FAISAL RAJPUT DOTAI

1. INTEGRAL UNIVERSITY
LUCKNOW
Department of chemistry
HPLC An Important Analytical Technique
Presented by
FAISAL RAJPUT
Roll No. 1801025009

3. Introduction
Principle
Types Of HPLC Separation
Instrumentation
Advantage
Disadvantage
Application

4. INTRODUCTION
Developed by HUBER in 1969
HPLC commonly called High Performance Liquid Chromatography
HPLC is a chromatographic technique used to separate a mixture
of compound in Analytical Chemistry and Biochemistry with
the purpose of identify , quantify or purify the individual
components of the mixture
It can mainly divided two type
 Normal phase( Mobile Phase )
 Reversed phase( stationary Phase)
1 . MOBILE PHASE They consist of water organic solvent and
mixture of organic solvent . They are continuously flown like river
water and carried sample, example Methanol ,Acetone, Acetonitrile,
water .
2. STATIONARY PHASE They consist of large no of particles like
Silica .
They are constant in a place and the separation occurs in this
place example C-18 ,C-12

5.  Principal of HPLC:-
 HPLC is separation technique based on a solid
stationary phase and a liquid mobile phase. Separation
is achieved by adsorption partition or ion exchange
process.
 Principal:
 Adsorption
 Partition

6. TYPES OF HPLC SEPERATION
Normal Phase HPLC
This method separates analysts on the basis of polarity. the
stationary phase is usually silica and typical mobile phases are
hexane, methylene chloride, chloroform, diethyl ether, and
mixtures of these
Reverse Phase HPLC
The stationary phase is nonpolar (hydrophobic) in nature, while
the mobile phase is a polar liquid, such as mixtures of water and
methanol or acetonitrile.

7. Adsorption
In Between Normal and Reversed. Separation of moderately
polar analysts using adsorption onto a pure stationary phase
(e.g. alumina or silica)
Ion Exchange Chromatography
Separation of organic and inorganic ions by their partitioning
onto ionic stationary phases bonded to a solid support.
Size Exclusion Chromatography
Also called as Gel filtration chromatography. Separation of
molecules based on their size.

8. INSTRUMENTATION

9. Solvent system
The solvents or mobile phases used must be passed
through the column
Mobile phase reservoirs: Several reservoirs –
Use to carry mobile phase
Degassing: Remove dissolved gas - interfering
detection
Protect band spreading
Vacuum pumping.
Dust removal: Dust interference with detection,
column clogging,
Isocratic elution: constant composition
Gradient elution: different solvent systems during

10. Pumping system
High pressure pump to push the mobile phase through
the column.
 Requirements for HPLC pump.
 High pressure (up to 6000 psi)
 pulse free, prevents remixing of solutes
 control flow rate from 0.1 to 10 mL/min
Types of HPLC pumps
1) Reciprocating pumps: Most commonly used.
Disadvantage: pulses from single and double
piston.
2) Syringe pumps: Generates pulse free output.
Disadvantage: Limited mobile

11. Sample injection system
A device to inject the sample in to mobile phase
Limit of precision of HPLC
Sample size: 0.5 ~ 500 μL
No interference with the pressure
Sample loop, 1 ~ 100 μL,
Auto sampler: inject continuously
Volume uptake: 1 μL – 1 mL
Controlled temperature environment

12. Column
In which the separation will takes place
Column work as stationary phase holder in
HPLC (Heart of HPLC)
Analytical column: Column length 5cm to
30 cm
Column diameter 2mm to 50 mm and
particle
size 1μ to 20μ
Microcolumn: High speed and minimum
solvent consumption
Guard column: protects analytical column,
remove particulate matter and contamination
Temperature control: 0.1 °C - 150 °C.

13. Detectors
 The detector can detect the individual
molecules that elute from the column and
convert the data into an electrical signal
 A detector serves to measure the amount of
those molecules
 The detector provides an output to a recorder
or computer that results in the liquid
chromatogram
 Detector is selected based on the analyze or
the sample under detection

14. several kinds of detector
 NMR,IR.
 Electrochemical detector.
 Uv visible detector.
 Fluorescence detector.
 Refractive index.
 Photodiode array detector

15. IR detectors:
• filter instrument or FTIR
• Similar cell (V, 1.5 ~ 10 μL and b, 0.2 ~ 1.0mm)
• Limit: no suitable solvent, special optics
• FT-IR allows for spectrum records of flowing systems
analogous to the diode array system.
• Water/alcohols can be major interferences to solute
detection
• LOD 100 ng

16. U.V detectors:
Based on electronic transitions within molecules.
 Most common type of detector for LC
 Fixed wavelength, Hg lamp 254 nm (π => π*)
 Tunable wavelength, selectable for specific wavelengths,
monochromators or filters. Still limited to single
wavelengths.
 - 1 pg LOD
 Solvent limitations with UV-vis abs. Detectors
 Z-shape, flow-through cell (V, 1 ~ 10 μL and b, 2 ~ 10
mm)

17. Recorder
Recorders are used to recorded the electrical signal from
detector.

18. Advantage
 Separations of volatile and non volatile
components
 Quick analysis
 High resolution
 More reproducibility
 Analysis of very complex mixture
 High performance
 Accuracy and precision

19. Disadvantage
 High cost
 Complex to operate

20. Application
 Qualitative analysis
 Checking the purity of a compound Presence of impurities
 Quantitative analysis
 Multi component analysis or determination of mixture of
drugs
 Isolation and identification of mixture of compound
 Purification of compound
 Environmental application
 Biochemical separation , forensic test.
 Biopharmaceutical studies.
 Isolation & identification of drug.
 Cosmetic analysis
 Diagnostic studies
 Residual pesticide identification

21. THANK
YOU