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Experiment 4:

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

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Experiment 4:

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    1. 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. 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.

    3. 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

    4. NITROANILINE STRUCTURES

    5. POLARITY OF NITROANILINES

    6. ANALYTE POLARITY VS. STATIONARY PHASE

    7. THIN LAYER CHROMATOGRAPHY

    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

    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)

    12. HPLC STANDARD CHROMATOGRAMS

    13. HPLC SAMPLE CHROMATOGRAM

    14. TLC VS. HPLC RESULTS

    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. TABLE 4.1

    17. HPLC Chromatograms

    18. TABLE 4.2

    19. 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!

    20. 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!

    21. LABORATORY NOTEBOOK (Pre-lab)

    22. LABORATORY NOTEBOOK (In-lab)

    23. 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!

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