1. TLC and HPLC of Nitroanilines
Experiment 4:
TLC and HPLC of Nitroanilines

2. 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 based on TLC Rf and HPLC Rt.
To investigate the effect of changing solvent system polarity on TLC Rf values and HPLC retention times.

3. Before coming to lab…
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:

4. NITROANILINE STRUCTURES

5. POLARITY OF NITROANILINES
LARGER m = MORE POLAR
SMALLER m = LESS POLAR

6. ANALYTE POLARITY VS. STATIONARY PHASE
Bulk Solvent (Mobile Phase)
Polar analyte binds to the SiO2 sites, so it sticks and moves slowly
ANALYTE
SOLVENT
Bulk Solvent (Mobile Phase)
Nonpolar analyte doesn’t bind to SiO2 sites so it doesn’t stick and moves quickly

7. THIN LAYER CHROMATOGRAPHY
Supplies for TLC Analysis
Preparing TLC Plate
Applying solutions to TLC plate

8. 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.

9. THIN LAYER CHROMATOGRAPHY Calculation of Rf values
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)

10. 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.

11. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
Sample loaded here
SiO2 packed inside tube
HPLC chromatogram produced

12. HPLC STANDARD CHROMATOGRAMS
o-nitroaniline standard
SOLVENT: 50:50 hexane/ethyl acetate
Rt: min
p-nitroaniline standard
SOLVENT: 50:50 hexane/ethyl acetate
Rt: min

13. HPLC SAMPLE CHROMATOGRAM
Nitroaniline sample mixture
SOLVENT: 50:50 hexane/ethyl acetate
o-nitroaniline Rt: min
p-nitroaniline Rt: min
Compound
Retention
Times of Standards
Times of Sample
o-nitroaniline
1.074
p-nitroaniline
1.382
1.394
By comparison of sample retention times to standard retention times, the active ingredients can be identified.

14. Note: A high TLC Rf value = a low HPLC retention time!
TLC VS. HPLC RESULTS
Note: A high TLC Rf value = a low HPLC retention time!

15. FOR MORE INFORMATION...
Please refer to Appendices E and F in the back of your laboratory manual for further explanation of theory behind chromatography.

16. 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.

17. (Show measurements for solvent front and all spots in cm)
TABLE 4.1: TLC Results
TLC Rf Data
Compound
Rf values in
Solvent System 1
80:20
hexane/
ethyl acetate
Solvent System 2
50:50
Solvent System 3
100% ethyl acetate
o-nitroaniline
Same #
p-nitroaniline
mixture
TLC
Diagrams
(Show measurements for solvent front and all spots in cm)
X X X
o mix p
o mix p
X X X
o mix p
2 decimal places ONLY!
Don’t forget to give cm measurements for each spot and that of solvent front!

18. HPLC Chromatograms o-nitroaniline standard SOLVENT:
50:50 hexane/ethyl acetate
Rt: min
p-nitroaniline standard
SOLVENT:
50:50 hexane/ethyl acetate
Rt: min
Nitroaniline sample mixture
SOLVENT:
50:50 hexane/ethyl acetate
o-nitroaniline Rt: min
p-nitroaniline Rt: min
Nitroaniline sample mixture
SOLVENT:
80:20 hexane/ethyl acetate
o-nitroaniline Rt: min
p-nitroaniline Rt: min

19. TABLE 4.2: HPLC Results Use data from p. 37 HPLC Rt Data Compound
STANDARD
Rt (min)
SAMPLE
Solvent
System 1
System 2
System 3
o-nitroaniline
p-nitroaniline
Use data from p. 37

20. 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!

21. 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!

22. Effect of increasing solvent system polarity…
IN LAB QUESTIONS… (The following questions should be answered in laboratory notebook.)
Complete the following table in your laboratory notebook:
TLC
HPLC
Stationary phase
Mobile phase
Indication of
Purity
Identity
Polar
analyte
Nonpolar
Effect of increasing solvent system polarity…

23. IN LAB QUESTIONS… (The following questions should be answered in laboratory notebook.)
List the interm0lecular forces present in each of the compounds below.
Which compound would have a greater affinity for the stationary phase in liquid chromatography?
How would this affect the TLC Rf value?
How would this affect the HPLC retention time?
Which compound would have a greater affinity for the mobile phase in liquid chromatography?
How would this affect the HPLC retention time of this compound?