This document discusses high-performance liquid chromatography (HPLC), which is a widely used technique for separating and analyzing components in mixtures. It describes the basic components and principles of HPLC, including pumps to pass a pressurized liquid and sample mixture through a column containing stationary phase particles. The components interact differently with the stationary phase and are separated into bands that are then detected and analyzed. Common detectors described are UV-visible, fluorescence, and electrochemical detectors. The document also discusses various modes of operation like isocratic and gradient elution and types of columns and stationary phases used.

1. ARCHANA.K.R
PHYSICAL SCIENCE

2. HPLC
 It is the most versatile & widely used type of
elution chromatography.
 The technique is used by chemists to
separate and determine species in a variety
of organic, inorganic and biological
materials.

3. i. HPLC can be defined as the technique for the
separation of components of mixtures by differential
migration through a column containing a
microparticulate solid stationary phase.
ii. To obtain satisfactory flow rates, the liquid must be
pressurized.
iii. Originally it was referred to as high pressure liquid
chromatography.But now a days the term high
performance liquid cromatography is preferred.

4. CLASSIFICATION
 Based on the type of stationary phase or separation
mechanism,
• Partition chromatography
• Adsorption chromatography
• Ion exchange chromatography
• Size exclusion chromatgraphy
• Affinity chromatography
• Chiral chromatography

5. PRINCIPLE
•HPLC relies on pumps to pass a pressurized liquid and a
Sample mixture through a column filled with a sorbent.
•The components of the sample mixture are separated from
each other due to their different degrees of interaction with
the sorbent particles.

6. INSRTUMENTATION
The components of a typical apparatus for
HPLC are,
 Mobile phase reservoirs and solvent treatment
System
 Pumping systems
 Sample injection system
 Columns for HPLC
 Detectors

7. •The block diagram showing the components of a typical apparatus for HPLC
Fig(a)

8. 1.MOBILE PHASE RESERVOIRS AND SOLVENT
TREATMENT SYSTEM.
A modern HPLC apparatus is eqipped withone or more
glass reservoirs,each of which contains 500 mL or more
of a solvent.
Provisions are often included to remove dissolved
gases and dust from the liquids.(Degassers)
Sparging is a process in which dissolved gases are
swept out of a solvent by bubbles of an inert, insoluble
gas.

9. ISOCRATIC AND GRADIENT ELUTION
An isocratic elution in which the solvent composition
remains constant.
A gradient elution in HPLC is one in which the
composition of the solvent is changed continuously or
in a series of steps.
In gradient elution, two or sometimes more solvent
systems that differ significantly in polarity are used.
Gradient elution frequently improves separation
efficiency.

10. 2. PUMPING SYSTEMS
The pumping system is one of the most important
features of an HPLC.
The main features of a good pumping system are,
 It is capable of outputs of at least 3.4*107Pa(5000p.s.i)
 Pulse-free output.
 There must be a flow delivery of at least 10cm3 min-1
for normal analysis.
 Resistance to corrosion by a variety of solvents.

11. There are three major types of pumps;
 Screw-driven syringe type:-produce pulse free
delivery whose flow rate is readily controlled.
Recipocating pump :-most widely used type of
pump.
Pneumatic pump:-These pumps operate by the
introduction of high pressure gas in the pump, and
the gas in turn forces the solvent from the pump
chamber into the column.

12. RECIPROCATING PUMP

13. HOW IT WORKS?...........
•The device consist of a small cylindrical chamber that is
filled and then emptied by the back- and- forth motion of
a piston.
•The pumping motion produces a pulsed flow that must
be subsequently damped.
•Advantages of reciprocating pump are,
1. Small internal volume
2. High output pressure(up to 10,000psi)
3. Ready adaptability to gradient elution
4. Constant flow rates

14. 3. SAMPLE INJECTION SYSTEM
• The most widely used method of sample introduction in
liquid chromatography is based on a sampling loop.

15. •This devices are an integral part of some liquid
chromatography equipment.
•Interchangeable loops are available to provide a choice
of sample sizes ranging from 5 to 500
•The loop is first filled with sample from a syringe while
the mobile phase flows directly to the column.
•By turning a handle to rotate the body of the valve,the
mobile phase is diverted through the loop thus injecting
the sample on to the top of the column with out stopping
the flow .

17. 4.COLUMNS FOR HPLC
•Columns used for HPLC are generally made of stainless
steel.
•Most columns range in length from 10 to 30 cm &have
inside diameters of 2 to 5mm
•Column packings typically have particles size of 3 to 10
micrometers.
•The most common packing for liquid chromatography is
prepared from silica particles.Other packing material
include alumina particles,ion-exchange resins....

18. 5.DETECTORS
Detectors are based on a selective response for the solute,such
as UV- absorbance or fluorescence ,or on a bulk property of the
mobile phase which is modified by the solute,such as refractive
index.
Charecteristics of detectors
Rapid and reproducible response to solutes
High sensitivity, i.e, able to detect very low levels of
solute
stability in operation
A cell design that does not entrap air bubbles that outgas
from the mobile phase at the end of the column.
A signal directly proportional to solute concentration

19. TYPES OF DETECTORS
UV-visible
Absorbance
Fluorescence Electro chemical
•Fixed wave length
•Variable wavelength
•Photo diode- array

20. REFERENCE
• Instrumental methods of chemical analysis-
Gurudeep.R.Chatwal & Sham.K.Chand
• Fundamentals of analytical chemistry-S.A.Skoog,West&Holler
• Internet