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

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

Supplies for TLC

Analysis

Preparing TLC Plate

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.

Visualize plate.

Circle spots.

Calculate TLC Rf value for each spot.

o mix p

o mix

filter paper

p

THIN LAYER CHROMATOGRAPHY

Calculation of Rf values

"o" "p"mixxx x

Solvent Front

Origin

Distance solvent migrated = 5.0 cm

Distance "o" migrated = 3.0 cm

Distance "p" migrated = 0.8 cm 0.8 cm

3.0 cm

Rf (B) =

Rf (C) =

Rf (U1) =

Rf (U2) =

= 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

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)

SiO2 packed inside tube

Sample loaded here

HPLC chromatogram produced

HPLC STANDARD CHROMATOGRAMS

o-nitroaniline standardSOLVENT: 50:50 hexane/ethyl acetateRt: 1.074 min

p-nitroaniline standardSOLVENT: 50:50 hexane/ethyl acetateRt: 1.382 min

HPLC SAMPLE CHROMATOGRAM

CompoundRetention

Times of StandardsRetention 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

TLC VS. HPLC RESULTS

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!

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 Rf 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 4.1TLC Rf Data

Compound

Rf values in Solvent

System 180:20

hexane/ethyl acetate

Rf values in Solvent

System 250:50

hexane/ethyl acetate

Rf values in Solvent System

3100% ethyl acetate

o-nitroaniline Same #

p-nitroaniline Same #

mixture

TLC Diagrams

(Show measurements for solvent front and all spots in cm)

X X X o mix p

X X X o mix p

X X X o mix p

Don’t forget to give cm measurements for each spot and that of solvent front!

2 decimal places ONLY!

HPLC Chromatogramso-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 minp-nitroaniline Rt: 1.394 min

Nitroaniline sample mixture

SOLVENT: 80:20 hexane/ethyl acetate

o-nitroaniline Rt: 1.534 minp-nitroaniline Rt: 3.059 min

TABLE 4.2

HPLC Rt Data

Compound

STANDARD

Rt (min)

SAMPLE

Rt (min)Solvent System

1

SAMPLE

Rt (min) Solvent System

2

SAMPLE

Rt (min) Solvent System 3

o-nitroani

line

p-nitroani

line

Use data from p. 37

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 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)

• REFERENCE TO PROCEDURE (Must include…)•full title•Edition•authors•page numbers where actual procedure can be found

Compound MW (g/mol)

bp (Co) d (g/mL)

HAZARDS

AcetonehexaneEthyl acetateo-nitroaniline

XXX XXX

p-nitroaniline

XXX XXX

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.