SIC1011

ORGANIC CHEMISTRY
SAFETY AND BASIC
LABORATORY TECHNIQUES

SEM 1 2021/2022

NAME : NOR ATIYAH BINTI IDRIS

MATRICULATION NUMBER: U2103417
EXPERIMENT: C2 SEPARATION BY CHROMATOGRAPHIC
TECHNIQUES
LECTURE NAME: DR. MUHAMAD AQMAL BIN OTHMAN
OCCURANCE:4
Title : Separation by chromatographic techniques
Objective:
✓ To separation of compounds by column chromatography
✓ To visualisation of separation by TLC using UV-detection
Result :

Flask 1 2 3
Colour of liquid colourless Colourless Yellow

Figure 1: thin layer chromatography of C1 – F3 Figure 2: thin layer chromatography of

Under naked eyes C1 -F3 under UV light at long wave
Table 1 : the result of 𝑅𝑓 𝑣𝑎𝑙𝑢𝑒 of spot C1 , C2 and F1 – F3

Compound C1 C2 F1 F2 F3
Distance travelled 6.7 4.4 6.7 6.5 4.5
by substance ,x
(cm)
Distance travelled 8.0
by solvent front, y
(cm)
𝑥 0.8375 0.55 0.8375 0.8125 0.5625
𝑅𝑓 𝑣𝑎𝑙𝑢𝑒 ,
𝑦

ANALYSIS AND DISCUSSION

In analytical chemistry, chromatography is a process for separating components of a

mixture. To get the process started, the mixture is dissolved in a substance called the mobile
phase, which carries it through a second substance called the stationary phase. Thermo
Fisher Scientific - Site Down. (2019, October 17). The types of chromatography is column
chromatography and thin layer chromatography. Column chromatography method for
separating mixtures of substances in which a liquid or gaseous solution of the mixture is
caused to flow through a tube packed with a finely divided solid, which may be coated with
an adsorbent liquid, or through a long capillary tube bearing a thin film of adsorbent liquid;
the components of the mixture separate because they travel through the tube at different
rates, determined by the degree to which each is retarded by interacting with the stationary
material.
Column chromatography can divide by two which is mobile phase and stationary phase.
The mobile phase is an inert gas that the sample is injected into that will carry it through the
stationary phase, which is normally a solid. The hexane solution is analyzed as it the mobile
phases interacts and moves through the stationary phase. (WorkplaceTesting. (2018, May
25)). The role of mobile phase is transport of the sample through the separation column and
subsequently to the detector for identification of the separated component. TLC plate in
chromatography is thin layer chromatography (TLC) is an affinity- based used to separate
compound in a mixture. In TLC, the stationary phase is thin adsorbent material layer like
silica gel. TLC is a chromatography technique used to separate non- volatile mixtures. Thin
layer chromatography can be used to monitor the progress of a reaction , identify
compounds present in a given mixture , and determine the purity of a substance.
(Application of thin layer chromatography for qualitative analysis of gunpowder in purpose
of life prediction of ammunition. (2019, January 9)).

There are 5 spot of TLC plate. From 5 spot only 2 spot can see with naked eyes but
another can see under UV light in long wave . The first spot is pyrene (C1) , second spot
consists of m-nitroaniline (C2) , the third is fraction 1 and forth is fraction 2 the last fifth
consist fraction 3. The fraction 1 and fraction is the same compound. Fraction 3 is the
different compound but have similar 𝑅𝑓 𝑣𝑎𝑙𝑢𝑒 with C2.

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒

𝑅𝑓 =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡
 The use of 𝑅𝑓 used to identify unknown chemicals if they can be compared to a range of
reference substances. The 𝑅𝑓 value of C1 is 0.8375 and the 𝑅𝑓 value of C2 is 0.55 .For
this result 𝑅𝑓 value of C1 higher than C2 , so the C1 less polar that C2. This is because
pyrene less polar than m-nitroaniline. The reason is the structure of pyrene only
carbon and hydrogen atom present in compound while structure of m – nitroaniline
have nitrogen atom and oxygen. Nitrogen and oxygen have bigger atomic number
than carbon and hydrogen. C1 less polar so it use mobile phase and moves up faster
on the TLC plate. The conclude C1 have large travel distance by the substance.

