m.pharm, 1st year, GNIPST

1. Discuss HPLC Shortly
(HIGH PERFORMANCE
LIQUID
CHROMATOGRAPHY)
By Bachaspati Jana

2. CHROMATOGRAPHY
1. It is define as it is analytical method in which separation of active constituent in complex
mixture, and the mixture was distributed in two phases e.g. Stationary phase and mobile
phase is known as chromatography.
2. It is technique is used for separation ,purification identification and extraction of compound.
3. It is method it can consist of two phases stationary phase and mobile phase.
4. Stationary phase is constant phase or column packaging material.
5. Mobile phase is moveable phase.
6. The basic principle of chromatography is base on Absorption and partition
chromatography.
7. Adsorption chromatography – The affinity of molecules towards stationary phase is known
as Adsorption chromatography.
8. Partition chromatography – The molecule can moves in two phases of liquid is known as
partition chromatography.
9. It is important for qualitative and quantitative analysis.

3. TYPES OF CHROMATOGRAPHY
• Base on modes of chromatography
1. Normal phase mode
2. Reversed phase mode
• Based on principle of separation
1. Adsorption chromatography
2. Ion exchange chromatography
3. Partition chromatography
4. Size exclusion
• Based on elution technique
1. Isocratic separation
2. Gradient separation
• Based on the scale of operation
1. Analytical HPLC
2. Preparative HPLC
• Base on the type of analysis
1. Qualitative analysis
2. Qualitative analysis

4. PRINCIPLES OF LIQUID CHROMATOGRAPHY
Time

5. TYPES OF LIQUID CHROMATOGRAPHY
LC mode Packing
material
Mobile phase Interaction
Normal phase
chromatography
Silica gel N-Hexane/IPE Adsorption
Reversed phase
chromatography
Silica- MeOH/Water Hydrophobic
Size exclusion
chromatography
Porous
polymer
THF Gel
permeation
Ion exchanger
chromatography
Ion exchanger
gel
Buffer sol. Ion exchange
Affinity chromatography Packings with
ligand
Buffer sol. Affinity

6. HPLC
 HPLC is an abbreviation for High Performance Liquid Chromatography. ... Only compounds
dissolved in solvents can be analyzed with HPLC. HPLC separates compounds dissolved in a
liquid sample and allows qualitative and quantitative analysis of what components and how
much of each component are contained in the sample.
 High Pressure Liquid Chromatography.
 High Priced Liquid Chromatography
 It is column chromatography
 It is modified from of gas chromatography, it is applicable for both Volatile as well as Non
volatile compound.
 It can mainly divided by two types 1. Normal Phase HPLC 2. Reversed phase HPLC
 It having a high resolution and separation capacity.

8. TERMINOLOGIES FOR
HPLC
 HPLC : High performance Liquid Chromatography ; High Pressure
LC
 Now, before we go in depth of principle, lets have a basic look at
few terms as follow;
 Resolving Power ; The extent of separation of the compounds
present in the mixture across the column.
 Theoretical plates : An imaginary division of the column into
equalling plates.

9. PRINCIPLE OF HPLC
• The table shows relation between various parameters of HPLC.
• Trendline
• Stationary phase have small particulate size and high surface areas.
• Columns: 20 cm or less
• Mobile phase pumped at higher pressures of 200Bar, 3000 psi.
• Flow rates : 1 - 3 cm3 per min
Column length(increase) No. of theoretical plates per
unite area(increase)
Resolving power(increase) Column length(increase)
Particle Size(decrease) Surface area (increase)

10. HPLC SYSTEM

11. COMPOSITION OF A LIQUID CHROMATOGRAPHY SYSTEM
1. Solvent
2. Solvent Delivery System (pump)
3. Injector
4. Sample
5. Column
6. Detectors
7. Waste collector
8. Recorder (Data Collection)

12. Computer
Detector
Solvent
reservoirs &
degassing
Column
Injector
Pump

13. PUMP
• The role of the pump is to force a liquid (called the mobile phase) through the
liquid chromatography at a specific flow rate, expressed in millilitres per min
(mL/min).
• Normal flow rates in HPLC are in the 1- to 2-mL/min rage.
• Typical pumps can reach pressures in the rage of 6000-9000 psi (400-to 600-
bar).
• During the chromatographic experiment, a pump can delivery a constant
mobile pump can deliver a constant mobile phase composition (isocratic) or
an increasing mobile phase composition (gradient).

14. Injector
• The injector serves to introduction the liquid sample into flow
stream of the mobile phase.
• Typical sample volumes are 5 to 20 microliters(μL)
• The injector must also be able to withstand the high pressure of
the liquid system.
• An auto sampler is the automatic version for when the user many
sample to analyse or when manual injection is not practical.

15. Column
• Considered the “heart of chromatograph” the Colum's stationary phase
separates the sample components of interest using various physical
and chemical parameters.
• The small particles inside the column are what cause the high back
pressure at normal flow rates.
• The pump must push hard to move the mobile phase through the
column and this resistance cause a high pressure within the
chromatograph.

16. Detector
• The detector can see(detect) the individual molecules
that come out(elute)f rom the column.
• A detector serves to measure the amount of those
molecules so that the chemist can quantitatively analyse
the sample components.
• The detector provides an output to a recorder or
computer that results in the liquid chromatogram(e.g.
the graph of the detector response).

17. Computer
• Frequently called the data system,
• The computer not only controls all modules of the HPLC
instrument but it takes the signal from the detector and
uses it to :
1. Determine the time of elution(retention time) of the
sample components (qualitative analysis) and
2. The amount of sample (quantitative analysis).

18. HPLC USES
This technique is used for –
Chemistry and biochemistry research analysing complex mixtures
Purifying chemical compounds
Developing process for synthesizing chemical compounds
Isolating natural products, or predicting physical properties.
It is also used in quality control to ensure the purity of raw materials, to
control and improve process yields, to qualify assays of final products, or
to evaluate product stability and monitor degradation.
In addition,
It is used for analysing air and water pollutants, for monitoring materials
that may jeopardize occupational safety or health, and for monitoring
pesticide levels in the environment.