EXERCISE 4:

Chromatography

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 Chromatography
 general term applied
for a number of
techniques used for
analyzing, identifying
and separating a
mixture of
compounds.

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 Principle
 a liquid or gaseous solution of sample
called MOBILE PHASE is passed through
an adsorbent called STATIONARY
PHASE
 separation is achieved due to selective
interaction of chemicals with both
stationary and mobile phases

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 Principle
 the interactions of the chemicals with the
stationary and mobile phases depend on
their physical properties (absorptivity,
solubility, charge, size, boiling point, etc.)

 differences in these properties cause

different compounds to be more or less
securely held either in the mobile phase or
the solid phase
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 Application
 Analytical technique – purpose is to gather
information on the amounts and mobilities
of the components of the mixture

 Preparative technique – purpose is to

purify the compound by separating it from
other components of the mixture

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TYPES OF CHROMATOGRAPHY
 Partition Chromatography
 due to differences in the solubility of the
sample in the stationary and mobile phases
 STATIONARY PHASE- liquid supported on
the surface of a solid
 MOBILE PHASE- liquid/gas which is insoluble
in the stationary phase

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TYPES OF CHROMATOGRAPHY
 Example:
 Paper chromatography
 Liquid-liquid partition
chromatography
 Stationary phase –
thin film of water on a
strip of filter paper
 Mobile phase –
solvent system

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TYPES OF CHROMATOGRAPHY
 Adsorption Chromatography
 Governed by surface adsorption phenomena
 STATIONARY PHASE – solid
 MOBILE PHASE – liquid

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TYPES OF CHROMATOGRAPHY
 Example:
 Thin layer
chromatography
 Solid-liquid adsorption
chromatography
 Stationary phase –
uses a strip of
glass/plastic coated
with a thin layer of
alumina or silica gel
 Mobile phase – solvent
system

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 In general…
 The rate at which a compound ascends
depends on the properties of the
adsorbent, solvent and compound
 Polarity is the property of each of these
three factors which has the most influence
on the rate of ascension

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 In general…
 Since the solvent and the adsorbent are
the same for all components of the
mixture, separation is based on
differences in polarity of the compounds
in the mixture

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 USES OF TLC AND PAPER
CHROMATOGRAPHY
1. Check purity of the compound
2. Determine the number of compounds in a
mixture
3. Follow course of reactions
4. Choose solvents for column
chromatography
5. Quantitative analysis

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Course of Reaction

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 STEPS IN THE ANALYSIS
 1. Selection of a solvent system
proper choice of solvent is critical to
separation of a given mixture
- generally, the more polar a solvent is, the
more effective it is at eluting both polar and
nonpolar compounds
- polar solvent more effectively competes with
the compounds for adsorption on the
relativelypolar surface of the adsorbent

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 STEPS IN THE ANALYSIS
 1. Selection of a solvent system
- best solvent for a particular application
must be determined by a trial and error
process
- usually a mixture of solvents or solvent
systems

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 STEPS IN THE ANALYSIS
 1. Selection of a solvent system

A B C

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 STEPS IN THE ANALYSIS
 A – is solvent system not polar enough,
little separation will result

 B - solvent system is too polar; all

components will travel about equally well,
little separation will result

 C – solvent system appears to be

satisfactory
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 STEPS IN THE ANALYSIS
 Other important points in the selection of the
solvent system
 must have a large selectivity in desorbing the
compounds
• better and faster separation

 must dissolve the compounds to be separated

• to effect the separation
 STEPS IN THE ANALYSIS

 must not dissolve the adsorbent

• loss of stationary phase

 must be inert
• better and faster separation
 STEPS IN THE ANALYSIS

 must not react irreversibly with the adsorbent

or compounds to be separated
• alteration of the structure of the desired
compounds

 relatively nontoxic and inexpensive

 STEPS IN THE ANALYSIS
 2. Preparation of the
developing chamber
 can be a specially
designed chamber, a
jar with a lid or beaker
with a watchglass on
top
 container is filled with
solvent to a depth of
0.5 cm

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 STEPS IN THE ANALYSIS
 2. Preparation of the developing chamber
 developing container should be saturated with
the vapors of the eluting solvent to prevent
the plate from drying out during the run
 line part of the inside of the container with
filter paper, which will serve as wick to
saturate the atmosphere in the jar
 for good and fast resolution

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 STEPS IN THE ANALYSIS
 3. Spotting
 it is important not
to spot too much
or too little
sample
 Size of spots: < 1-
2 mm in diameter
 to avoid tailing
and overlapping
of spots
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 STEPS IN THE ANALYSIS
 3. Spotting
 use a volatile solvent such as hexanes,
ethyl acetate or methylene chloride to
prepare the solutions of compounds for
spotting
 for it to evaporate quickly so as not to interfere
with process

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 STEPS IN THE ANALYSIS
 4. Development of chromatogram
 position of paper – vertical; should not
touch the sides of chamber
 solvent level – should be below the spot
 not to wash away components of spots or the
spots will dissolve off
 chamber – must be tightly covered to
maintain equilibrium inside

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 STEPS IN THE ANALYSIS
 4. Development of chromatogram
 solvent will rise up the TLC plate by capillary
action
 solvent front should not exceed paper/plate
since Rf value cannot be computed

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 STEPS IN THE ANALYSIS
 5. Analysis/Visualization
 use of UV, iodine, ninhydrin, etc.

