96-WELL SPE PLATES

96 well plates with small wells (so-called

microtiter plates) are widely used for
processing large number of samples in
automated instruments.
 Principle behind extraction is same.
Chromatography

Chapter 19
 Chromatography
Chromatography is the science which studies
the separation a mixture of molecules based
on differences in their structure and/or
composition.
The word was first derived from the Greek word chroma meaning colour
and graphy = analysis
therefore it means Color Analysis
 Used by scientists to:
Analyze : To examine the mixture or structure or something
especially by separating it into its parts.
Purify : To make something pure by removing substances that
are not wanted out of another substances that contains it.
Identify : To recognize something and to determine the
identity of mixture
 History

Mikhail Tswett, Russian, 1872-1919

Botanist
In 1906 Tswett used to chromatography to separate
plant pigments
He called the new technique chromatography
because the result of the analysis was 'written in
color' along the length of the adsorbent column
Chroma means color and graphein means to write
The first chromatography by Tswett
 Importance

Chromatography has application in every

branch of the physical and biological
sciences
12 Nobel prizes were awarded between 1937 and
1972 alone for work in which chromatography
played a vital role
Chromatography is a physical method of separation in which
the components to be separated are distributed between
two phases
one of which is stationary (stationary phase) while the other
(the mobile phase) moves through it in a definite direction.
The chromatographic process occurs due to differences in the
distribution constant of the individual sample components.
ANALOGY
 Chromatography
Is a technique used to separate
and identify the components of a
mixture.
Works by allowing the molecules present in the
mixture to distribute themselves between a
stationary and a mobile medium.

Molecules that spend most of their

time in the mobile phase are carried
along faster.
 Aim of chromatography
The compounds of interest has to be separated
from matrix components.
The signal to noise ratio must be as high as
possible.
The time consumption of analysis must be
short.
 Uses of Chromatography

Real-life examples:

Pharmaceutical Company- determine the amount of

each chemical component found in a new product.

Hospital- detect blood or alcohols levels in a patient

blood stream.

Environmental Agency- determine the levels of

pollutants in the water supply.
 Stationary phase

Definition: Fixed in place either in a column or on a planar surface.

Function: Act as adsorbent

Adsorbent : atoms that accumulate on the surface of the material

Types of stationary phase :

Particles : Porous or solid
Walls of Tubes: Capillary Tube
Fibrous Material: Paper
Examples

14
 Mobile phase

In the form of:

Gas, liquid or supercritical fluid

Supercritical fluid:
mobile phase is a fluid above and close to its
critical temperature and pressure.

15
Diagram of Simple Liquid Column Chromatography
DIAGRAM OF S IMPLE LIQUID COLUMN CHROMATOGRAPHY

Solvent(mobile or
moving phase)
OOOOOOOOOOO
A+ B+ C Sample
OOOOOOOOOOO
(A+B+C)
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOA OOOO
OOOOOOOOOO Column OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO Solid Particles OOOOOOOOOO
OOOOOOOOOOO (packing material- OOOOOB OOOO
OOOOOOOOOO stationary phase) OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOC OOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO

Eluant (eluate)
 Elution: The process of extracting a substance that is
adsorbed to another by washing it with a solvent.
Eluent : Fluid entering a column (solvent used in
elution)
Eluate : The Fluid exiting the column