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 R f and HPLC R t.

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 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 R f value, and compared to R f values of standard solutions, can be used to identify compounds.

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

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 on the same sheet, run at the same time.  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 (TLC vs. Normal Phase HPLC) Note: A high TLC Rf value = a low HPLC retention time!

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

HPLC CHROMATOGRAMS OF NITROANILINE STANDARDS 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 OF NITROANILINE SAMPLES CompoundRetention 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

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!