High Performance Liquid

Chromatography (HPLC)
Liquid Chromatography (LC)

A sample mixture is eluted with a mobile phase (liquid) through a

column packed with a solid particles which may may or may not be
coated with another liquid

The solute separates on the column via interactions based on different

physical mechanisms.
 Types of Liquid Chromatography

Adsorption Partition Size Exclusion Affinity Ion Exchange

Competition Competition Molecular “Lock and Key” Competition

between liquid between liquid sieving mechanism between liquid
mobile phase mobile phase and ionic
and solid and “liquid” stationary phase
adsorbent stationary phase

The classification of chromatographic modes is based

according to the retention mechanism
Column chromatography – an example of the equipment used in low-performance
liquid chromatography

Solvent reservoir

Column head
Column

Column packing

Porous glass plate

 Sample is usually applied directly to the top of the column.

 Detection is by fraction collection with later analysis of each fraction
 High Performance Liquid Chromatography (HPLC)

Instrument Components

• The mobile phase is pumped through

the column by a pump

 Solvents must be degassed to

eliminate formation of bubbles
 Role of the pump is to deliver the solvent (mobile
phase) into the column

Solvent composition

Isocratic: It can be a constant mobile phase composition

(isocratic) (the composition remains unchanged during the
analysis.

Gradient: Composition of the mobile phase changes during the

analysis analysis.
 Normal Phase HPLC

Various solvents can be

used based on the best
polarity ratio of the solvent
mixture in the separation -
the solvents are the
“mobile phase” ..Gradient syt.

This system is “isochratic” –

Uses constant solvent system
Electrochromatograms showing the comparison of isocratic and gradient
elution for the separation of the 16 PAHs.
Injector:

•The injector serves to introduce the liquid sample

into the flow stream of the mobile phase.
May be auto-sampler or manual
 HPLC column
Normal Phase and Reversed Phase
• Normal phase: polar stationary phase and a less polar
solvent. Separates on the basis of polarity – gradient
elution's start with a non-polar solvent and move gradually
to a more polar solvent. The polar analytes are the most
retained.

Example: Silica gel (stationary phase) and organic solvent

mobile phase such as hexane, chloroform….
Polar
• Reversed Phase (C8, C18): the stationary phase is nonpolar or
weakly polar and the solvent is polar (Water +
methanol/acetonitrile/ THF).

silica gel -C18

Gradient elutions usually start with a predominantly aqueous mobile

phase and move gradually to a more organic mobile phase.
For non polar with various carbons

Based on hydrophobicity (approximates carbon number) Separation based

on the differential solubility in aqueous and organic media

CH3

H3C CH3
7 Carbons
8 Carbons
6 Carbons

Time
Column packing particle
Diameter
• Has a greater effect on resolution than length
• Small particles – short analysis times
• Short columns with small particles ideal
• 5m is standard size
• 3m better, but restricted range of packings
available
• Downside is high back pressure and issues with
retention of small particles inside the column,
blockages
By reducing the particle size diameter, not only is the efficiency of
the column increased (plate height (H) is reduced), but the
optimum linear velocity at which the minimum plate height is
achieved is also increased
 The concepts of HPLC...
• Capacity or Retention factor
k’R = (tR - tm) / tm = (VR - Vm) / Vm
• Selectivity =t2 - t0) / (t1 - t0)
= (V2 - Vm) / (V1 - Vm) = k’2 / k’1

• Efficiency (plate height) H= L / N

N = number of theoretical plates