Solvent line

Carotenes (Yellow orange)

Stationary
phase
Xanthophylls (Yellow)

Chlorophyll a (Blue green)

Chlorophyll b (Greenish yellow)
Origin of pigments
Separation of Mobile
phase

Mixtures
 What is Chromatography?
Chromatography is a technique for separating
mixtures into their components in order to analyze,
identify, purify, and/or quantify the mixture or
components.

• Analyze
Separate • Identify
• Purify
• Quantify
Mixture Components

Chapter 4: Techniques in Biochemical Analysis 2

 Definition
 Chromatography is a physical separation method of
components in a sample by distribution of the components
between two phases, one that is stationary (stationary
phase) and one that moves (mobile phase)

 The stationary phase is the substance which is fixed in

place for the chromatography procedure and is the phase to
which solvents and the analyte travels through or binds to

 The mobile phase is the analyte and solvent mixture which

travels through the stationary phase
Illustration of Chromatography
Stationary Phase

Separation

Mobile Phase

Mixture Components
Affinity to Stationary Affinity to Mobile
Components
Phase Phase
Blue ---------------- Insoluble in Mobile Phase

Black  

Red  

Yellow          
Chapter 4: Techniques in Biochemical Analysis 4
Chapter 4: Techniques in Biochemical Analysis 5
 Paper Chromatography (PC)
 The mobile phase is a solvent mixture, e.g. water & ethanol

 Trial and error establishes the best solvent for good resolution

 The mixture under analysis is placed in a tiny, concentrated dot

near the bottom of the paper
Paper Chromatography (PC)
 The paper is hung with the
bottom dipped in solvent, which
rises up the paper to come in
contact with the mixture

 As the solvent rises further up

the paper, the components are
separated as they are swept
along

 The strip of paper is called a

chromatogram
Paper Chromatography (PC)
 Identification of the components is based
on Retention Factor (Rf) values
 Chromatography
Start

While chlorophyll is the chief pigment responsible for

photosynthesis in green plants, many plants contain
other colored pigments as well
These chlorophyll and colored pigments may be
separated according to their various chemical charges
by a technique known as chromatography
In this technique, a mixture of plant pigments is
separated by placing a drop or two of pigment on a Finish
special paper called chromatography paper which is
dipped in a chemical allowing the different plant
pigments to move based on their charges
A picture of a completed chromatography may be
viewed in the graphic at the right