Introduction to HPLC

What is chromatography
What is HPLC
HPLC Components
Working principle
Applications

Chromatography
Invented by Mikhail Tswett in 1900
Plant pigments

Chromatography

Colors
CaCO3

What is HPLC?
High Performance Liquid
Chromatography
HPLC is really the automation of traditional
liquid chromatography under conditions
which provide for enhanced separations
during shorter periods of time, utilizing
very small particles, small column
diameters, and very high fluid pressures.

Advantages of HPLC
High performance liquid chromatography is
basically a highly improved form of column
chromatography. Instead of a solvent being
allowed to drip through a column under
gravity, it is forced through under high
pressures of up to 400 atmospheres. That
makes it much faster.
The other major improvement over column
chromatography concerns the detection
methods which can be used. These
methods are highly automated and
extremely sensitive.

Interaction Between Solutes,

Stationary Phase, and Mobile
Phase
Differences in the interactions between the
solutes and stationary and mobile phases
enable separation.

Solute

Degree of adsorption,
solubility, ionicity, etc.

Stationary
phase

Mobile phase
6

Stationary Phases
Polar (Normal Phase):
Silica, alumina
Non-Polar (Reversed Phase)
C18, C8

The Mobile Phase

Normal chromatography
Hexane; dichloromethane
Increasing strength

Reverse phase chromatography

water; methanol; acetonitrile
Increasing strength

Diagram of HPLC
Injector

Mixer

Pumps

Column

Detector

Solvents

Waste

Components of HPLC
1.
2.
3.
4.

Solvent Reservoir
Pumps
Sample Injection System
Guard Columns and main
column
5. Detectors
6. Data Processing
7. Waste

Solvent Reservoir
Mobile phase
isocratic elution proportion of the
solvents remains the same throughout
the operation
gradient elution solvent proportion can
be varied during separation
To carry sample into the column

Pumps
To produce an appropriate pressure
to push solvent into the column.
Single pump, binary pump, tertiary
pump, quaternary pump

Sample Injection System

sample valve
Syringe/injector

Syringe :
manual
Autoinjector

A fixed-volume loop of between 1

200 l (20 l is often used as
standard)

Guard column
Placed prior to column
For the safety of column
Particles collect in an HPLC columns inlet
frit or in the first millimeter of the packed
column, can cause degradation of analysis
such as peak tailing and split peaks.
Another source of problems are
compounds that irreversibly bond to the
stationary phase and are often injected
into analytical columns. These compounds
cause permanent damage to columns that
are not protected by a guard column.

Columns
straight, 15 to 250 cm in length; 2 to 5
mm i.d.
packing - silica gel, alumina, Celite

HPLC Detectors
UV/Vis
Diode Array Detector (DAD)
Refractive index
MS
Fluorescence

Data Processing
Using specific software that is connected
to HPLC machine
Receive the information from HPLC
machine and present it as a graph
The graph describes about qualitative data
(retention time) and quantitative data
(area under curve)

Separations
Injector

Mixer

Separation in based upon differential

migration between the stationary and
mobile phases.
Stationary Phase - the phase which
remains fixed in the column, e.g. C18,
Silica
Mobile Phase - carries the sample
through the stationary phase as it
moves through the column.

Pumps

Column

Detector

Solvents

Waste

High Performance Liquid Chromatograph

18

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

High Performance Liquid Chromatograph

19

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

20

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

21

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

22

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

23

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

24

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

25

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

26

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

27

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

28

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

29

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

30

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

31

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

32

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

33

time

Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection
Column

Detector

Solvents

34

time

The Chromatogram
tR- retention time - determines sample identity
tR

tR
mAU

Area or height is proportional

to the quantity of analyte.

Injection
35

time

Application of HPLC
1. Pharmaceuticals industry

To control the drug stability

Quantity of drug determination from

pharmaceutical dosage forms, ex. Paracetamol
determination in panadol tablet

Quantity of drug determination from biological

fluids, ex: blood glucose level
2. Analysis of natural contamination
- Phenol & Mercury from sea water
3. Forensic test
- Determination of steroid in blood, urine &
sweat.
- Detection of psychotropic drug in plasma

Application of HPLC
4. Clinical test
- Monitoring of vitamin D levels in the
patients.
5. Food and essence manufacture
- sweetener analysis in the fruit juice
- preservative analysis in sausage.

advantages
1. Needs a small sample with a high
accuracy and precise
2. Non-destructed sample during
operation compared to GC.

Disadvantages
Need a skill to run the instruments
Solvents consuming

Thank you!