3. Mixtures & Compounds
Mixture – Two or more substances that are mixed together, but not
chemically combined.
Examples of mixtures ...
Air – mixture of gases
Bowl of cereal – mixture of cereal and milk
Soda pop – mixture of soda syrup, water, and CO2 gas
Fog –water suspended in air
Kool-Aid – mixture of water, sugar, and flavor crystals
Examples of compounds ...
Salt –Sodium and chlorine combined chemically
Water –Hydrogen and oxygen combined chemically
Carbon Dioxide – Carbon and oxygen combined
chemically
Compounds – Two or more elements that are chemically combined.
4. Solutions
Solutions are mixtures in which one substance is dissolved in another.
Solutions have two parts: solute and solvent
The solute is the substance that is dissolved.
The solvent is the substance that does the dissolving
Solubility - A measure of how much of a given substance will dissolve in a
liquid.
A substance that does not dissolve in water is called insoluble.
A substance that does dissolve in water is called soluble.
5. ADVANTAGES OF CHROMATOGRAPHY
OVER OTHER METHODS
Separation of mixtures of chemicals having closer
physical or chemical properties.
Gentle method.
Separation in even micrograms is possible.
Simple, rapid and efficient technique.
6. History
• The subject of Chromatography was introduced into scientific
world in a very modest way by M. Tswett in 1906.
• He employed a technique to separate various pigments such as
chlorophylls and xanthophylls by passing the solution of these
compounds into the glass column which was packed with finely
divided calcium carbonate.
• After the later, Thompson and Way had realized the Ion
Exchange properties of soils.
• Almost after three decades, in 1935 Adams and Holmes
observed the Ion Exchange characteristics in crushed
phonograph. This observation opened the field for preparation
of Ion Exchanged resins.
• The concept of Gas-Liquid Chromatography was first
introduced by Martin and Synge in 1941.
7. • They were also responsible for the development in Liquid-
Liquid chromatography.
• In 1944, from Martin laboratory, the separation of amino acid
by paper chromatography was reported.
• In 1952, the importance of the chromatography was observed
when both Synge and Martin were awarded with Nobel Prize.
• In 1959, a technique known as Gel Filtration chromatography
was observed which is used to separate low molecular weight
substances from high molecular substances.
• In 1960, further improvement in liquid chromatography led to
the development of High Performance Liquid
Chromatography.
• The following decade of 1970 improvement in the field of
adsorption chromatography in the form of Affinity
chromatography which was mainly based on biological
interactions.
8. • A new field was originated which was supercritical fluid
chromatography.
• Supercritical fluid chromatography is a hybrid of gas and
liquid chromatography and combine advantageous feature
of the both gas and liquid chromatography.
• It will not be wrong to say that the entire twentieth century
can be named as the century of chromatography.
9. 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.
10. What attracts the scientists
to Chromatography?
Chromatography is a way to
separate two components based
on a specific characteristics.
What makes chromatography
so useful...
The results are reproducible
with better accuracy than the
other techniques.
Chromatography can separate
more complex mixtures than the
other techniques.
Chromatography is less time
consuming and cheaper.
11. Gas Chromatography
Used to determine the chemical composition of
unknown substances, such as the different
compounds in gasoline shown by each
separate peak in the graph below.
Paper
Chromatography
Can be used to separate the
components of inks, dyes, plant
compounds (chlorophyll), make-
up, and many other substances
Liquid
Chromatography
Used to identify unknown plant
pigments & other compounds.
Thin-Layer
Chromatography
Uses thin plastic or glass trays to
identify the composition of pigments,
chemicals, and other unknown
substances.
Examples of Chromatography
12. 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.
Separate
• Analyze
• Identify
• Purify
• Quantify
ComponentsMixture
13. Illustration of Chromatography
Components
Affinity to Stationary
Phase
Affinity to Mobile
Phase
Blue ---------------- Insoluble in Mobile Phase
Black
Red
Yellow
Mixture Components
Separation
Stationary Phase
Mobile Phase
14. Uses for 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
15. Uses for Chromatography
Real-life examples of uses for
chromatography:
• Pharmaceutical Company – determine amount of
each chemical found in new product
• Hospital – detect blood or alcohol levels in a
patient’s blood stream
• Law Enforcement – to compare a sample found at
a crime scene to samples from suspects
• Environmental Agency – determine the level of
pollutants in the water supply
• Manufacturing Plant – to purify a chemical
needed to make a product
17. ADSORPTION
Based on relative polarities .
Compounds having high affinity towards the stationary
phase travel slower
Compounds having lesser affinity towards the
stationary phase travel faster
No two components have same affinity for a
combination of stationary phase, mobile phase and other
conditions.
18. PARTITION
Based on relative solubility.
Solutes will be distributed according to their partition
coefficients.
Components which are more soluble in the stationary
phase – Travel slower
Components which are less soluble in the stationary
phase –Travel faster
No two components have same partition coefficient for
a particular combination of stationary phase, mobile
phase and other conditions.
19. ION EXCHANGE
Stationary phase contains fixed charged groups and
mobile counter ions
Counter ion exchange with ions of solute
Reversible exchange of ions takes place between
similar charged ions of solute in the mobile phase and
that of an ion exchange resin.
20. SIZE EXCLUSION
Retention depends on the extend to which the solute
molecules are trapped in the pores of inert stationary
phase.
This depends on size of molecules.
Smaller molecules diffuses into the pores of the
stationary phase – Travel slower.
Larger molecules do not enter the pores of stationary
phase – Travel faster.
21. Classification Of Chromatography
On the basis of interaction of
solute to the stationary
phase
On the basis of
chromatographic
bed shape
On the basis of physical
state of mobile
phase
Adsorption
Chromatography
Partition
Chromatograph
y
Ion Exchange
Chromatography
Size Exclusion
Chromatography
Two
Dimensional
Three
Dimensional
Thin Layer
Chromatography
Paper
Chromatography
Column
Chromatography
Liquid
Chromatography
Gas
Chromatography
Super Critical
Fluid
Chromatograph
y
23. Terminology
• Influent – The liquid entering the column.
• Effluent – The liquid living the column.
• Elution – The process by which the adsorbed ions are
removed from the column.
• Eluent – The solution used for elution.
• Eluate – The solution obtained as result of elution.
24. HOW TO CHOOSE A METHOD
Polarity of the sample.
Solubility and volatility of sample.
Resolution required.
Concentration of analyte.
Detection limit.
Physical and chemical properties of sample.
