Experiment 5: COLUMN CHROMATOGRAPHIC PURIFICATION OF NITROANILINES

Objectives  To learn the separation technique of column chromatography.  To separate constitutional isomers of o-nitroaniline from p-nitroaniline using this technique.  To analyze the purity of the isolated compounds using TLC and HPLC.

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:  Please note that this is an example of how column chromatography experiments are performed, however our procedure may be slightly different.

TLC VS. COLUMN CHROMATOGRAPHY Polar components (b) adsorb more strongly to the polar silica gel and elute after the less polar components, which move more quickly with the non-polar (relative to silica gel) solvent.

COLUMN CHROMATOGRAPHY: General Procedure  Prepare column  Load column  Develop column  Collect fractions  Analyze fractions

OVERVIEW  Run column to separate the nitroaniline compounds from one another.  Collect the compounds in separate test tubes as they elute from the column.  Perform a TLC experiment to determine which test tubes contain pure o-nitroaniline and which contain pure p-nitroaniline.  Combine pure p-nitroaniline fractions in one container, combine pure o-nitroaniline fractions in another.  Evaporate solvent to concentrate samples.  Prepare an HPLC vial of each sample and submit for analysis.

COLUMN CHROMATOGRAPHY: Preparing the column… MATERIALS: o 50 mL SOLVENT 1 o 30 mL SOLVENT 2 o 20 mL SOLVENT 3 o 20 cc SiO 2 gel

COLUMN CHROMATOGRAPHY: Preparing the column… Mix slurry Add slurry Drain solvent

COLUMN CHROMATOGRAPHY: Loading the column… Add concentrated sample solution with spiraling motion Rinse sides of column with solvent to remove excess sample Allow column rinse to load into silica gel

COLUMN CHROMATOGRAPHY: Developing the column… SLOWLY add solvent to column to elute compounds

COLUMN CHROMATOGRAPHY: Collecting fractions… o Once individual fractions are collected, you will perform a TLC experiment to determine which test tubes contain which nitroaniline compound. o You should prepare ONE TLC plate, with up to 7 lanes per plate. o Remember to apply the original sample mixture solution as your standard.

COLUMN CHROMATOGRAPHY: Analyzing fractions by TLC… o In order to evaluate the success of the separation after the column, TLC analysis is performed. o By applying the original nitroaniline mixture solution (STD) along with the sample solutions from the individual fractions collected, the fractions containing pure compounds can be identified. o Once the pure fractions are identified, they will be combined in an effort to produce a pure sample of each nitroaniline compound for further analysis. o Since the UV detector on the HPLC is more sensitive than the human eye, HPLC analysis will be performed on the samples prepared after TLC analysis to ensure the correct fractions were combined during the experiment.

TABLE 5.1 Compound Standard R f Value R f value Fraction #1 R f value Fraction #2 R f value Fraction #3 R f value Fraction #4 R f value Fraction #5 R f value Fraction #6 o- nitroaniline p- nitroaniline R f values are unit less and 2 decimal places ONLY! Individual fractions may contain a single compound or both compounds as the column proceeds. Fractions containing a single compound are considered PURE fractions, while those containing both compounds are considered MIXED (IMPURE) fractions.

HPLC SAMPLE PREPARATION  Place a small amount of crystals of each purified sample into a small auto analyzer vial. Add 1 mL of HPLC solvent.  Place sample vial into vial slot in sample tray and sign out on vial slot sheet.  Any samples with visible solid in them will be DISCARDED!

HPLC SAMPLE PREPARATION HPLC sample prep area located in balance room To operate dispenser on solvent bottle, pull up on plunger and push down SLOWLY!

TABLE 5.2 Compound StandardsComponent #1Component #2 R t (min) R t (min) HPLC % Area R t (min) HPLC % Area o-nitroaniline p-nitroaniline Samples containing a single compound are considered PURE samples, while those containing both compounds are considered MIXED (IMPURE) samples. Be sure to attach both sample chromatograms to final lab report!

SAFETY CONCERNS  Ethyl acetate and hexane are flammable. Never use these solvents around an open flame or hot hot plate.  o-nitroaniline and p-nitroaniline are toxic if ingested or inhaled. Wear safety goggles all times during the experiment!  GLOVES are available upon request.

WASTE MANAGEMENT  Place all liquid waste into the container marked “LIQUID ORGANIC WASTE”.  Place used TLC capillaries in the broken glass container.  Place TLC plates in yellow solid waste trashcan under the supply hood.  Leave the columns containing SiO 2 gel suspended in hood.

For Next Lab…  Lab Quiz #1 will be given at the beginning of lab.  We will do Experiment 3 after the lab quiz.  There is NO pre-lab notebook entry for Experiment 3!