Experiment 5: Chromatography

1
Experiment 5 Chromatography

• Reading Assignment
• Experiment 5, pages 42 - 50
• Technique 19, pages 756 777
• Technique 20, pages 777 792
• The following two references provide information
  on reverse-phase chromatography (C-18 silica)
• Technique 21, 794
• Technique 12, pages 689 - 692

2
Experiment 5

• This is a two-day experiment. We will be working
  in pairs
• Day One
• Part A (Thin-Layer Chromatography)
• Part B (Selecting the Correct Solvent for
  Thin-Layer Chromatography)
• Day Two
• Part D (Column Chromatography)
• We will skip Part C

3
How Does Chromatography Work?

• Chromatography is a method for separating the
  components of a mixture by differential
  adsorption between a stationary phase and a
  mobile (moving) phase

4
Different Kinds of Chromatography

• Liquid chromatography (includes column
  chromatography, thin-layer, and HPLC)
• Stationary phase silica, alumina, etc.
• Mobile phase (moving phase) organic solvents
• Important properties polarity
• Gas chromatography
• Stationary phase a film of a polymer or a wax.
  The film must have a high boiling point
• Mobile phase gas (Helium is the usual carrier
  gas)
• Important properties boiling point

5

• Thin-layer chromatography and column
  chromatography and are different types of liquid
  chromatography.
• The mobile (moving) phase is a liquid. The
  stationary phase is usually silica or alumina.
  This phase is very polar.
• The principle of operation is the same!

6
Stationary Phase Silica (SiO2)
7
Stationary Phase Alumina
Acidic -Al-OH Neutral -Al-OH
-Al-O- Basic -Al-O-
8
Thin Layer Chromatography
The surface of the plate consists of a very thin
layer of silica on a plastic or aluminum
backing. The silica is very polar. This is the
stationary phase. Spot the material at the
origin (bottom) of the TLC plate. Place the
plate into a glass jar with a small amount of a
solvent in the glass jar. This solvent acts as
the moving phase. Remove the plate from the
bottle when the solvent is close to the top of
the plate. Visualize the spots. Non-polar
compounds will be less strongly attracted to the
plate and will spend more time in the moving
phase. This compound will move faster and will
appear closer to the top of the plate. Polar
compounds will be more strongly attracted to the
plate and will spend less time in the moving
phase and appear lower on the plate.
9
Thin-Layer Chromatography A Two-Component
Mixture
s
o
l
v
e
n
t

f
r
o
n
t
Less polar!
c
o
m
p
o
n
e
n
t

B
s
o
l
v
e
n
t

f
r
o
n
t
c
o
m
p
o
n
e
n
t

B
More polar!
c
o
m
p
o
n
e
n
t

A
c
o
m
p
o
n
e
n
t

A
mixture
o
r
i
g
i
n
o
r
i
g
i
n
Increasing Development Time
10
Important hint!
Be sure to remove the TLC plate when it appears
that the solvent front isnt moving! Reason
the solvent is evaporating as it moves up the
plate. Results If you dont remove the plate
all of the spots will appear near the top of the
plate!!!!! This isnt a pretty sight and makes it
difficult to get good Rf values!
11
Visualization Method

• The previous slide shows colored spots. Most of
  the time, the spots wont show unless they are
  visualized!
• Vizualization is a method that is used to render
  the TLC spots visible.
• A visualization method can be
• Ultraviolet light
• Iodine vapors to stain spots
• Colored reagents to stain spots
• Reagents that selectively stain spots while
  leaving others unaffected.

12
Thin-Layer Chromatography Determination of Rf
Values
Rf of component A dA dS Rf
of component B dB dS
The Rf value is a decimal fraction, generally
only reported to two decimal places
13
Thin-Layer Chromatography Qualitative Analysis
14
Part A of Experiment 5
Prepare a TLC plate with five lanes. Spot the
plate with pure fluorene, fluorenone and
fluorenol as indicated on page 81 of the text in
the first three lanes. Spot the plate with an
unknown mixture containing the above compound(s)
in lane four. Now spot the plate with a
reference mixture of the three compounds in lane
five. Develop the plate in a screw cap glass
bottle using CH2Cl2 Visualize the spots using
iodine vapor
15

• Which one of these compounds is the least polar?
• Which one of these compounds is the most polar?
• What would be the relative order of separation on
• the TLC plate remembering that CH2Cl2 is not
  very
• polar?

16
Part B of Experiment 5

• In this part of the experiment you will be
  assigned one of the
• mixtures listed in the footnote on page 83.
• Your task is to determine which of the three
  solvents listed
• will provide the best separation on the TLC
  plate.
• Benzoin and benzil in acetone, methylene chloride
  and
• hexane
• 2) Vanillin and vanillyl alcohol in acetone,
  50-50 toluene/
• ethyl acetate and hexane
• 3) Diphenylmethanol and benzophenone in acetone,
• 70-30 hexane/acetone and hexane
• Note carefully different solvents are used with
  each
• part. Be sure to use the correct solvent!

17
Uses of TLC

• To determine how many components there are in a
  mixture (is it really pure?)
• To determine the best solvent conditions for
  separation on a column
• To identify the substances being studied
• To monitor the composition of fractions collected
  from column chromatography
• To monitor the progress of a reaction

18
Uses of TLC
What solvent should you use to separate the
following compounds?

• Acetone
• Hexane
• Acetic acid

19
Uses of TLC
A student tries to separate a series of
carboxylic acids using hexane. Nothing
separates. Why? What should you do next?
20
Uses of TLC
A student tries to separate a mixture of
aliphatic and aromatic hydrocarbons using
acetone. The components all appear at the
solvent front on the TLC plate. What was wrong
and what should be done next?
21
Column Chromatography
The stationary phase (column packing) in the
column is very polar! Polar compounds are
going to be attracted to the polar column
packing by hydrogen bonding or dipole-dipole
attractions. Polar compounds are going to move
slowly! Non-polar compounds are going to come
off the column first, while the polar compounds
are going to come off the column last. Usually,
one starts will a less polar solvent to
remove the less polar compounds, and then you
slowly increase the polarity of the solvent to
remove the more polar compounds.
22
Principles of Separation on a column
23
Principles of Separation
24
Principles of Separation
25
Principles of Separation
26
Principles of Separation
27
Principles of Separation
28
Principles of Separation
29
REMEMBER

• The stationary phase is POLAR
• The more polar component interacts more strongly
  with the stationary phase
• The more polar component moves more slowly.
• The non-polar component moves more rapidly.

30
Reverse phase chromatography
Silica is alkylated with long chain hydrocarbon
groups, using 18 carbons long. This is usually
referred to as C-18 silica.
31
Reverse Phase column chromatography

• The stationary phase (column packing) is now
  NON-POLAR
• Non-polar compounds will move more slowly because
  they are attracted to the column packing.
• The more polar component moves more quickly down
  the column.
• Polar solvents, such as water and methanol are
  used in reverse phase chromatography
• Used mainly in columns, such as HPLC

32
How does reverse phase chromatography compare to
normal phase chromatography?
33
Normal phase Column Chromatography
The column packing in the column is very polar!
Polar compounds are going to be attracted to
the polar column packing by hydrogen bonding or
dipole-dipole attractions. Polar compounds are
going to move slowly! Non-polar compounds are
going to come off the column first, while the
polar compounds are going to come off column
last. Usually, one starts will a less polar
solvent to remove the less polar compounds, and
then you slowly increase the polarity of the
solvent to remove the more polar compounds.