Chromatography
Chromo: color
Graph: to write
In the mid 1900s used to separate and
analyze naturally occurring pigments like
those in leaves.
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Real life uses for
Chromatography
Law Enforcement – to compare a sample found at a crime scene to
samples from suspects
Environmental Agency – determine the kinds of pollutants and level of
pollutants
Pharmaceutical Company – determine amount of each chemical found
in new product
Hospital – detect levels of chemicals in a patient’s blood stream
Manufacturing Plant – to purify a chemical needed to make a product
Solvent front
Plate: stationary phase
Pigments in the mixture
Solvent: mobile phase
Definition of Chromatography
Definition:
Chromatography separates components of a
mixture by their distinctive attraction to the mobile phase
and the stationary phase.
The mixture looks homogenous before procedure but is made of
more than one component
Separates: spreads out for analysis or to make a pure sample
Two phases -- one moves and one stays still
Phases
Compound is placed on a stationary phase
Made of paper, beads, solid
On Monday made of silica gel plate
Mobile phase passes through the stationary phase
Liquid or gas
On Monday made of solvent 50% acetone/ H2O
Some compounds in mixture spend more time in mobile
phase than others and therefore move faster/ farther
What determines how long the pigment spends in the mobile phase?
Polarity
Within covalent molecules, atoms share electrons
If the atoms share equally the molecule is non-polar
Typical of molecules made up of carbon and hydrogen
If the atoms do not share electrons equally,
then portions of the molecule are charged.
These molecules are called polar.
Polarity and Solubility
In general: Like dissolves like
More polar substances dissolve best in polar
solvents.
Less polar substances dissolve best in nonpolar solvents.
Solvent front
Plate: stationary phase
Pigments in the mixture
Solvent: mobile phase
Molecular View of Chromatography
Stationary phase –
molecules are here
when they are not
dissolved
Mobile phase –
molecules are here
when they are
dissolved
Blue molecules spends the most time
in mobile phase because they are the
most polar. Red molecules spends the
most time in stationary phase
because they are the least polar.
Rf is not calculated with
distance between
pigments or distance
between pigments and
solvent front.
D2 = distance to solvent
front
D1 = distance to pigment
Rf = D1/D2
= 6.2cm/12.4cm
= 0.5
Rf = retention factor
= ratio of distance travelled
Illustration of Chromatography
Stationary Phase
Separation
Mobile Phase
Mixture
Components
Components
Affinity to Stationary
Phase
Affinity to Mobile
Phase
Blue
----------------
Insoluble in Mobile Phase
Black


Red


Yellow

        
Optimizing information
• Careful choice of solvent and stationary phase.
Separation visible at 50%-70%
• View under UV and ambient light to see all pigments.
0%
20%
50%
70%
Concentration of Isopropanol
100%
To make your lab work…
•
•
•
•
Mark plate in pencil
Don’t chip plate
Avoid getting hand oil on plate
Spot pigments above level of solvent
Types of
of Chromatography
Chromatography
Types
• Paper Chromatography – separates dried liquid samples
with a liquid solvent (mobile phase) and a paper strip (stationary
phase)
• Thin-Layer Chromatography – separates dried liquid
samples with a liquid solvent (mobile phase) and a glass
plate covered with a thin layer of alumina or silica gel
(stationary phase)
•Liquid Chromatography – separates liquid samples
with a liquid solvent (mobile phase) and a column
composed of solid beads (stationary phase)
• Gas Chromatography – separates vaporized samples
with a carrier gas (mobile phase) and a column
composed of a liquid or of solid beads (stationary
phase)