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 MAIN PRINCIPLE BEHIND
CHROMATOGRAPHY
 Normal phase chromatography
 A mode of chromatography carried
out with a polar stationary phase
and a nonpolar mobile phase
 More polar compound, more affinity to
stationary phase, lower Rf value
 Less polar compound, more affinity to the
mobile phase, higher Rf value
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 MAIN PRINCIPLE BEHIND
CHROMATOGRAPHY
 Reversed phase chromatography
 A mode of chromatography carried
out with a nonpolar stationary
phase and a polar mobile phase
 Less polar compound, more affinity to
stationary phase, lower Rf value
 More polar compound, more affinity to the
mobile phase, higher Rf value
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 Retardation factor, Rf
 Distance that the spot of a particular
compounds moves up the paper or plate
relative to the distance travelled by the
solvent front.
 Rf = distance travelled by the compound
distance travelled by the solvent
** same Rf = most likely but not
necessarily the same compound
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 Retardation factor, Rf
 The Rf for a compound is a constant from
one experiment to the next only if the
chromatography conditions are also
constant.

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SAMPLE PROBLEM

 Solve for the Rf

value of A and B

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 SAMPLE PROBLEM
 Calculate the Rf values of the compounds
present in the chromatogram and arrange
the compounds in increasing polarity.
(NOTE: Chromatogram resulted by using
a nonpolar stationary phase and a polar
mobile phase and assume comparable
MM).
 Determine the identity of the unknown (U1)
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 100 mm

80 mm

45 mm 44
mm
30 mm
20 mm

A B C D U1

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 RETARDATION FACTOR (Rf)
Factors affecting Rf values
1. solvent
solvent and solvent mixtures have
different polarities
a compound travels at different rates
in solvents of different polarities
2. adsorbent
different polarities for different
adsorbents
 RETARDATION FACTOR (Rf)
Factors affecting Rf values
3. thickness of adsorbent
if adsorbent is so thick, some sample
may be adsorbed

4. distance travelled by the solvent

relatively different Rf values
 RETARDATION FACTOR (Rf)
Factors affecting Rf values
5. amount of compound used
thick/big spot
tailing
uneven separation

6. temperature
 EXPERIMENT PART
 Separation of green leaf pigments by
paper chromatography
 Acetone – dissolve the pigments present in
the leaf samples
 Solvent system able to separate a greater
number of components is the more
appropriate mobile phase

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 EXPERIMENT PART
 Probable identity of
spots
 Chlorophyll a
(blue-green, more
intense than
chlorophyll b)
 Chlorophyll b
(green)
 Xanthophylls
(yellow)

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 EXPERIMENT PART
 Analysis of components dyes of black ink
by TLC
 Black inks are mixtures of colored pigments
- not all inks having the same color have the
same components
 Two ink brands may be similar if the
chromatogram are the same (example, same
number of component colors); otherwise, they
are different.
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EXPERIMENT PART

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 EXPERIMENT PART
 Identification of amino
acid by TLC
 Visualizing agent:
ninhydrin – reacts with
amino acid to form a
purple complex
 proline gives a yellow-
orange color instead

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 EXPERIMENT PART
- Molar Mass: 165 g/
mol

O
- Neutral amino acid

H2 N OH - Highest Rf value since

phenylalanine moderate molecular
weight and less polar
than tyrosine and
aspartic acid
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 EXPERIMENT PART
-Molar Mass: 181 g/ mol

HO NH2 -Neutral amino acid

O OH - Rf value between
phenylalanine and
tyrosine
aspartic acid since
heavier than Phe and
less polar than Asp
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 EXPERIMENT PART
- Molar mass: 133 g/mol

O
- Acidic amino acid
OH
HO - although it has the least
MM, it is the most polar
NH2 O among the three, hence it
aspartic acid has the highest affinity to
the stationary phase

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 EXPERIMENT PART
 In terms of molecular weight
 Higher MW, less distance travelled, lower Rf
 Lower MW, more distance travelled, higher Rf
 In terms of polarity:
 More polar amino acid, more affinity to
stationary phase, lower Rf value
 Less polar amino acid, more affinity to mobile
phase, higher Rf value

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 Seatwork
 Compounds A, B, C and D were separated
by reversed-phase chromatography. The
resulting chromatogram is shown in the
next slide.
 Determine the solvent front in centimeters.
 Calculate Rf of compounds A, B, C and D.
Show complete solution.
 Arrange the following compounds
according to decreasing polarity
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 END

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