CHROMATOGRAPHY

Chromatography

 It is a technique used to separate and identify the

components of a mixture.
 It works by allowing the molecules present in the
mixture to distribute themselves between a stationary
and mobile medium.
 Molecules that spends most of their time in the mobile
phase are carried along faster.
 A laboratory technique that separates components
within a mixture by using the differential quantities of
the components for a mobile medium and for a
stationary absorbing medium through which they
pass.  Can be used to separate the components of inks and
Terminologies dyes (pens, marker, clothing, candy shells) and even
the pigments of plants.
 Differential – showing a difference, distinctive
 Affinity – natural attraction or force between things Retardation Factor (Rf)
 Mobile Medium – gas or liquid that carries the  The distance the pigments traveled up the strip and the
components (mobile phase) distance water traveled. The RF of each of the colors
 Stationary Medium – the part of the apparatus that that are present in a sample needs to be calculated.
does not move with the sample (stationary phase)
Rf = distance moved by substance
distance moved by solvent front
 Substances that are very soluble in liquid, Rf will be
close to 1.
 Substances that are rather insoluble in liquid, Rf will be
close to 0.
 Molecules in mixtures have different characteristics
(size, solubility), they travel at different speeds when
pulled along a piece of paper by a solvent (water).
 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.

Types of Chromatography

1. Paper Chromatography
 Separates dried liquid samples with a liquid
solvent (mobile phase) and a paper strip
(stationary phase).
 Principles:
- Capillary Action: movement of a liquid
through a porous material due to the forces of
adhesion, cohesion, and surface tension. The
liquid can move up the filter paper because its
attraction to itself is stronger than the force of
gravity.
 - Solubility: degree to which a material (solute) 1. A gas mixture called the Mobile Phase
dissolves in a solvent. Solute dissolves in composed of an inert carrier gas (usually
solvents that have similar properties (like Helium) and various other chemical
dissolves like). This allow different solutes to components in the vapor phase introduced at
be separated by different combination of the Injection Port pass through a non-
solvents. moving material called the…
- Separation of components depends on both 2. Stationary Phase, usually tightly packed into
their solubility in the mobile phase and their a copper tube called the Column which
differential affinity to the mobile phase and separates the gas stream into its particular
the stationary phase. components based on how quickly they move
through the stationary phase. The
components reach a…
3. Detector at different times, the detector
generates an electric signal which is sent to
a…
4. (parang may phrases na natakpan ng picture
sa handouts)
5. Since many chemical substances are solids or
liquids at room temperature, the stationary
phase- mobile phase must be heated to a
higher temperature in order to vaporize the
components of the mobile phase. Thus, an
2. Thin-layer Chromatography external heating device or…
 Uses an absorbent material on flat glass or 6. Oven is needed. The temperature of the oven
plastic plates must not be above the boiling points of the
 Used to detect pesticide or insecticide residues components but rather above the dew point
on food of the least volatile component.
 Used in forensics to analyze the dye 7. As the mobile phase passes through the
composition of fibers stationary phase, the different components of
 Most samples are not colorized and need to be the mobile phase will dissolve into the liquid
visualized with a UV lamp film of the stationary phase.
 The solubility of the gases in the liquid phases
depends on several 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 less
time dissolved in the liquid phase, whereas
gases from less volatile liquids will spend more
time
 The less time a gas spends dissolved in the
liquid phases, the more time it spends flying
along with the carrier gas. Thus, it moves
through stationary phase more quickly.

3. Gas Chromatography
 Used in airports to detect bombs and in
forensics in different ways.
 Used to analyze fibers on a person’s body and
analyze blood found at a crime scene.
 Helium is used to move a gaseous mixture
through a column of absorbent material.
 Process:
4. Liquid Chromatography
 A sample mixture passed through a column
packed with solid particles which may or may
not be coated with another liquid.
 With the proper solvents, packing
conditions, some components in the sample
will travel the column more slowly than
others resulting in the desired separation. The separation mechanism in LSC is based on the
 Used in the world to test water samples to competition of the components of he mixture sample
look for pollution in lakes and rivers. for the active sites on an absorbent such as Silica Gel
 Used to analyze metal ions and organic
compounds in solutions. 2. Liquid/Liquid or Partition Chromato-
 Uses liquid which may incorporate insoluble graphy
molecules. A. Normal Phase LLC
B. Reverse Phase LLC

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.

3. Ion-Exchange Chromatography

 Types of Liquid Chromatography:

1. Liquid/Solid or Adsorption Chromato-
graphy
A. Normal Phase LSC Separation in ion-exchange chromatography is
B. Reverse Phase LSC based on the competition of different ionic
compounds of the sample for the active sites on the
ion-exchange resin (column packing).

4. Gel Permeation or Exclusion

Chromatography

It is a mechanical sorting of molecules based on the

size of the molecules in solution. Small molecules can
permeate more pores and are, therefore, retained
longer than larger molecules.
High Performance Liquid Chromatography (High Pressure
Liquid Chromatography, HPLC)

 A form of column chromatography used frequently in

biochemistry and analytical chemistry to separate,
identify, and quantify compounds.
 It 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 reaction times of the
molecules.
 Retention Time varies depending on the interactions
between the stationary phase, molecules being
analyzed, and the solvent(s) used.

Chromatography

 Normal Phase Chromatography 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 of Orange Juice Compound