For the F1 , F2 and F3 , we can see on TLC plate. This show of mixture of pyrene
and mixture of m- nitroaniline. The 𝑅𝑓 value of F1 is 0.8375. The 𝑅𝑓 value of F2 is
0.8125. The 𝑅𝑓 value of F3 is 0.5625. For this result 𝑅𝑓 value of F3 lower than
𝑅𝑓 value of F1, so F3 have higher polar than F1. The higher value of 𝑅𝑓 cause the
compound move slow attracted to the stationary phase and take a longer time on the
TLC plate . The distance travel is the shorter.

From the analysis of 𝑅𝑓 value of F1 is higher than 𝑅𝑓 𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 F2 .the higher

value of 𝑅𝑓 is higher that show the lower polarity of the compound and the faster
move to the stationary phase on TLC plate. The compound less polar are more
attracted to the the mobile phase. From this we can see larger distance travelled by the
substance F1. The 𝑅𝑓 value of C1 and F1, F2 is the same so that mean the compound
is pyrene meanwhile the 𝑅𝑓 value of C2 and F3 is quick near so that mean the
compound is m- nitroaniline.

The error of this experiment is can’t let the column dry because it can cause bubble
in the column , so make sure always put enough solvent. Another than that, always
rinse the capillary tube to make sure the solution do not mix and the result with be
separated spot. Don’t put to much wood cotton because it can hold the solvent
through it. So it put enough for the cotton wood.
QUESTION AND ANSWER

1.

i) The liquid or gas that flows through a chromatography system moving the materials to be
separated at different rates over the stationary phase.

ii) The phase over which the mobile phase passes in the technique of chromatography.

iii) The distance travelled by a given component divided by the distance travelled by the solvent
front.

2.

𝑅𝑓 =

𝑅𝑓 of X = = 0.81

𝑅𝑓 of Y = = 0.31

𝑅𝑓 of Z = = 0.44

3. Suitable chromatography technique to separate mixture of amino acid is thin layer

chromatography. Common amino acid have carboxyl group and an amino group bonded to the same
carbon atom. Each of the differ in their side chain or R groups which vary in structure size and
electric charge. The interaction of the amino acid with the stationary phase like silica varies
depending on their R group. The amino acid that interact strongly with silica will be carried by the
solvent to a small distance whereas the one less interaction will be moved further.

4. In column chromatography, it has wider range of mobile phase compared to TLC. No limit for
quantity as any amount of mixture can be separated by column chromatography. The stationary
phase if TLC is small in size compared to column chromatography. In column chromatography, the
separated analytes can be reused.

5. ortho-nitroaniline>meta-nitroaniline>para-nitroaniline
SUMMARY

Based this experiment we use chromatography method which is column chromatography and thin
layer chromatography to separate compound mixture. If increase 𝑅𝑓 value of the compound so
the compound less polar cause consists of hydrogen and carbon. The result F1 and F2 is
pyrene and F3 is m-nitroaniline.
REFERENCES

1. Aryal, S. (2021, February 4). Column Chromatography. Microbe Notes.

https://microbenotes.com/column-chromatography/
2. WorkplaceTesting. (2018, May 25). Mobile Phase. WorkPlaceTesting.Com.
https://www.workplacetesting.com/definition/1293/mobile-
phase#:%7E:text=The%20mobile%20phase%
3. Application of thin layer chromatography for qualitative analysis of gunpowder in
purpose of life prediction of ammunition. (2019, January 9). International Journal of
Biosensors & Bioelectronics. https://medcraveonline.com/IJBSBE/application-of-
thin-layer-chromatography-for-qualitative-analysis-of-gunpowder-in-purpose-of-
life-prediction-of-
ammunition.html#:%7E:text=TLC%20is%20a%20chromatography%20technique,t
he%20purity%20of%20a%20substance.
4. column chromatography | chemistry. (2016, February 27). Encyclopedia Britannica.
Retrieved December 27, 2021, from https://www.britannica.com/science/column-
chromatography
5. Thermo Fisher Scientific - Site Down. (2019, October 17). Thermo Fisher
Scientific. Retrieved December 27, 2021, from
https://www.thermofisher.com/blog/ask-a-scientist/what-is-chromatography/