HPLC

High Performance Liquid

Chromatography

or

High Pressure Liquid

Chromatography (HPLC)
It is a separation technique used to:
• identify

• quantify and

• purify the individual components of the

mixture.
High-Performance LiquidChromatography
• Mobile Phase: Liquid

Based on the principle/ Separation mechanism HPLC utilizes

different types of stationary phases:

• Stationary Phase Separation Mechanism

- Ion exchange resin Ion exchange
- Chemically modified resin Affinity
In traditional liquid chromatography:
 External pressure is zero.

 Mobile phase passes through the column by under

the influence of gravitational force.

In HPLC:
The pressure applied is significantly higher i.e. 50–500 bar

Small sample amount is separated

Fixed volume of samples are used: 20 / 50 / 100 / 200 µl .

Dimensions of HPLC column dimensions are also small:

2.1 – 4.6 mm diameter, and
30–250 mm length
HPLC columns are packed with
smaller matrix particles (2–30 micrometer).
 Instrumentation
1. Solvent Reservoirs
2. Pump
3. Sample Injector
4. Column(s)
5. Detector
6. Data System
Parts of HPLC
 HALMINTON SYRINGE

It is specially designed syringe for HPLC.

Ideal for manual and automated HPLC injections.

There are made of:

• stainless steel or
• glass or
• borosilicate
ensures a chemically inert fluid path.
 Rheodyne injector or loop injector

•Used for sample injection in HPLC ,

•Has 6 port switching valves (Accessing points)

Has two distinct operations:

 Load mode (step – 1)
Inject mode (step – 2)
In load mode
sample from the syringe is
injected into the loop

In injection mode:
• the loaded sample
• is left into the flow of mobile phase

Excess sample is transferred to drain.

HPLC has loops of fixed volume like 20, 50, 100
and 200 µl .

Therefore,
when a sample of 25 µl is injected
into a loop of 20 µl,
then only 20 µl is passed
into the column
and
remaining is drained out.
In HPLC:
•injection volume is in the range of microlitres.

•Injection sample should be free of

particulate matter.

•Injection sample should be free of air

bubbles.

•Mobile phase passes through the column

under the influence of pressure.
 Mobile Phase Reservoirs
• Inert containers
• Provided with filters
• Solvent is degassed
 HPLC Pump
• Body is made of - Stainless steel
• inert toward solvents to be used
• Delivers high volumes (flow rates) of
solvent (to 10 ml/min)
• Delivers accurate flow
• Delivers in presence of high pressure
(50 – 500 Bar)
 HPLC Pumps: 3 types
• Reciprocating pumps
• Syringe pumps
• Constant pressure pumps
 Reciprocating Pumps
• PLUNGER plays important role in the movement of solvent.
• The left side / right side movement of the plunger sucks
the fluid into the cylinder.
• The upward / downward movement of the plunger pushes
the liquid out of the discharge valve.
 Syringe pumps
Used, when the mobile phase is required in very
small amounts
and
Flow rate of solvent (mobile phase):
Regulated for long periods (i.e. slow flow rate).
constant flow rate
 Constant Pressure Pump

• To apply constant high pressure.

• There is a constant flow rate
 Sample Introduction
TWO methods:

1. fixed volume
loop size is constant

2. variable volume
loop size variable
 Auto Injectors
• In Auto-injector or Auto-sampler
– Loading of sample
is done automatically
– Injection of sample
• More expensive
• Much more convenient to use.
• Upto 100 samples along with the standards can be
processed.
 Columns
Columns are made of:
• stainless steel tubing and
• heavy-walled glass tube.
 Column packing material

column packing materials / particles / spherical beads have

an average diameter of approximately 2–30 µm.

They are typically:

• silica-based
or
• alumina-based material
 Guard Columns
 increases the life of the column
by removing particulate contaminants from the solvents.

 composition of the guard-column packing is similar to

that of the column (column packing material size is same).

 when the guard column has become contaminated, it is

repacked or discarded and replaced with a new one.
 HPLC detectors

Detect
the solute particles
present in the eluent
that comes
from the
HPLC column.
Detectors should possess certain properties like:

• It should not REACT with mobile phase and

• it should not REACT with solute.

• Compatible for all types of compounds under testing.

 Should have good sensitivity i.e. able to detect compounds

at very low concentration in the ranges below
micro-grams / nano-grams.

• It should be unaffected:
by variation in temperature and
flow rate.
Based on the method or principle used - the
detectors are of 4 types:

•UV detectors,

•Fluorescent detectors,

•Electrochemical detectors

•Photo diode-array detectors (PDA).

 UV detector

detector work on the spectroscopy principle.

sample detection
depends on
absorption of UV ray energy
by the sample
UV detectors

These are commonly used detectors.

Capable to detect very wide range of
compounds.

The sensitivity ranges till microgram quantity of

estimation.
 Fluorescent detectors

The detector is suitable for compounds which

can produce florescence.

These detectors are very sensitivity i.e.

Compounds are measurable till
nanogram quantities.
 Electrochemical detector

This detector is specially suitable to estimate

oxidisable & reducible compounds.

The principle is that when compound is either

oxidized or reduced, the chemical reaction
produces electron flow.

The electron flow is measured as current

which is the function of type and quantity of
compound.
 Electrochemical detector

This detector has super sensitivity

which ranges to picograms
measurement.
 Photo diode-array detectors (PDA)

Principle is similar to UV detectors.

wide range of wavelengths at one time
because it is provided with polychromator.

Advantage is:

• higher sensitivity: microgram

quantity of the sample.
Computer operated software

HPLC system is connected to computer having

related HPLC software.

Used to set required pressure &

Used to set reqiured volume of the mobile phase.
APPLICATION

It is one of the sophisticated tool in the field of

science because of its:
 high sensitivity nature
 ability to evaluate samples of minute
concentration (nanogram and picogram).

In diagnosis, Research and Pharma industry.

Research labs which are involved in medical,
biological, chemical field wherein, the
analysis and quantification of molecules.

Pharma industry – Identification of new

chemical molecules.
 HPLC analysis in clinical diagnosis

Estimation of metabolites
from plasma, cerebrospinal fluid and urine
samples.