Chapter 7.1 CHM

CHM256
CHAPTER 7
BASIC CHROMATOGRAPHY
Learning Outcomes
At the end of the chapter, student should be able to explain:
Principle of chromatography
Types of chromatography - on the basis of interaction

of the analyte with stationary phase
Types of chromatography based on techniques

- Planar chromatography and column chromatography
Applications of planar and column chromatography

PRINCIPLE OF
CHROMATOGRAPHY
• Samples to be analysed are usually a mixture of many components in a complex matrix.
mixture
• For samples containing unknown compounds, the components must be separated from
each other so that each individual component can be identified by other analytical
methods.
• A mixture can be separated using the differences in physical or chemical properties of the
individual components.
• Example of physical properties that are useful for separations are density and size.
• Some useful chemical properties by which compounds can be separated are solubility,
boiling point, and vapor pressure.
CLASSICAL SEPARATION METHODS
• Masking
• Precipitation
• Distillation
• Solvent extraction
• Ion exchange
• Chromatography
• Electrophoresis
CHROMATOGRAPHY
✔ Chromatography is a technique used to separate the components
of a mixture based on the rates at which they are carried through a
stationary phase by a gaseous or liquid mobile phase
✔ All types of chromatography contain a stationary phase (solid or

liquid) to adsorb or absorb the mixture being separated and a
mobile phase (liquid or gas) which passes over the stationary phase
that carry the component.
✔ The two phases are: Absorb:To take (something) in through or

as through pores
1) the stationary phase
2) the mobile phase Adsorb:to attract and retain other material
on the surface;
USES OF CHROMATOGRAPHY
Chromatography is used by scientists to:
• Analyze
– examine a mixture, its components, and their relations to one
another.
• Identify
– determine the identity of a mixture or components based on
known components.
• Purify
– separate components in order to isolate one of interest for
further study.
• Quantify
– determine the amount of the a mixture and/or the components
present in the sample.
PRINCIPLE OF CHROMATOGRAPHY
1. Sample is dissolved in a solvent and washed through a stationary
phase (SP) by a mobile phase (MP)
2. Competition between the sample molecules adsorbed by the

stationary phase and dissolved by the MP results in separation.
3. Substances that are more soluble in the MP and less adsorbed by the
SP move faster and further.
4. Speed of the substance passes through depends on the nature of the

stationary phase and the eluent.
5. Components in the mixture separate out as the mobile phase moves

through the stationary phase.
Terms: • Retention time
Time taken for a component in the sample to pass
through the column.
• Chromatogram
Visual output of the chromatograph. Different peaks
or patterns on the chromatogram correspond to
different components of the separated mixture.
X-axis - retention time

Y-axis – signal corresponding to the
response created by the analytes existing
in the system
Types of chromatography on the basis of
interaction of the analyte with stationary phase
Adsorption Partition
Ion exchange Size exclusion

1) ADSORPTION
CHROMATOGRAPHY
o Definition: A chromatography where the analytes are separated

based on their different degree of adsorption on a solid stationary
phase.
o In the adsorption process, the solute molecules are held on or

attached on the surface of the stationary phase which is solid.
o The separation of solute depends on the different in polarity.
o Because of the different degree of intermolecular attraction, some

components of the mixture will be more attracted or adsorbed to
the stationary phase.
o Substances that are strongly adsorbed to SP move through
the SP much more slowly than those that are weakly
adsorbed. The stationary phase is a polar solid, usually finely
divided silica and alumina
o The separation of the solutes depends on the difference in

their polarity. The more polar solutes are more readily
adsorbed onto the SP than the less polar solutes. Thus this
polar solute will move slower.
o While the less polar solute will move together with less polar
or non-polar MP
o Mobile phase may be a liquid (LSC) or a gas (GSC).

ADSORPTION
CHROMATOGRAPHY
Stationary
phase: solid
Mobile
phase: liquid
solute
o Example:
1. Thin-layer chromatography (TLC)
- stationary phase: plane (a solid
supported on an inert plate)
- mobile phase: liquid (solvent)
2. Column chromatography
- Stationary phase: silica gel (solid)
- Mobile phase: solvent (liquid)
3. Gas solid chromatography

- Stationary phase: solid
- Mobile phase: gas
2) PARTITION
CHROMATOGRAPHY
❖ Partition is the distribution (by dissolving) of the components between 2 immiscible
phases. Separation of the components will based on relative solubitlity of the
components in the mobile phase and stationary phase
❖ Also known as liquid- liquid chromatography where the liquid SP and liquid MP are
immiscible (do not mix)
❖ Separation is based on solute partitioning between two liquid phase (relative

solubility).
❖ Stationary phase is a liquid supported on an inert solid (surface of packing particles).
❖ Mobile phase may be a liquid (LLC) or gas (GLC).

Some compounds dissolve in one solvent. Some compounds dissolve in the other
solvent. That way the compounds in the mixture become separated into two groups.
PARTITION CHROMATOGRAPHY
Mobile phase:
liquid
Stationary solute
phase: liquid
supported on
solid
❖ Examples:
i. High performance liquid chromatography (HPLC)
ii. Paper chromatography
iii. Gas chromatography (GC)
HPLC GC Paper chromatography

❖ Two main types of partition chromatography based on the type
of stationary phase:
• normal-phase liquid chromatography
• reversed-phase liquid chromatography
Normal-phase chromatography Reversed-phase chromatography

✔ Stationary phase: polar ✔ Stationary phase: nonpolar
stationary phase (e.g., stationary phase (e.g., carbon
methanol on silica gel). chain bonded to silica)
✔ Mobile phase: nonpolar

✔ Mobile phase: polar mobile
mobile phase (e.g., hexane). phase. (e.g acetic acid)
✔ Separation: Favours retention
of polar compounds and ✔ Separation: Nonpolar solutes
elution of nonpolar are retained more and polar
compounds. solutes more readily eluted.
Polar solvents have large dipole moments (aka “partial
charges”); they contain bonds between atoms with very
different electronegativities, such as oxygen and hydrogen.
Non polar solvents contain bonds between atoms with

similar electronegativities, such as carbon and hydrogen
(think hydrocarbons, such as gasoline)
TYPES OF CHROMATOGRAPHY
BASED ON TECHNIQUES
•PLANAR CHROMATOGRAPHY
•COLUMN CHROMATOGRAPHY (CC)
TYPES OF CHROMATOGRAPHY
COLUMN AND PLANAR
CHROMATOGRAPHY

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CHM256

Types of chromatography - on the basis of interaction

Types of chromatography based on techniques

Applications of planar and column chromatography

✔ All types of chromatography contain a stationary phase (solid or

✔ The two phases are: Absorb:To take (something) in through or

2. Competition between the sample molecules adsorbed by the

4. Speed of the substance passes through depends on the nature of the

5. Components in the mixture separate out as the mobile phase moves

X-axis - retention time

Ion exchange Size exclusion

o Definition: A chromatography where the analytes are separated

o In the adsorption process, the solute molecules are held on or

o The separation of solute depends on the different in polarity.

o Because of the different degree of intermolecular attraction, some

o The separation of the solutes depends on the difference in

o Mobile phase may be a liquid (LSC) or a gas (GSC).

3. Gas solid chromatography

❖ Separation is based on solute partitioning between two liquid phase (relative

❖ Stationary phase is a liquid supported on an inert solid (surface of packing particles).

❖ Mobile phase may be a liquid (LLC) or gas (GLC).

HPLC GC Paper chromatography

Normal-phase chromatography Reversed-phase chromatography

✔ Mobile phase: nonpolar

Non polar solvents contain bonds between atoms with

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