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!