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.