This document provides an overview of high performance liquid chromatography (HPLC). It discusses the basic components and principles of HPLC, including the stationary and mobile phases, various modes (normal and reverse phase), and instrumentation. The objective is to understand the purification and analytical applications of HPLC. The document outlines the experimental procedures for HPLC separation and analysis using chromatograms. Advantages include high performance and automation, while disadvantages include cost and difficulty with co-elution. HPLC has various applications in fields like drug discovery, food analysis, forensics, and clinical research.

1. HPLC
CHROMATOGRAPHY
Submitted to.-Dr.Bijender Singh
Associate professor, department of
biotechnology,cuh
Presented by-Mahesh Kumar
Roll.no.-191506
Deparment of biotechnology,cuh.

2. INTRODUCTION
The term hplc is given by Csabe Horvath.
This is the type of liquid chromatography.
This is known as high performance liquid
chromatography.
Also known as high pressure liquid
chromatography.
It’s is used for identification, quantification
and separation.
It has two types-
A.Normal phase HPLC
B.Reverse phase HPLC

3. MODESOF HPLC
Normal phase chromatography-This
method separates analytes on the basis of
polarity.
Stationary phase –silica(Hydrophilic)
Mobile phase(Non polar) –Hexane,
chloroform.
Reverse phase chromatography-
Stationary phase –hydrophobic
Mobile phase-polar
Mixture 10%+water 90%

4. OBJECTIVE
To understand the mechanism of
purification, function and
importance in analytical field.
To validate the developed method as
par ICH guidelines.

5. PRINCIPLE
HPLC is based on adsorption as well
as partition.
When mixture of components are
introduced into a Hplc column, they
travel according to their relative
affinities towards the stationary
phase.
The components which has more
affinities towards the Adsorbent,
travel slower.
The components which has less
affinities towards the stationary
phase,travels faster.

6. COMPONENTS
 1.Stationery phase-Here , column is used as
stationary phase.
 This is a tube like structure.
 This is made up with stainless steel and plastic.
 The column is properly packed in which a solid
Adsorbent material is contain.
 Example-Silica.
 2.Mobile phase –This is a liquid solution where
water is 90%and organic solvent is 10%.
 3.Sample-it may be blood, bacteria or fungi and
any plant material which we want to analyze.

7. INSTRUMENTATIO
N
 Solvent reservoir.
 Degasser.
 Injector.
 Column.
 Oven.
 Detector.
 Computer system.
 Data processor.
 Recorder.
 Collecting tube.
 Pump.
 Gradient device.

9. FUNCTIONOF
INSTRUMENTS
 1.Solvent reservoir-Here contains solvent and mobile phase.
 This is bottle like structure.
 2.Degassor – this will remove dissolved gas ,and avoiding bubble
formation.
 3.Gradient device-this allows automatically generation of column
before the separation.
 4.Pump-it should be capable of delivery the mobile phase , pressure
under 3000Psi to 5000Psi.
 5.Sample injector-used to inject the sample.
 Range-0.1-100ml., pressure-400Psi.
 6.Detector-used for detection of analyte.
 It may be –Ir detector,UV detector , electrochemical detector.
 7.Data processor-this is send the data from detector to recorder.
 8.Recorder-keep the record of exact value of separated molecules.
 Column-used as a stationary phase.
 This should be 300mm long.
 Diameter-2-5mm.

10. EXPERIMENTAL
PROCEDURES
1.Firstly ,we pass the buffer through a column.
We equilibrate the column to ready for purification.
Here we don’t pass the sample,only pass buffer,after that, detector
detects the signal to a computer system,and there will be developed a
straight line .
2.After that,we load the sample with mobile phase,after that, detector
detects the hydrophilic molecules first and on the system, there will
generate a peak,this peak will be hydrophilic molecules.
Detector is attached with a collecting tube.
The elute will be hydrophilic molecules first time.
Hydrophilic molecules will form a bond with resin .
Resin in hydrophilic in nature.
By this binding, Hydrophobic molecules move slow in the column.
So for this problem,we will apply here concentration gradient.
Here concentration gradient means that we will decrease the
concentration of water and increase the concentration of organic
solvent.

11. CONTINUOUS....
So, slowly-slowly polarity of mobile phase
changes.
As a result,bonded molecules elutes
slowly-slowly and they pass out through
column and detector detects the
hydrophobic molecules and send the
signal to the computer and this will
generate the second peak .
Eluted Molecules collected in collecting
tube.
Here , on computer system ,there will be
formation of a graph.
This is called chromatogram.

13.  Here in this graph ,on the x-axis,there will be retention time and y-axis,there
will be concentration or absorbence.
 By this graph ,we can calculate the area of peak.
 Peak area, generally tell as the concentration of molecules.
which molecules have high peak area , concentration will be more those
molecules.
By this graph ,we can know that how the peak is quantified.what is the
concentration of peak ,we can know by this graph.
Here we also know about the retention time.
Retention time-the amount of time it takes for the compound to pass through
the column.
By this graph ,we can also analyze the RRF value.
RRF value-known amount of a component/size of the peak
Also we determine the retention factor(k)-
Time sollute spends in stationary phase /time solute spends in mobile phase

14. ADVANTAGES
AND
DISADVANTAGES
Advantages-
 1.High performance.
 2.simple and fast.
 3.Good accuracy and precision rate.
 4.Automated procedure and data handling.
 5.Repetative analysis using same column.
Disadvantages-
 1.High cost.
 2.Co-elution is difficult.
 3.Requring large quantities of expensive organics.
 4.low sensitivity for certain compounds.
 5.Not universal.

15. APPLICATION
 Used in Drug discovery.
 Used in water purification.
 To study environment issues.
 Drug metabolism study.
 Proteomics analysis.
 Used in food industry.
 Used in forensic purpose.
 Used in clinical industry.
 Used for cosmetic analysis.
 Used to determine structure.
 Determination of retention time.

16. CONCLUSION
HPLC play a critical role in analysis of
pharmaceutical products.
The new technology in chemistry
and instrumentation provides more
and more information per unit of
works as HPLC begins to fulfill the
promise of increased speed,
resolution and sensitivity predicted
for liquid chromatography.

17. REFERENCE
 Alkesh Pradhan, chochin university of science and technology.
 School of industrial fisheries.
 www.water.com
 www.chemguide.co.uk
 www.science direct.com.
 hiq.linde-gas.com
 www.knaeur.net
 chem.libretexts.org

18. Thankyou