Experiment 4: TLC and HPLC of Nitroanilines
Jan 03, 2016
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
NH2
NO2
NH2
NO2
p-nitroaniline
MF: C6H6N2O2
MW: 138.13 g/mol
mp: 147-151oC
hazards: Toxic if ingested
Toxic if inhaled
uses: Organic dyes
dipole moment: 3.00 D
o-nitroaniline
MF: C6H6N2O2
MW: 138.13 g/mol
mp: 70-74 oC
hazards: Toxic if ingested
Toxic if inhaled
uses: Organic dyes
dipole moment: 2.27 D
POLARITY OF NITROANILINES
N
N
N
N
p-nitroanilinedipole moment (): 3.00 D
o-nitroanilinedipole moment (): 2.27 D
H HO
O
H H
O O
LARGER = MORE POLAR SMALLER = LESS POLAR
ANALYTE POLARITY VS. STATIONARY PHASE
Si O
OH
Si
OH
O Si
OH
O Si
OH
O Si
OH
O Si
OH
Silica (Stationary phase)
Si O
OH
Si
OH
O Si
OH
O Si
OH
O Si
OH
O Si
OH
Silica (Stationary phase)
Bulk Solvent (Mobile Phase)
ANALYTE
SOLVENT
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)
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 CU D
A B CU
filter paper
D
THIN LAYER CHROMATOGRAPHY
Supplies for TLC
Analysis
Preparing TLC Plate
Applying
solutions to TLC
plate
THIN LAYER CHROMATOGRAPHY
Calculation of Rf values
A B CU
x xx 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
Rf (A) =
Rf (B) =
Rf (C) =
Rf (U1) =
Rf (U2) =
2.0 cm5.0 cm
= 0.40
= 0.60
= 0.16
= 0.60
= 0.16
3.0 cm5.0 cm
0.8 cm5.0 cm
3.0 cm5.0 cm
0.8 cm5.0 cm
Dx
Rf (D) = = 0.804.0 cm5.0 cm
4.0 cm
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)
x
a
b
c
a b c
0 Time
Normal Phase (SiO2)
Normal Phase (SiO2) TLC
Note: A high TLC Rf value = a low HPLC retention time!
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
SiO2 packed inside tube
Sample loaded here
HPLC chromatogram produced
HPLC CHROMATOGRAMS OF NITROANILINE
STANDARDSo-nitroaniline standard
SOLVENT: 50:50 hexane/ethyl acetateRt: 1.074 min
p-nitroaniline standardSOLVENT: 50:50 hexane/ethyl acetateRt: 1.382 min
HPLC OF NITROANILINE SAMPLES
Compound Retention Times of Standards
Retention Times of Sample
o-nitroaniline 1.074 1.074
p-nitroaniline 1.382 1.394
By comparison of sample retention times to standard retention times, the active ingredients can be identified.
Nitroaniline sample mixtureSOLVENT: 50:50 hexane/ethyl acetateo-nitroaniline Rt: 1.074 minp-nitroaniline Rt: 1.394 min
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!