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

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: http://www.youtube.com/watch?v=yoYECMP_1iI&feature=related

NITROANILINE STRUCTURES

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

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

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.

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)

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) Sample loaded here SiO2 packed inside tube HPLC chromatogram produced

HPLC STANDARD CHROMATOGRAMS o-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

HPLC SAMPLE CHROMATOGRAM Nitroaniline sample mixture SOLVENT: 50:50 hexane/ethyl acetate o-nitroaniline Rt: 1.074 min p-nitroaniline Rt: 1.394 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.

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!

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.

(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!

HPLC Chromatograms o-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 min p-nitroaniline Rt: 1.394 min Nitroaniline sample mixture SOLVENT: 80:20 hexane/ethyl acetate o-nitroaniline Rt: 1.534 min p-nitroaniline Rt: 3.059 min

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

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!

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…

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?