressure , the flow rate would be insufficient and efficiency will be less.
High performance liquid chromatography
INSTRUMENTATION

Schematic representation of High Performance Liquid Chromatography

1. Solvent reservoir

• HPLC instrument is equipped with one more glass reservoirs.

• Isocratic elution : an elution with a single solvent or solvent mixture of constant composition.
• Gradient elution : two solvent systems that differ significantly in polarity are used.

2. PUMP
• A pump sucks the mobile phase from the solvent reservoir and pushes it through the system.
• Requirements for a liquid chromatographic pump
The generation of pressures upto 6000 psi
Pulse free output
flow rates ranging from 0.1 to 10 ml/min
resistance to corrosion by variety of solvents
• Two major types of pump are used in HPLC instruments
 Reciprocating pump
 Screw-driven syringe pump
3. Sample injector

• The sample solution is usually introduced into the flowing mobile phase at or near the head of the column

4. Column
• Length = 5 to 25 cm
Inside diameter = 3 to 5 mm
particle size = 3 to 5 μm
• HPLC columns are usually made from stainless steel , although
glass and polymer tubing such as PEEK are used.
• There are two types of column
Homogenous column
bonded column
• The separation takes place at the column
• Each separated components get eluted at the end of the
column and after elution each component that is eluted Column used for HPLC
passes into the detector.
• Stationary phase : Silica , alumina , the synthetic resign polystyrene Column used for HPLC
divinyl benzene.
5. Detector

• The detector senses the presence of the individual components as they leave the column.
• The detector converts a change in effluents into an electric signal that is recorded by the data system.
• There are different kinds of detectors such as absorbance , fluorescence , electrochemical , mass
spectrometric and refractive index detectors.

 UV Detector
• UV detector is categorized as absorbance detector.
• During analysis sample goes through a clear colourless
cell called flow cell. When UV light is irradiated on the
flow cell , sample absorbs a part of uv light.
• The intensity of UV light observed by the mobile phase
and eluent containing sample will differ.
• By measuring this difference , the amount of sample can
be determined.
UV detector for HPLC
• Types UV detector

Fixed wavelength detector

Variable wavelength detector

 Applications

High performance liquid chromatography is widely used for many clinical , forensic , environmental and

Industrial applications.

HPLC has replaced many gas chromatography procedures for forensic drug screening analysis.
CONCLUSION

High performance liquid chromatography is a crucial analytical technique widely used for separating and

Quantifying components in diverse mixtures.

HPLC’s precision and sensitivity enable the detection to trace amounts , ensuring results.

HPLC plays a vital role in quality control , especially in pharmaceutical manufacturing.

REFERENCE

• Instrumental methods of chemical analysis , Gurudeep R Chatwall , Sham K Anand.

• Analytical chemistry , Sixth edition , Gary D Christian.

• Skoog and West’s Fundamentals of Analytical chemistry , ninth edition , Stanly R Crouch , F James Holler.

• PG patashala.
THANK YOU