CHM 312

Fall 2008

CHROMATOGRAPHY
THIN LAYER CHROMATOGRAPHY
(TLC)
Thin layer chromatography (TLC) is an important
technique for identification and separation of mixtures
of organic compounds. It is useful in:

Identification of components of a mixture (using appropriate
standards)
following the course of a reaction,
analyzing fractions collected during purification,
analyzing the purity of a compound.
In TLC, components of the mixture are partitioned
between an adsorbent (the stationary phase, usually
silica gel, SiO
2
) and a solvent ( the mobile phase) which
flows through the adsorbent.
THIN LAYER CHROMATOGRAPHY
THIN LAYER CHROMATOGRAPHY
In TLC, a plastic, glass or aluminum sheet is coated
with a thin layer of silica gel.

A very small amount of a solution of the substance
to be analyzed is applied in a small spot with a
capillary tube, ~1cm from the bottom of the
TLC plate
The TLC is developed in a chamber
which contains the developing solvent
(the mobile phase). A truncated filter
paper placed in the chamber serves to
saturate the chamber with mobile phase.

A B C U D
A B C U
filter paper
D
As the mobile phase rises up the TLC plate by
capillary action, the components dissolve in the
solvent and move up the TLC plate.
Individual components move up at different rates,
depending on intermolecular forces between the
component and the silica gel stationary phase and
the component and the mobile phase.
THIN LAYER CHROMATOGRAPHY
The stationary phase is SiO2 and is very polar.

It is capable of strong dipole-dipole and H-bond donating and accepting
interactions with the analytes (the components being analyzed).

More polar analytes interact more strongly with the stationary phase in move
very slowly up the TLC plate.

By comparison, the mobile phase is relatively nonpolar and is capable
of interacting with analytes by stronger London forces, as well as by dipole-
dipole and H-bonding.

More nonpolar analytes interact less strongly with the polar silica gel and more
strongly with the less polar mobile phase and move higher up the TLC plate.

http://www.instructables.com/id/EW1YDCYF4REC0IU/
Once the solvent is within ~1-2 cm of the top of
the TLC sheet, the TLC is removed from the
developing chamber and the farthest extent of
the solvent (the solvent front) is marked with a
pencil.

The solvent is allowed to evaporate from the
TLC sheet in the hood.

The spots are visualized using a UV lamp.

A fluorescent compound, usually Manganese-
activated Zinc Silicate, is added to the adsorbent
that allows the visualization of spots under a
blacklight (UV254). The adsorbent layer will
fluoresce light green by itself, but spots of analyte
quench this fluorescence and appear as a dark spot.


THIN LAYER CHROMATOGRAPHY
http://orgchem.colorado.edu/hndbksupport/TLC/TLCprocedure.html
THIN LAYER CHROMATOGRAPHY - Visualization

As the chemicals being separated may be
colorless, several methods exist to visualize
the spots:

Visualization of spots under a UV
254
lamp. The
adsorbent layer will thus fluoresce light green by
itself, but spots of analyte quench this
fluorescence.

Iodine vapors are a general unspecific color.

Specific color reagents exist into which the TLC
plate is dipped or which are sprayed onto the
plate.

Once visible, the R
f
value of each spot can be
determined
Chromatogram of 10 essential oils,
Stained with vanillin reagent.

THIN LAYER CHROMATOGRAPHY
Calculation of Rfs
The R
f
is defined as the distance the center of the spot moved divided
by the distance the solvent front moved (both measured from the origin)

A B
C U
x x x x
Solvent Front
Origen
Distance solvent
migrated = 5.0 cm
Distance A
migrated = 3.0 cm
Distance B
migrated = 2.0 cm
Distance C
migrated = 0.8 cm
0.8 cm
3.0 cm
R
f
(A) =
R
f
(B) =
R
f
(C) =
R
f
(U
1
) =
R
f
(U
2
) =
2.0 cm
5.0 cm
= 0.40
= 0.60
= 0.16
= 0.60
= 0.16
3.0 cm
5.0 cm
0.8 cm
5.0 cm
3.0 cm
5.0 cm
0.8 cm
5.0 cm
D
x
R
f
(D) = = 0.80
4.0 cm
5.0 cm
4.0 cm
THIN LAYER CHROMATOGRAPHY
Calculation of Rfs
The R
f
is defined as the distance the center of the spot moved divided
by the distance the solvent front moved (both measured from the origin)

A B
C U
x x x x
Solvent Front
Origen
Distance solvent
migrated = 5.0 cm
Distance A
migrated = 3.0 cm
Distance B
migrated = 2.0 cm
Distance C
migrated = 0.8 cm
0.8 cm
3.0 cm
R
f
(A) =
R
f
(B) =
R
f
(C) =
R
f
(U
1
) =
R
f
(U
2
) =
2.0 cm
5.0 cm
= 0.40
= 0.60
= 0.16
= 0.60
= 0.16
3.0 cm
5.0 cm
0.8 cm
5.0 cm
3.0 cm
5.0 cm
0.8 cm
5.0 cm
D
x
R
f
(D) = = 0.80
4.0 cm
5.0 cm
4.0 cm
R
f
values can be used to aid in the identification of a
substance by comparison to standards.

The R
f
value is not a physical constant, and
comparison should be made only between spots on
the same sheet, run at the same time.


Two substances that have the same R
f
value may be
identical; those with different R
f
values are not
identical.

