Chromatography
Organic Chemistry Laboratory
By: Joseph Martin Mikula III
Chromatography Basics

Definition- Any of various laboratory techniques
used to separate mixtures of substances into their
components. It may be used for analysis or for
purification of organic compounds.
Chromatography Basics
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Stationary Phase- The liquid or solid that stay
doesn't move.
Mobile Phase- The liquid or gas that moves and
contains the components to be separated.
The mobile phase carries the components through
the stationary phase and the components separate
based on the different affinities of substances as they
travel through the stationary phase.
Theory of Chromatography
Stationary Phase: silica, aluminium oxide, various
synthetic resins
Mobile Phase:
organic liquid (solvent),
helium gas
Organic Analysis
Qualitative procedures determine the identity the
compounds in the sample.
Quantitative procedures determine how much of each
component is in the sample.
Types of Chromatography

Thin Layer Chromatography

Column Chromatography

Gas Chromatography

Ion Exchange Chromatography

Size Exclusion Chromatography

Affinity Chromatography

Paper Chromatography
Column Chromatography
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Separates mixtures based off of polarity

The stationary phase is silica gel or alumina

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The mobile phase is a solvent like methylene chloride, ethyl
acetate, or hexanes.
Polar molecules interact with the silca gel and move slowly
through the column, while nonpolar and slightly polar
molecules move faster.
These different interactions with the stationary phase cause
the molecules to elute at different rates.
Polar molecules will elute faster with a polar solvent such as
ethyl acetate.
Column Chromatography
Column Chromatography

Procedure: First Part-Isolating Sample

Put two tea bags in 50mL of distilled water and boil

After two minutes at a boil turn off heat and allow to cool

Remove the tea bags and squeeze them out

Add 20mL of 5% HCl

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Extract solution with 4 10mL increments of methylene
chloride (don't shake, but swirl)
Add sodium sulfate and filter into a 100mL round bottom
flask
Add 0.2 grams silica gel and evaporate
Column Chromatography

Procedure: Second Part-Building Column

Obtain 10 x 150-mm column and 0.7g of silica gel

Put glass wool in bottom of column

Add 0.5cm of sand

Add silica gel

Add sample

Add 0.5cm of sand again
*each layer should be gently tapped to get an even
layer
Column Chromatography

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Procedure: Third Part-Running Column
Add 3 5mL increments of methylene chlride trying not to
disturb column

Add 2 5mL increments of ethyl acetate

Collect 5mL increments from bottom of column in test tubes

Run thin layer chromatography to find which increment
contains caffeine against known caffeine

Take 5mL increments with caffeine and evaporate

Record mass and find melting point
Gas Chromatography

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Separates molecules by vaporizing them into gas
phase, which then travel through tubing containing a
stationary phase at different rates.
The mobile phase is typically helium.
The stationary phase is a high boiling point liquid
absorbed onto a solid-solid diatomaceous earth and
liquid usually a waxy polymer.
How fast a particular compound travels through the
column will depend on how much of its time is spent
moving with the gas as opposed to being attached to
the liquid in some way.
Gas Chromatography
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The time it takes a molecule to come out the end of
the tube is called the retention time.
High solubility and high boiling points lead to
longer retention times.
High column temperature leads to shorter retention
times and less separation, while low column
temperature give longer retention times, but better
separation.
Gas Chromatography
Chromatograph - The printed record of the separation.
May be used for both qualitative and quantitative analysis.
Retention Time - The time required for a
component to emerge from the column
from the time of injection.
Gas Chromatography
Gas Chromatography
nitration product group B
, 27-Oct-2009 + 14:39:21
nitration b
100
1: Scan EI+
TIC
7.64e9
4.27;65;330075584
Area
4.92
137
263832976
%
8.59
165
80129664
7.67
165
17600620
4.66
91
19141724
0
3.40
3.90
4.40
4.90
5.40
5.90
6.40
6.90
7.40
7.90
8.53
165
16575386
8.40
8.90
Time
9.40
Chromatograms
GC/MS
Mass Spectrometry
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A mass spectrometer ionizes the compound by
knocking an electron out of compound.
This gives the compound an overall positive charge
and it will fragment into other charged positively
charged ions.
In a mass spectrum graph the most stable ion
fragment is the most abundance and appears as the
tallest peak- called the base ion peak
The other ion fragments are recorded as relative
abundance to the base ion peak.
Mass Spectrometry


Also, on the graph the peak with the largest mass
value is the unfragmented compound and therefore
equal to the molecular weight.
It is called the parent ion peak.
Mass Spectrometry
Mass Spectrometry
nitration product group B
, 27-Oct-2009 + 14:39:21
nitration b 447 (4.234)
100
4.88e8
65 120
91
%
63
137
178 233 275
0
R:942
100
%
337 381
519
716
818 884 932
Nist 72838: BENZENE, 1-METHYL-2-NITROHit 1
65 120
92
39
137
0
m/z
96
196
296
396
496
596
696
796
896
Thin Layer Chromatography

Uses a silica plate: glass plate with a thin layer of
silica on it.

Silica is the stationary phase and is very polar.

A solvent is the mobile phase.

How well the molecule dissolves in the solvent and
affinity to the silica determines the distance it will
travel up the plate.
Thin Layer Chromatography

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Origin line - the location of the initial placement of a spot
of the compound (marked in pencil).
Solvent front - how far the solvent moved up the plate.
Spots will be between solvent front and origin line. If
colorless, they can be revealed with uv light (requires
fluorescence in silica) or iodine vapor.
The retardation factor (Rf) can be calculated to quantify the
distance traveled by a compound.
Thin Layer Chromatography
TLC is only qualitative,
not quantitative.
TLC Chromatogram
Rf value = distance component traveled
distance solvent traveled
Rf value for Quinine = 0.4
Rf value for Heroin = 0.8
Thin Layer Chromatography

Procedure:

Dissolve 10mg of four compounds in a few drops methanol in a test tube

Take 10mg of an unknown and dissolve it in a few drops methanol in a test tube

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Obtain 2-3 TLC plates and draw a line in pencil 1cm up from an end on each (no pen
why?)- This is the origin line
Put a drop from an open ended capillary tube onto the penciled line creating lanes
A technique to use is place a drop let it dry then put another drop in same spot let it
dry and repeat several times
Put TLC plate in chamber with solvent (make sure solvent level is not higher than
origin line
Pull out of chamber when solvent front is 0.5cm from top and immediately mark
Allow plate to dry and put it in iodine chamber for 30 seconds and then expose to uv
light circling the spots that appear
Calculate Rf values and deduce compounds or compound in unknown