Experiment 4:
TLC and HPLC of Nitroanilines
Objectives



To learn the separation techniques of Thin
Layer Chromatography and HPLC
chromatography.
To use these techniques to separate and
identify o-nitroaniline and p-nitroaniline in
sample mixture.
To identify the compounds based TLC Rf
and HPLC Rt.
NITROANILINE STRUCTURES
NH 2
NH 2
NO 2
NO 2
p-nitroaniline
MF: C6H6N2O2
o-nitroaniline
MF: C6H6N2O2
MW: 138.13 g/mol
mp: 147-151oC
MW: 138.13 g/mol
mp: 70-74 oC
hazards: Toxic if ingested
Toxic if inhaled
hazards: Toxic if ingested
Toxic if inhaled
uses: Organic dyes
dipole moment: 3.00 D
uses: Organic dyes
dipole moment: 2.27 D
POLARITY OF NITROANILINES
H
O
N
N
H
H
N
H
O
N
O
O
p-nitroaniline
dipole moment (): 3.00 D
LARGER  = MORE POLAR
o-nitroaniline
dipole moment (): 2.27 D
SMALLER  = LESS POLAR
ANALYTE POLARITY VS.
STATIONARY PHASE
Polar
analyte
binds to the
SiO2 sites,
so it sticks
and moves
slowly
Nonpolar
analyte
doesn’t bind
to SiO2 sites
so it doesn’t
stick and
moves quickly
Bulk Solvent
(Mobile
Phase)
OH
Si
OH
O Si
OH
O Si
OH
O Si
OH
O Si
OH
O Si
ANALYTE
Silica
(Stationary phase)
SOLVENT
Bulk Solvent
(Mobile
Phase)
OH
Si
OH
O Si
OH
O Si
OH
O Si
Silica
(Stationary phase)
OH
O Si
OH
O Si
THIN LAYER
CHROMATOGRAPHY

Sample and standards are applied on origin line of
POLAR TLC plate.

TLC Plate is placed in a developing chamber
containing a nonpolar, organic solvent.

The substance dissolves in the solvent, and is
carried up the plate. The polarity of the substance
determines how far up the plate the substance
travels.

Once developed, the spots are visualized.

The spots are marked with a pencil, and the
distances traveled by the spots are measured.

The distance the unknowns spot has traveled is
called the Rf value, and compared to Rf values of
standard solutions, can be used to identify
compounds.
A
B U C D
filter paper
A
B U C D
THIN LAYER CHROMATOGRAPHY
Supplies
for TLC
Analysis
Preparing TLC Plate
Applying
solutions
to TLC
plate
THIN LAYER CHROMATOGRAPHY
Calculation of Rf values
2.0 cm
5.0 cm
= 0.40
Rf (B) = 3.0 cm
= 0.60
Rf (A) =
Solvent Front
5.0 cm
Distance solvent
migrated = 5.0 cm
4.0 cm
Rf (C) = 0.8 cm = 0.16
Distance A
migrated = 3.0 cm
5.0 cm
Distance B
migrated = 2.0 cm
3.0 cm
Rf (D) = 4.0 cm = 0.80
5.0 cm
Distance C
migrated = 0.8 cm
Origen
x
A
x x x x
B U C D
0.8 cm
Rf (U1) = 3.0 cm = 0.60
5.0 cm
Rf (U2) =
0.8 cm
5.0 cm
= 0.16
The Rf value is defined as the distance the center of the spot moved
divided by the distance the solvent front moved (both measured from
the origin)
THIN LAYER CHROMATOGRAPHY
Rf values



Rf values can be used to aid in the
identification of a substance by comparison
to standards.
The Rf 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 Rf
value may be identical; those with
different Rf values are not identical.
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
(TLC vs. Normal Phase HPLC)
Normal Phase (SiO2) TLC
Normal Phase (SiO2)
a
b
a
0
b
c
Time
c
x
Note: A high TLC Rf value = a low HPLC retention time!
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Sample
loaded
here
SiO2
packed
inside
tube
HPLC
chromatogram
produced
HPLC CHROMATOGRAMS OF
NITROANILINE STANDARDS
o-nitroaniline standard
SOLVENT: 50:50 hexane/ethyl acetate
Rt: 1.074 min
p-nitroaniline standard
SOLVENT: 50:50 hexane/ethyl acetate
Rt: 1.382 min
HPLC OF
NITROANILINE SAMPLES
Nitroaniline sample mixture
SOLVENT: 50:50 hexane/ethyl acetate
o-nitroaniline Rt: 1.074 min
p-nitroaniline Rt: 1.394 min
By comparison of
sample retention
times to standard
retention times,
the active
ingredients can be
identified.
Compound
Retention
Times of Standards
Retention
Times of Sample
o-nitroaniline
1.074
1.074
p-nitroaniline
1.382
1.394
FOR MORE INFORMATION...

Please refer to Appendices E and F
in the back of your laboratory
manual for further explanation of
theory behind chromatography.
SAFETY CONCERNS



Nitroanilines are toxic if inhaled or
ingested. Use gloves at all times during the
experiment!
All solvents used in today’s experiment are
flammable, eye, and skin irritants. Be
sure to wash your hands before leaving the
laboratory.
Safety goggles are required!
WASTE MANAGEMENT



Place waste solvent from TLC sample
preparation and TLC developing chambers
into container labeled, “Organic Waste
(TLC)”.
Place all used TLC capillary tubes in the
broken glass container.
TLC chambers should be left with the lids
removed in the lab drawer. Do not clean
with soap, water, OR acetone!