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 R f and HPLC R t.  To investigate the effect of solvent system polarity on TLC R f 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:

NITROANILINE STRUCTURES

POLARITY OF NITROANILINES LARGER  = MORE POLARSMALLER  = LESS POLAR

ANALYTE POLARITY VS. STATIONARY PHASE Bulk Solvent (Mobile Phase) ANALYTE SOLVENT Polar analyte binds to the SiO 2 sites, so it sticks and moves slowly Nonpolar analyte doesn’t bind to SiO 2 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 R f value for each spot.

THIN LAYER CHROMATOGRAPHY Calculation of R f values The R f 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 R f values  R f values can be used to aid in the identification of a substance by comparison to standards.  The R f 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 R f value may be identical; those with different R f values are not identical.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) SiO 2 packed inside tube Sample loaded here HPLC chromatogram produced

HPLC STANDARD CHROMATOGRAMS o-nitroaniline standard SOLVENT: 50:50 hexane/ethyl acetate R t : min p-nitroaniline standard SOLVENT: 50:50 hexane/ethyl acetate R t : min

HPLC SAMPLE CHROMATOGRAM Compound Retention Times of Standards Retention Times of Sample o-nitroaniline1.074 p-nitroaniline By comparison of sample retention times to standard retention times, the active ingredients can be identified. Nitroaniline sample mixture SOLVENT: 50:50 hexane/ethyl acetate o-nitroaniline R t : min p-nitroaniline R t : min

TLC VS. HPLC RESULTS 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.

TABLE 4.1 TLC R f Data Compound R f values in Solvent System 1 80:20 hexane/ ethyl acetate R f values in Solvent System 2 50:50 hexane/ ethyl acetate R f values in Solvent System 3 100% ethyl acetate o-nitroanilineSame # p-nitroanilineSame # 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 Chromatograms Nitroaniline sample mixture SOLVENT: 100% ethyl acetate o-nitroaniline R t : min p-nitroaniline R t : min Nitroaniline sample mixture SOLVENT: 50:50 hexane/ethyl acetate o-nitroaniline R t : min p-nitroaniline R t : min Nitroaniline sample mixture SOLVENT: 100% ethyl acetate o-nitroaniline R t : min p-nitroaniline R t : min Nitroaniline sample mixture SOLVENT: 80:20 hexane/ethyl acetate o-nitroaniline R t : min p-nitroaniline R t : min

TABLE 4.2 HPLC R t Data Compound STANDARD R t (min) SAMPLE R t (min) Solvent System 1 SAMPLE R t (min) Solvent System 2 SAMPLE R t (min) Solvent System 3 o-nitroaniline p-nitroaniline 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 ONLY. Wikipedia is unacceptable) REFERENCE TO PROCEDURE (Must include…) full title Edition authors page numbers where actual procedure can be found CompoundMW (g/mol)bp (C o )d (g/mL)HAZARDS Acetone hexane Ethyl acetate o-nitroanilineXXX p-nitroanilineXXX

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 R f 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.

FOR NEXT LAB…  The pre-lab lab notebook entry for Experiment 5 will be due at the beginning of lab!  Experiment 4 Final Lab Report, along with yellow in-lab notebook pages due at the beginning of lab!