Introduction to
Chromatography
STEM
What is Chromatography?
Derived from the Greek word Chroma
meaning color, chromatography provides a
way to identify unknown compounds and
separate mixtures
Chromatography is a method of
physically separating mixtures of
gases, liquids, or dissolved
substances.
Chromatography can be used to
identify drugs, poisons and many
other substances.
Separation is determined by the
molecular size and/or charge
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Mixtures & Compounds
Mixture – Two or more substances that are mixed
together, but not chemically combined.
Examples of mixtures ...
Air – mixture of gases
Soda pop – mixture of soda syrup, water, and CO2 gas
Kool-Aid – mixture of water, sugar, and flavor crystals
Compounds – Two or more elements that are
chemically combined.
Examples of compounds ...
Salt –Sodium and chlorine combined chemically
Water –Hydrogen and oxygen combined chemically
Carbon Dioxide – Carbon & oxygen combined chemically
Applications of Chromatography
Forensics
Research
Pharmaceutical
industry
• All forms of chromatography involve
Two Phases
• 1. stationary (absorbent) phase
–the material on which the separation
takes place
• 2. mobile phase
- the solvent transports the sample
• Types of Chromatography - 5 of them
5 Types of Chromatography…
Thin Layer
Paper
HPLC
Gas
Column
Chromatography Application:
Matching of Ink Types
Chromatography can be used to
physically separate the components of
inks. There are many types, such as:
 HPLC—high-performance liquid
chromatography
 TLC—thin-layer chromatography
 Paper Chromatography
Gas Chromatography
- a gas separates the components
- they are then ionized and an electrical
signal is recorded
- mobile phase - carrier gas
- stationary phase
- thin film of liquid
HPLC
• High Performance Liquid Chromatography
(HPLC)
• - done at room temp. - advantageous for
chemicals that are heat sensitive or volatile example LSD
• - a liquid (mobile phase) is pumped through a
column containing fine particles (stationary
phase)
TLC
• Thin - Layer Chromatography (TLC)
• -A plate is coated with a granular gel usually silica gel or aluminum oxide
(stationary phase)
• - the substance to be separated is carried
up the plate by capillary action - the
substance with most affinity for the plate
will rise the farthest.
Paper Chromatography
Same as TLC but paper is
used as stationary phase
Electrophoresis
• - Similar to TLC but the substance is
separated through a gel by electric current
• - Due to different size and charge
substances will move across the plate at
different speeds.
Paper Chromatography
Reflects Differences in Components such as:








Raw material
Weight
Density
Thickness
Color
Watermarks
Age
Fluorescence (radiation emitted by exposure to ultraviolet light)
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Chromatography as Evidence
 Class characteristics may include
general types of pens, pencils or paper.
 Individual characteristics may include
unique, individual handwriting
characteristics; trash marks from
copiers, or printer serial numbers.
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Paper Chromatography of Ink
Two samples of
black ink from two
different
manufacturers
have been
characterized
using paper
chromatography.
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Process of Paper
Chromatography
(Click to advance)
Retention Factor (Rf)
 A number that represents
how far a compound
travels in a particular
solvent
 It is determined by
measuring the distance
the compound traveled
and dividing it by the
distance the solvent
traveled.
Solve for Rf
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Calculations
DistanceSubstance
Rf 
DistanceSolute
Black Ink
Blue Dye
4.3
R 
1
f
4.3
3
R 
 0.697
f
4.3
Red Dye
1.6
R 
 0.3271
f
4.3
Orange Dye
Your mission, should you choose to
accept it, is to……..
Analyse several ink samples
to connect them to a suspect
Paper Chromatography
Sample: writing implements (pen, marker,
etc.
Stationary Phase: chromatography paper
Mobile Phase: water, alcohol, acetone
So what will happen?
Each dye will travel up the paper at
different speeds
The speed depends on the solubility of
the dye in the regent and its interaction
with the paper
The dyes are composed of different
molecules with different characteristics
Example: Calculation of Results
Calculation of Results
Analysis = Calculate an Rf value for each
spot.
Rf = Distance from start to the middle of
a spot
Distance from start to finish point of the
regent