1. Chromatography.
Sibtain Ahmed, PhD Biochemistry

3. The term “chromatography” is derived from Greek, chroma
meaning, “colour,” and graphein meaning “to write.”

4. Chromatography
• All types of chromatography are based on a
very simple principle. The sample to be
examined (called the solute) is allowed to
interact with two physically distinct entities-a
mobile phases and a stationary phase.

5. Chromatography
• Chromatography is a technique of separating individual
compounds from a mixture of compounds.
• Mobile phase (Liquid/gas that flows through the supporting
medium).
• Stationary phase (a layer or coating on supporting medium
that interacts with the analyte). It can be solid or liquid.
• Supporting medium: A solid surface on which the stationary
phase is bound or coated.

6. Chromatography
Chromatography is a technique for
 separating mixtures of compounds
 identifying unknown compounds
 establishing the purity or concentration of compounds
 monitoring product formation in the pharmaceutical and biotechnology
industries
• Chromatography is widely used by forensic teams to analyse blood and
urine samples for drugs, for paint analysis, and for testing for the presence
of explosives. Most chromatography uses modern instrumentation and
involves placing the sample to be analysed on support (paper or silica) and
transporting it along a mobile phase. The mobile phase can be a liquid
(liquid chromatography) or a gas (gas chromatography).

7. Separation Techniques
• Most of the samples are mixtures of different substances.
• In a mixture, the analytical signal of one substance usually
affects the signal of another substance.
• Before performing qualitative or quantitative analysis of a
sample, the substances should be separated from each other.
• If the separation of the substances is performed by using a
mobile phase and a stationary phase, this separation technique
is called chromatography.

8. Chromatography
• Stationary phase and mobile phase are two
immiscible mediums. (Solid/liquid, liquid/gas,
liquid/liquid, etc.)
• The duty of the stationary phase is to keep and retain
the substances. The duty of the mobile phase is to
push and move the substances.
• The working principle of chromatography is based on
the interaction of substances with stationary phase
and mobile phase.

9. Chromatography Terms
• Analyte: is the component to be separated during
chromatography.
• Chromatogram is the visual output of the
chromatography. Eluent: The mobile phase.
• Elution time: The time taken for a solute to pass
through the system.
• A solute with a short elution time travels through the
stationary phase rapidly, i.e. it elutes fast

14. QUESTIONS
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