CHML211 Exp. 5 Column Chromatographic Separation of Nitroanilines IN-LAB ASSIGNMENT: (EACH STUDENT will record the following data in his/her laboratory notebook in a well organized manner during lab period. Yellow carbon copies will be submitted for grading along with Post-Lab assignment.) …..max score = 20 pts. 6. Data/Calculations (The following information should be recorded directly into laboratory notebook.) a. b. c. d. e. f. g. h. Volume of nitroaniline sample used Amount of silica gel used Volume and identity of all column developing solvent systems TLC diagram(s) including all cm measurements TLC developing solvent system All Rf value calculations HPLC sample solvent identity HPLC vial slot # for each sample submitted 7. In-lab Questions (The following questions should be answered in laboratory notebook.) a. List the 5 steps of the general procedure for column chromatography. b. Column chromatography is used to separate compounds in a mixture. Why is TLC analysis performed on the fractions collected during the column, and how is this information useful prior to HPLC analysis? CHML211 Exp. 5 Column Chromatographic Separation of Nitroanilines POST-LAB ASSIGNMENT: (EACH LAB GROUP will submit one copy of a typewritten, paragraph style report addressing all of the points listed below. Must be written using PAST TENSE, PASSIVE VOICE. ) …..max score = 50 pts. 8. Experimental (Write 1-2 paragraphs including all of the following. Do NOT present a bulleted outline.) Describe the process used to prepare, load, and develop the column. Include names and specific volume/mass of any compounds used during this process. Describe the process used to analyze the fractions by TLC. Include name and specific volume of TLC developing solvent used, method of detection used to visualize spots, and how particular fractions were selected to combine for further analysis. Describe process of preparing the compounds for HPLC analysis, including solvent used for HPLC sample preparation and actual HPLC vial slots used. Describe the process of analyzing the HPLC results, including how compounds in each sample were identified, and how the purity of each sample was determined. 9. Results (Complete tables, then copy/ paste the completed tables into your document.) Standard Rf Value Compound Table 5.1 TLC Results Sample Rf values Fraction Fraction Fraction Fraction #1 #2 #3 #4 Fraction #5 Fraction #6 o-nitroaniline p-nitroaniline Compound Table 5.2 HPLC Results Standard Component #1 Rt Rt HPLC (min) (min) % Area Component #2 Rt HPLC (min) % Area o-nitroaniline p-nitroaniline 10. Discussion (Write 1-2 pages addressing all of the following points.) Based on HPLC analysis of the o-nitroaniline sample, is there any indication of p-nitroaniline present? Give the name and area % value(s) of any compound in this sample chromatogram. Based on HPLC analysis of the p-nitroaniline sample, is there any indication of o-nitroaniline present? Give the name and area % value(s) of any compound in this sample chromatogram. Based on the statement above, was pure o-nitroaniline isolated? And pure p-nitroaniline? Briefly explain. Include a short comment addressing what could be done differently to improve the experimental results, if repeated. Be sure to attach your HPLC sample chromatograms to your final lab report!