Chromatography

Week 15

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.

Detailed Definition:
Chromatography is a laboratory technique that
separates components within a mixture by using
the differential affinities of the components for a
mobile medium and for a stationary adsorbing
medium through which they pass. We can use chromatography to separate the
components of inks and dyes, such as those
Terminology: found in pens, markers, clothing, and even candy
• Differential – showing a difference, shells. Chromatography can also be used to
distinctive separate the colored pigments in plants.

• Affinity – natural attraction or force

between things

• Mobile Medium – gas or liquid that

carries the components (mobile phase)

• Stationary Medium – the part of the

apparatus that does not move with the
sample (stationary phase)
Illustration of Chromatography
RF (Retardation Factor)
Because molecules in mixtures have different
characteristics (such as size and solubility), they
travel at different speeds when pulled along a
piece of paper by a solvent (water).

For example, black ink contains several colors.

When the water flows through a word written in
black, the molecules of each one of the colors
behave differently, resulting in a sort of
“rainbow” effect with the colors separating out at
different rates.
We can determine the RF (or retardation factor)
by measuring the distance the pigments traveled
up the strip and the distance the water traveled.
You will need to calculate the RF for each of the
colors that are present in a sample.
For substances that are very soluble in the liquid
Rf will be close to ....
1 into solvents that have similar properties. (Like
For substances that are rather insoluble in the
liquid Rf will be close to ....
0 differential affinity to the mobile phase and the
Principles of Paper Chromatography
Capillary Action – the movement of liquid within
the spaces of a porous material due to the forces
of adhesion, cohesion, and surface tension. The
liquid is able to move up the filter paper because
its attraction to itself is stronger than the force of
gravity.
Solubility – the degree to which a material
(solute) dissolves into a solvent. Solutes dissolve
dissolves like) This allows different solutes to be
separated by different combinations of solvents.
Separation of components depends on both their
solubility in the mobile phase and their
stationary phase.

Types of Chromatography

1. Paper chromatography
2. Thin-Layer Chromatography
3. Gas chromatography
4. Liquid Chromatography

Paper Chromatography
Thin Layer Chromatography
- separates dried liquid samples with a
- uses an absorbent material on flat glass or
liquid solvent (mobile phase) and a paper
plastic plates.
strip (stationary phase)
- It is used to detect pesticide or insecticide
residues in food.
- Thin-layer chromatography is also used in
forensics to analyze the dye composition
of fibers.
- Most samples are not colored and need to
be visualized with a UV lamp.
 which separates the gas stream into its
particular components based on how “quickly”
they move through the stationary phase. The
components reach a...

Detector at different times, the detector

generates an electrical signal which is sent to a...

here are two proper sized spots, viewed under a

UV lamp
Gas Chromatography
- is used in airports to detect bombs and is
used in forensics in many different ways.
- It is used to analyze fibers on a persons
body and also analyze blood found at a
crime scene.
- Helium is used to move a gaseous mixture
through a column of absorbent material.
• Since many chemical substances are
solids or liquids at room temperature, the
stationary phase-mobile phase must be
heated to higher temperature in order to
“vaporize” the components of the mobile
phase. Thus an external heating device
or...
• Oven is needed. The temperature of
the oven need not be above the boiling
points of the components but rather
above the “dew point” of the least volatile
component

A gas mixture, called the Mobile Phase,
composed of an inert “carrier” gas (usually
Helium) and various other chemical components
in the vapor phase introduced at the Injection
Port pass through a non-moving material, called
the...
• The solubility of the gases in the liquid
Stationary Phase, usually tightly packed into a
copper tube called the Column…
As the mobile phase passes through the
stationary phase, the different
components of the mobile phase will
dissolve into the liquid film of the
stationary phase.
phases depends on a number of factors,
but one of the most important is the
volatility of the liquid from which the
component vapor drives.
 • Gases from low boiling liquids will spend The 4 basic liquid chromatography modes are
less time dissolved in the liquid phase, named according to the mechanism involved:
whereas gasses from less volatile liquids
 1.Liquid/Solid Chromatography (adsorption
will spend more time in solution with the
chromatography)
liquid phase.
A. Normal Phase LSC
• The less time a gas spends dissolved in
the liquid phases, the more time it spends B. Reverse Phase LSC
flying along with the carrier gas. Thus it
moves through the stationary phase  2.Liquid/Liquid Chromatography (partition
more quickly. chromatography)

A. Normal Phase LLC

Liquid Chromatography B. Reverse Phase LLC

- A sample mixture is passed through a  3.Ion Exchange Chromatography

column packed with solid particles which
 4.Gel Permeation Chromatography (exclusion
may or may not be coated with another
chromatography)
liquid.
- With the proper solvents, packing MOBILE PHASE LIQUID

conditions, some components in the

sample will travel the column more slowly FORMAT
Liquid-Liquid Liquid-Solid
Chromatography
Chromatography

than others resulting in the desired (Partition) (Adsorption)

separation. STATIONARY Liquid Solid

PHASE
- is used in the world to test water samples
to look for pollution in lakes and rivers. Normal Phase Reverse Phase Normal Phase Reverse Phase

- is used to analyze metal ions and organic Mobile Phase - Mobile Phase -
Nonpolar Polar
compounds in solutions. Stationary phase -
Polar
Stationary phase -
Nonpolar

- uses liquids which may incorporate

insoluble molecules. Normal phase LS
Reverse phase LS
Diagram of Simple Liquid

Column Chromatography
Si - O - H

DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY

 Silica Gel
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

The separation mechanism in LSC is based on the

OOOOOOOOOO stationary phase) OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO

competition of the components of the mixture

OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOC OOOO
sample for the active sites on an absorbent such
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO as Silica Gel.
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO

Eluant (eluate)

Liquid-Liquid Chromatography

Types of Liquid Chromatography


ODPN(oxydipropionylnitrile)
Normal Phase LLC
Reverse Phase LLC
NCCH3 CH2 OCH2 CH2 CN(Normal)
CH3 (CH2 ) 16 CH3 (Reverse)
Gel-Permeation Chromatography is a mechanical
sorting of molecules based on the size of the
molecules in solution.
Small molecules are able to permeate more pores
and are, therefore, retained longer than large
molecules.

High Performance Liquid Chromatography

The stationary solid surface is coated with a 2nd
liquid (the Stationary Phase) which is immiscible
in the solvent (Mobile) phase.
Partitioning of the sample between 2 phases
delays or retains some components more than
others to effect separation.
Ion-Exchange Chromatography
(or High pressure liquid chromatography,
HPLC) is a form of column chromatography used
frequently in biochemistry and analytical
chemistry to separate, identify, and quantify
compounds. HPLC utilizes a column that holds
chromatographic packing material (stationary
phase), a pump that moves the mobile phase(s)
through the column, and a detector that shows
the retention times of the molecules. Retention
time varies depending on the interactions
between the stationary phase, molecules being
analyzed, and the solvent(s) used.

- +
SO3 Na

Separation in Ion-exchange Chromatography is

based on the competition of different ionic
compounds of the sample for the active sites on
the ion-exchange resin (column-packing).

Gel-Permeation Chromatography

Chromatography
 • Normal Phase Chromatograaphy uses a
polar stationary phase and a non-polar
mobile phase, and works effectively for
relatively polar analytes

• Reversed Phase Chromatography has a

non-polar stationary phase and an
aqueous, moderately polar mobile phase.

Result Chromatogram

Methods of Separation and Purification of

Organic Compounds

Study hardest….