High Performance Liquid Chromatography (HPLC)


An analytical separation technique that involves the high-pressure
flow of a liquid through a column that contains the stationary phase.

Stationary phase: Can be a solid (LSC) or a liquid (LLC)
 A mixture of compounds injected at one end of the column
separates as the compounds pass through.

 Separated compounds are detected electronically as they
elute at the other end of the column



1) Solvent or mobile phase

Major components
 Usually a mixture of an organic solvent (Ex. methanol, IPA)
and water

 Sometimes buffered – keeps solutes in electrically neutral form

 Solvent polarity affects the separation process
Mobile phase considerations 

Must be filtered (to prevent tiny solids from depositing at the
column head) and degassed

 Degassing is done by helium sparging
 Bubbles could interfere with detection




Role is to pump the solvent at a high pressure (usually from
1000 to 6000 psi) through the packed column



Sample introduction system

 Usually a loop injector – see image below
 Introduces the injected sample to the flowing mobile phase
 Automated injectors are common






A small metal tube (typically 5 to 30 cm long; 1-5 mm i.d.)
that contains the stationary phase
Instrumentation (Cont.)

 Role is to separate the components of a mixture



Column – Cont.
 Much shorter than columns used in GC — Why?
 Highly efficient separations achieved in HPLC due to
interactions of both m.p. and s.p. with the components of
a mixture
 vs. GC, where only the s.p. interacts with
 No need for long columns


Column Oven:

This allows the separation of individual sample components to be controlled and optimised. Columns come in two types: packed and capillary . Most gas chromatography today is performed using capillary columns, as they offer significant advantages for most samples



 Different design from those of GC detectors because the
components are dissolved in a liquid m.p. (vs. gas in GC)



HPLC Column Selection


Dependent on the:
(1) type of mixture being separated, and
(2) type of interaction with the s.p




What is Internal standard HPLC / GC- How to USE

Internal standard how we have to used? Internal standard 1. Quantatives analylsis 2. Purpose is 3. Selection process 4. Example is Quantatives analylsis main three parts • External standard • Internal standard • Standard addition External standard Here we find...