SEPARATION OF COMPOUNDS USING CHROMATOGRAPHIC TECHNIQUES

Objective:
1. To separate the compounds using chromatographic techniques
2. To test the purify of compounds separated using thin layer chromatography

Introduction:
Chromatography is an analytical technique that are commonly used in separating the mixture of
chemical substances into its individual components. There are many types of chromatography
such as liquid chromatography, gas chromatography, column chromatography, thin layer
chromatography etc.
The term chromatography comes from Greek which kromatos=colour and graphos=written.
Basically chromatography means writing in colour. Mikhail Tswett was the one who discovered
this technique in 1906.

Results:
Total distance of solvent travelled from bottom line in TLC plate = 7.3cm
distance travelled of the substances from the bottom line
Retardation factor, Rf = total distance of solvent travelled from bottom line in TLC plate

Sample Distance travelled by Retardation factor, Rf

substances (cm)

A 5.7 0.78

B 3.2, 5.7 0.44, 0.78

C 3.3 0.45

Pure pyrene 5.7 0.78

Pure m-nitroaniline 3.2 0.44

Discussion:
For this experiment, m-nitroaniline and pyrene which have different polarity was mixtured. In
the column chromatography,the separated mixture was collected. The column chromatography
allow the mixture to separate and being collected individually. Silica is used as stationary phase
in column chromatography. The most polar sample which is m-nitroaniline is retained in the
stationary phase longer. The least polar compound will elute from the column first, then followed
by each compound in order of increasing polarity. The yellow compound is obviously than clear
compound. This means that it must absorb more strongly to the silica gel than the clear solution.
The less polar spent most of its time in the solvent thus, washes through the column much faster.

Pyrene m-nitroaniline

Structure of pyrene shows the delocalized electron in the aromatic ring due to the lower polarity.
The pyrene was stable as the delocalizing electrons are evenly distributed. In addition, pyrene
does not have substituent. M-nitroniline have more substituent that gives more polarity. N and O
atoms are highly electronegative which tends to withdraw the electron from the benzene ring
towards the atoms. In conclusion, the structure of m-nitroaniline induces the polar property of the
compound and hence the polar of m-nitroaniline is higher than pyrene.

In thin layer chromatography, three samples were collected from the experiment. Two levels
were spotted on the TLC plate. Sample A contained pyrene while sample B contained both
pyrene and m-nitroaniline. Sample C contained m-nitroaniline only. By calculating the Rf value
on thin layer chromatography plate, we can conclude that higher polarity gave lower Rf value.

References:
1. Frederick George Mann, Bernard Charles Saunders, Practical Organic Chemistry. 1960
2. The Systematic Identification of Organic Compounds by Ralph L Shriner 8th edition.
3. http://www.chemguide.co.uk/analysis/chromatography/column.html
4. http://orgchem.colorado.edu/Technique/Procedures/Columnchrom/Columnchrom.html