COLUMN CHROMATOGRAPHIC PURIFICATION OF NITROANILINES 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.

THIN LAYER VS. COLUMN CHROMATOGRAPHY More polar components (b) adsorb more strongly to the polar silica gel (STATIONARY PHASE) and elute after the less polar components, which move more quickly with the relatively non-polar solvent (MOBILE PHASE).

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

COLUMN CHROMATOGRAPHY: Preparing the column…

COLUMN CHROMATOGRAPHY: Preparing the column… Mixing slurry Adding slurry Draining solvent

COLUMN CHROMATOGRAPHY: Loading the column… Adding sample solution with spiraling motion Rinsing sides of column with solvent Allowing column rinse to elute into silica gel

COLUMN CHROMATOGRAPHY: Developing the column… Adding solvent to elute compounds

COLUMN CHROMATOGRAPHY: Collecting fractions…

COLUMN CHROMATOGRAPHY: Analyzing fractions by TLC… Supplies for TLC analysis Drawing the origin line

OVERVIEW Run column to separate the nitroaniline compounds from one another. Collect the compounds in separate containers as they elute from the column. Perform TLC experiment on collected fractions to determine which contain pure compounds. Combine pure o-nitroaniline fractions and evaporate solvent to concentrate sample. Combine pure p-nitroaniline fractions and evaporate solvent to concentrate sample. Prepare an HPLC vial of each sample and submit for analysis. Analyze HPLC results to determine success of separation (purity).

HPLC SAMPLE PREPARATION Place a small amount of crystals of each purified sample into a small autoanalyzer 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! (Ask your lab instructor for assistance if you are unsure!)

TABLE 5.1: TLC Results Compound Standard Rf Value Rf value Fraction #1 #4 Fraction #5 Fraction #6 o-nitroaniline All Rf values are unit less and 2 decimal places ONLY! Some test tubes may NOT contain both compounds (which is GOOD). In this case, you will ONLY record Rf values for any compounds that appear. You will apply the ORIGINAL SAMPLE SOLUTION as your standard. This is where your STANDARD Rf values will come from! p-nitroaniline

TABLE 5.2: HPLC Results Compound Standards Component #1 (o-nitroaniline) Component #2 (p-nitroaniline) Rt (min) HPLC % Area o-nitroaniline p-nitroaniline

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 and gloves at all times during the experiment!

WASTE MANAGEMENT Place all organic solvent waste into container marked, “LIQUID WASTE”. Place any unused silica gel in container marked, “SOLID WASTE”. Place used TLC capillaries in the broken glass container. Drain excess solvent out of column into a container, empty solvent waste into appropriate container, then leave the column containing SiO2 gel suspended in hood.

IN-LAB QUESTION (The following question should be answered in your laboratory notebook.) List the 5 steps of the general procedure for column chromatography. 1. 2. 3. 4. 5.

IN-LAB QUESTION (The following question should be answered in your laboratory notebook.) Column chromatography is used to separate compounds in a mixture. Why is TLC analysis performed on the fractions collected during the column? How is this information useful prior to HPLC analysis?