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!