Experiment 4:

TLC and HPLC of Nitroanilines

 Objectives
To learn the analytical techniques of Thin Layer
Chromatography (TLC) 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.

To investigate the effect of solvent system

polarity on TLC Rf values and HPLC retention times.
 Before coming to lab
Prepare the Pre-lab notebook entry for
this experiment!

It is *highly recommended* that you

watch a short video prior to coming to
lab! Simply copy and paste the
following link into your address bar:
http://www.youtube.com/watch?v=yoYECMP_1iI&feature=related
NITROANILINE STRUCTURES
NH2 NH2
NO2

NO2
p-nitroaniline o-nitroaniline
MF: C6H6N2O2 MF: C6H6N2O2
MW: 138.13 g/mol MW: 138.13 g/mol
mp: 147-151oC mp: 70-74 oC
hazards: Toxic if ingested hazards: Toxic if ingested
Toxic if inhaled Toxic if inhaled
uses: Organic dyes uses: Organic dyes
dipole moment: 3.00 D dipole moment: 2.27 D
POLARITY OF NITROANILINES

H H H H
N N O
N
O

N
O O
p-nitroaniline o-nitroaniline
dipole moment (): 3.00 D dipole moment (): 2.27 D
LARGER = MORE POLAR SMALLER = LESS POLAR
 ANALYTE POLARITY VS.
STATIONARY PHASE
Bulk Solvent
(Mobile
Phase)

Polar
analyte OH OH OH OH OH OH
binds to the Si O Si O Si O Si O Si O Si
SiO2 sites,
so it sticks Silica ANALYTE
and moves (Stationary phase)
slowly SOLVENT

Bulk Solvent
(Mobile
Nonpolar Phase)

analyte
doesnt bind OH OH OH OH OH OH
to SiO2 sites Si O Si O Si O Si O Si O Si
so it doesnt
stick and
Silica
moves quickly (Stationary phase)
THIN LAYER CHROMATOGRAPHY
Supplies Preparing TLC Plate
for TLC
Analysis

Applying
solutions
to TLC
plate
THIN LAYER CHROMATOGRAPHY
(Running the Experiment)

Prepare TLC plate and TLC chamber.

Apply standard solutions and sample solutions to
plate.

Develop TLC plate in chamber.

o mix p

Visualize plate.
filter paper

Circle spots.
Calculate TLC Rf value for each spot.

o mix p
 THIN LAYER CHROMATOGRAPHY
Calculation of Rf values

Solvent Front

Rf (B) = 3.0 cm = 0.60

Distance solvent
5.0 cm
migrated = 5.0 cm

Distance "o"
migrated = 3.0 cm
Rf (C) = 0.8 cm = 0.16
5.0 cm

3.0 cm

Distance "p"

Rf (U1) = 3.0 cm = 0.60

migrated = 0.8 cm 0.8 cm

Origin
x x x 5.0 cm
"o" mix "p"
0.8 cm
Rf (U2) = = 0.16
5.0 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 run under the same experimental
conditions.

Two substances that have the same Rf

value may be identical; those with different
Rf values are not identical.
 HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)

Sample
loaded
here

SiO2
packed
inside
tube

HPLC
chromatogram
produced
 HPLC STANDARD
CHROMATOGRAMS
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 SAMPLE
CHROMATOGRAM
Nitroaniline sample mixture
SOLVENT: 50:50 hexane/ethyl acetate
o-nitroaniline Rt: 1.074 min
p-nitroaniline Rt: 1.394 min

Retention Retention
By comparison of
Compound Times of Standards Times of Sample
sample retention
times to standard o-nitroaniline 1.074 1.074
retention times,
p-nitroaniline 1.382 1.394
the active
ingredients can be
identified.
 TLC VS. HPLC RESULTS
Normal Phase (SiO2) TLC

a Normal Phase (SiO2)

a b c

0 Time
c

Note: A high TLC Rf value = a low HPLC retention time!

FOR MORE INFORMATION...
Please refer to Appendices E and F
in the back of your laboratory
manual for further explanation of
theory behind chromatography.
 OVERVIEW
Prepare 3 TLC plates.
Apply sample and standard solutions to each plate.
Prepare 3 TLC chambers containing 3 different solvent systems.
Develop TLC plates.
Visualize plates using UV lamp. Circle spots and calculate R f
values for each spot.
Sketch plates CLEARLY in lab notebook, and on Final Lab Report.
Record HPLC data for standards and samples using p. 37 in lab
manual.
 TABLE 4.1
TLC Rf Data
Rf values in Rf values in Rf values in
Solvent System Solvent System Solvent System
Compound 1 2 3
80:20 50:50 100% ethyl acetate
hexane/ hexane/
ethyl acetate ethyl acetate
o-nitroaniline Same #
2 decimal places ONLY!
p-nitroaniline Same #
mixture

Dont forget to give cm

measurements for each spot and
TLC
that of solvent front!
Diagrams
(Show measurements for solvent front
and all spots in cm)

X X X X X X X X X
HPLC Chromatograms
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

Nitroaniline sample mixture

SOLVENT:
50:50 hexane/ethyl acetate

o-nitroaniline Rt: 1.074 min

p-nitroaniline Rt: 1.394 min

Nitroaniline sample mixture

SOLVENT:
80:20 hexane/ethyl acetate

o-nitroaniline Rt: 1.534 min

p-nitroaniline Rt: 3.059 min
 TABLE 4.2
HPLC Rt Data

STANDARD SAMPLE SAMPLE SAMPLE

Compound Rt (min) Rt (min) Rt (min) Rt (min)

Solvent Solvent Solvent
System 1 System 2 System 3

o-nitroaniline

Use data from p. 37

p-nitroaniline
 SAFETY CONCERNS
Nitroanilines are toxic if inhaled or ingested.
Use gloves at all times during the experiment!

All solvents used in todays experiment are

flammable, eye, and skin irritants. Be sure
to wash your hands before leaving the
laboratory.

Safety goggles are required!

 WASTE MANAGEMENT
Place all liquid waste from TLC experiment
into container labeled LIQUID ORGANIC
WASTE.
Place all used TLC capillary tubes in the
broken glass container.
Place all TLC plates in yellow trashcan
under the supply hood.
TLC chambers should be left with the lids
removed in the lab drawer. Do not clean
with soap, water, OR acetone!
 LABORATORY NOTEBOOK
(Pre-lab)
OBJECTIVE (Must clearly state)
What is the goal of the experiment?
How will you accomplish this goal?
How will you determine if it worked?

TABLE OF PHYSICAL DATA (Complete the following table using

MSDS sheets from a site on WWW Links ONLY. Wikipedia is unacceptable)
Compound MW (g/mol) bp (Co) d (g/mL) HAZARDS
Acetone
hexane
Ethyl acetate
o-nitroaniline XXX XXX
p-nitroaniline XXX XXX

REFERENCE TO PROCEDURE (Must include)

full title
Edition
authors
page numbers where actual procedure can be found
 LABORATORY NOTEBOOK
(In-lab)

DATA/CALCULATIONS
TLC diagrams in all 3 solvent systems including cm measurements of all
spots and solvent front
Give an example of a TLC Rf calculation

EXPERIMENTAL PROCEDURE
In paragraph form, briefly describe the procedure that you actually
followed during the lab.
Paragraph must be written in PAST TENSE, PASSIVE VOICE.
Include any volumes or weights of chemicals used during the experiment.
Include any mistakes, accidents or observations if necessary.