High Performance Liquid Chromatography (HPLC)

Objective

At the end of the
lecture, student will be able to

• Explain the principle of Column chromatography

• Discuss the instrumentation of Column chromatography

• Discuss the application of Column chromatography in
various fields

• Isolate and estimate phytoconstituents from natural source

Contents

• Principle of column chromatography

• Stationary phase

• Selection of stationary phase

• Mobile phase

• Column characters

• Column packing

• Elution

• Detection

• Application

• Advantages and disadvantages

HPLC – High
Performance Liquid Chromatography

Difference
between Column Chromatography and HPLC

Parameter	Column chromatography	HPLC
Particle size  – stationary phase	60 – 200µ	Small – 3-20µ
Column size	Large	small
Column material	Glass	Mostly metal
Column packing pressure	Low pressure	High
Sample load	Low to medium(g/mg)	Low to very low(µg)
Column efficiency	Low	High
Cost	Low- hundreds	High – lakhs
Types of stationary phases used	Limited range	Wide range
Mode of separation	Preparative scale	Analytical and preparative scale

               

Types of
HPLC Techniques

• Based on modes of
chromatography

– Normal phase mode

– Reverse phase mode

• Based on principle
of separation

– Adsorption chromatography

– Ion exchange chromatography

– Size exclusion (or) Gel permeation chromatography

– Chiral phase chromatography

• Based on elution
technique

– Isocratic separation

– Gradient separation

• Based on the scale
of operation

– Analytical HPLC

– Preparative HPLC

• Based on the type
of analysis

– Qualitative analysis

– Quantitative analysis

HPLC
Instrumentation

• Solvent Reservoir (HPLC solvent reservoir systems)

• Pumps

• Pre Guard Column

• Sample injection system

• Columns

• Detector

• Recorder and integrators

Instrumentation of HPLC

HPLC
Solvent Reservoir systems

Pumps –
Solvent Delivery system

• Mobile phase – pumped – column – high pressure – 1000 –
3000psi

• Column – particle size – 5- 10µ

Ideal
characteristics of a Pump

• Non corrosive and compatible with solvent.

• Provide High pressure to push mobile phase

• Provide constant flow rate to mobile phase

• Should have reproducible flow rate

• Should not leak

• High pressure generated by pump should not lead to an
explosion

• It should be easy to dismantle and repair

Types of
Pumps

• Reciprocating pump

• Displacement pump

• Pneumatic pump

Reciprocating pump –

Constant flow rate

• Reciprocating piston – moves back and forth in hydraulic
chamber

• By movement of piston – solvent flow into column under
high pressure

• Piston moves backward – inlet valve open, exit valve
closes – mobile phase drawn into the main chamber (cylinder)

• Piston moves to the front – inlet valve closes, exit valve
opens

• Volume reduction in main chamber due to forward motion of
piston – mobile phase moves out of the exit valve under high pressure

Advantages

• Generate high output pressure (upto10000 poise)

• Ready adaptability to gradient elusion

• Provide constant flow rate

• Pressure generated is so high that any back pressure
generated in the column due to higher viscosity of stationary phase can be
easily overcome

Disadvantage

• Pulsed flow – produce a base line noise on the
chromatogram

Pulse
dampner

• Dampen the pulses – wavy baseline caused – pumps

Mixing unit

• Mix – solvents – different proportions – column

Solvent degassing

Solvents – pumped with high pressure – gas bubbles –
interfere – degassing

• Vacuum filtration – Not complete

• Helium purging – very effective – costly

• Ultrasonication – Ultrasonicator

Injector –
Sample injection system

Septum injection port

• Syringe – inject the sample through a self-sealing inert
rubber septum directly into the mobile phase

Stop flow septum less
injection

• Flow of mobile phase through the column is stopped for a
while

• Syringe is used to inject the sample

Rheodyne
injector / loop valve type

• Sample introduced without causing interruption to mobile
phase flow

• Volume of sample ranges between 2 µl to over 100 µl

Sample
injection system

• Operation of sample loop

– Sampling mode

– Injection mode

• Sample is loaded into an external loop in the micro volume
sampling valve – subsequently injected into the mobile   phase by rotation of the valve

Column

Guard Column

• Small quantity of adsorbent

• Improves the life of the column

• Prefilter – Particles that clog the separation column,

• Compounds and ions that could ultimately cause baseline
drift, decrease resolution, decrease sensitivity and create false peaks

• No separation

Column (Analytical)

Column material

• Stainless steel or heavy glass, polyethylene, PEEE (POLY
ETHER ETHER KETONE)

• Colum length – 5 – 30cm

• Stationary phase – 25 µm or less

Standard column

• Internal diameter 4 – 5 mm & length 10 – 30 cm

• Size of stationary phase is 3 – 5 µm in diameter

• Used for the estimation of drugs, metabolites,
pharmaceutical preparation and body fluids like plasma

Narrow bore column

• Internal diameter is 2 – 4 mm

• Require high pressure to propel mobile phase

• For high resolution analytical work -compounds with very
high Rt

Short fast column

• Length 3 – 6 cm for substances which have good affinity
towards the stationery phase

• Analysis time less (1- 4 min for gradient elusion & 15
– 120 sec for isocratic elusion)

Preparative column

• Used for analytical separation i.e. to isolate or purify
sample in the range of 10-100 mg form complex mixture

– Length: 25 – 100 cm

– Internal diameter: 6 mm or more

Bonded Column

• C18 – Octa decyl silane column

• C8 – Octyl column

• C4 – Butyl

• CN – Nitrile

• NH2 – Amino column

Packing

• Depends on the mechanical strength of stationary phase

• Particle size of the stationary phase

– Particles of greater then 20 µm – dry packing

– Particles of lesser then 20 µm – slurry packing / wet packing

Dry packing

– Particle size greater than 20 µm filled into vertical clamped
column in small quantity

– Deposition is done by tapping or vibrating the column

– Column is unclamped, tapped on the firm surface to obtain dense
and reproducible packing

Wet/Slurry Packing

• Particle size with diameter less then 20 µm can only be
placed wet as a suspension

• Suspension should be stable, should not sediment

Detectors

• Ultraviolet- visible detector

• Refractive index detector

• Flourimetric detector

Recorders
and Integrators

• Recorders – to record the response obtained from the
detector after amplification

• Record the baseline and all the peaks obtained, with
respect to time

• Retention time for all the peaks can be calculated

• Integrators – improved versions of recorder with data
processing capabilities

• They can record the individual peaks with retention time
height and width of peak, peak area, etc

Applications

• Qualitative analysis

• Checking the purity

• Quantitative analysis

• Stability studies

Summary

• Principle of HPLC – partition

• Solvent Reservoir (HPLC solvent reservoir systems) –
mixing unit – degassing of solvents

• Pumps – Pneumatic and reciprocating pumps are used

• Pre Guard Column acts as prefilter and made up of same
material of column

• Sample injection system – Rheodyne injector, septum
injection port

• Columns – Bonded column, packed columns and capillary
columns are used

• Detector – Ultraviolet- visible, refractive index and
Flourimetric detector