THIN LAYER CHROMATOGRAPHY R
f
s
Absorption of Solutes

The adsorption strength of compounds increases with increasing polarity of
functional groups, as shown below:

-CH=CH
2
, -X, -OR, -CHO, -CO
2
R, -NR
2
, -NH
2
, -OH, -CONR
2
, -CO
2
H.
(weakly adsorbed) (strongly adsorbed)
(nonpolar) (more polar)
THIN LAYER CHROMATOGRAPHY R
f
s
Elution Strength of Mobile Phase (e)
Elution strength is generally considered to be equivalent to polarity. A solvents
elution strength depends on Intermolecular Forces between the solvent and the
analytes and between the solvent and the stationary phase.

A more polar (or more strongly eluting solvent) will move all of the analytes to a
greater extent, than a less polar, weakly elution solvent.

For example, the elution strength of hexane is very low; e = 0.01.
the elution strength of ethyl acetate is higher; e = 0.45
the elution strength of ethanol is even higher; e = 0.68
Solvent MF
MW
Bp (
o
C)
Density (g/mL)
Hazards* Dipole Elution
Stength
(e)
Hexane
CH
3
(CH
2
)
4
CH
3

C
6
H
14

86.17
68.7
0.659
Flammable
Toxic
0.08 0.01
Toluene
C
6
H
5
CH
3

C
7
H
8
92.13
110.6
0.867
Flammable
Toxic
0.31 0.22
Diethyl ether
CH
3
CH
2
OCH
2
CH
3

C
4
H
10
O
74.12
34.6
0.713
Flammable
Toxic, CNS
Depressant

1.15 0.29
Dichloromethane
CH
2
Cl
2

CH
2
Cl
2

84.94
39.8
1.326
Toxic, Irritant
Cancer suspect
1.14 0.32
Ethyl Acetate
CH
3
CO
2
CH
2
CH
3

C
4
H
8
O
2

88.10
77.1
0.901
Flammable
Irritant
1.88 0.45
Acetone
CH
3
COCH
3

C
3
H
6
O
58.08
56.3
0.790
Flammable
Irritant
2.69 0.43
Butanone
CH
3
CH
2
COCH
3

C
4
H
8
O
72.10
80.1
0.805
Flammable
Irritant
2.76 0.39
1-Butanol
CH
3
CH
2
CH
2
CH
2
OH
C
4
H
10
O
74.12
117.7
0.810
Flammable
Irritant
1.75 0.47
Propanol
CH
3
CH
2
CH
2
OH
C
3
H
8
O
60.09
82.3
0.785
Flammable
Irritant
1.66 0.63
Ethanol
CH
3
CH
2
OH
C
2
H
6
O
46.07
78.5
0.789
Flammable
Irritant
1.70 0.68
Methanol
CH
3
OH
CH
4
O
32.04
64.7
0.791
Flammable
Toxic
1.7 0.73
Water
HOH
H
2
O
18.02
100.0
0.998
1.87 >1

Solvent Properties and Elution Strengths
Elution Strength of Mixed Solvents
The elution strength of the mixture is assumed to be the weighted average of the elution
strengths of the components:

e
o
net
= e
o
A
(mole % A) + e
o
B
(mole % B)
where: mole % A = (moles A) / (moles A + moles B)

Thus, to determine the e
o
net
of a solvent mixture, the molar ratio of the solvents must first
be calculated. For example, the e
o
net
of a solvent mixture prepared from 1.0 mL of ethyl
acetate plus 9.0 mL of hexanes is calculated as shown below:

e
o
net
= e
o
EtOAc [(moles EtOAc)/(moles EtOAc+moles hexane)] +
e
o
hexane [(moles hexane)/(moles EtOAc+moles hexane)]

where: moles EtOAc = [(volume EtOAc) (density EtOAc)] / [molecular weight of EtOAc]

thus: e
o
net
=
{0.45[(1.0mLEtOAc)(0.902g/mL)/(88.11g/mole)]+0.01[(9.0mLhexane)(0.659g/mL)/86.18g/mole)]}
{(1.0 mLEtOAc)(0.902g/mL)/88.11g/mole) + (9.0 mLhexane)(0.659g/mL)/86.18g/mole)}

and e
o
net
= 0.067
Resolution
The separation between two analytes on a
chromatogram can be expressed as the
resolution, Rs and can be determined using
the following equation:

Rs = (distance between center of spots)
(average diameter of spots)

In TLC, if the Rs value is greater than 1.0, the
analytes are considered to be resolved.

x x

Improving Resolution:

For two closely migrating components, optimum resolutions
are usually obtained when the R
f
s of both compounds
are between 0.2 and 0.5

* To Improve Rs, change the elution strength of the solvent
to optimize R
f
s
change e
o
net (= in capacity factor), all compounds will
be effected similarly.
Alter the composition of the solvent system so that the
components affinity for the mobile phase vs. the solid
phase are differentially changed (= change in
selectivity).
Changing the chemical nature of the solvent system,
such as changing a hydrogen bonding solvent to a
solvent which cannot hydrogen bond to the analyte, is
often the most effective.

** Improve Rs by decreasing the diameter of the
analyte spots. This can be achieved by applying
smaller and less concentrated spots.
http://orgchem.colorado.edu/hndbksupport/
TLC/TLCprocedure.html
Optimize R
f
s
TLC Stationary Phases
www.vwr.com
www.vwr.com
PREPARATIVE TLC (PTLC)
TLC - Optimizing for column chromatography
Optimum: 0.2 < R
f
< 0.5