DETERMINATION OF CAROTENOIDS AND CHLOROPHYLL BY

THIN LAYER CHROMATOGRAPHY

RESULT:
Solute Methanol Acetone Hexane
Distance of
solvent
front travelled,
start
finish (X)
travelled
Distance travelled (Y)
Rf (Y) Rf
travelled (Y) Distance
Distance
Carrot
Mangosteen
skin
Marker

Calculation:
Discussion:
Chromatography can be classified into two, column and plane chromatography. In this practical
session, we use planar chromatography (TLC). In this experiment, mangosteen skin carrot and
marker were extracted through column chromatography and the extraction was analysed by thin
layer chromatography (TLC). We made these three samples separately. For the first material, we use
carrot. As for the solute, we use acetone. Based on our observations, we can notice that acetone is
in contact with the stationary phase, some or all of the components of the mixture are adsorbed on
it.

When additional carrot pigments come in, some or all of the mixing dissolve and continue to move
until the acetone is almost completely adsorbed, we lift the chromatography layer (TLC) and
continue to measure the distance travelled by solvent and distance travelled by sample. After
making an estimate according to the formula....... we get the retention factor (Rf) for carrot is 0.53.
For the second sample, we used mangosteen skin. Just like the carrot, we did the procedure but we
used a different methanol solvent and thin layer chromatography for each sample. The retention
factor (RF) we got from mangosteen skin is 0.82.

Finally, the sample we use is a marker. For this sample we use hexane solvent. Because hexane does
not react too much with the sample, the marker is not like the other samples. We got results that
were not encouraging for the markers. The distance travelled by sample is very short which is 0.5cm
compared to carrot and mangosteen skin which can be 2.5cm and 3.7cm long. So that the distance
of solvent is almost completely adsorbed.
Finally, after making calculations, the retention factor for the marker is only 0.13. In conclusion,
among the three samples, mangosteen skin has the highest retention factor compared to the marker
that has the lowest retention factor that means higher retention factor the interaction between the
compound of interest and the surface is strong. A low value of the retention factor means the
interaction between the compound of interest and the surface is weak.

QUESTION
1. Identify which one is solid phase and mobile phase in TLC?

Solid phase – stationary phase

Mobile phase – moving phase

2. What is the Rf value for each of the pigments in your TLC plate?
Carrot – 0.53
Mangosteen skin – 0.82
Marker – 0.13

3. Based on Rf value obtained, please explain what is happening in your sample during
separation process in TLC and why?
As soon as the solvent is in contact with the stationary phase, some or all of the components of the
mixture are adsorbed on it and when additional mobile phases come in, some or all of the mixing
will dissolve and continue to move. This process of adsorption/solution continues throughout the
period of the column. The mixture will be separated in the column and its various components
will appear at different times. The three samples have different retention factors due to the
different components in the mixture having different attractions to the stationary and mobile
phases. The different attractiveness is due to the properties of the different components in the
mixture.

4. Which solvent mixture has the highest elution strength? State your answer why.
Hexane. Because hexane can produce good recoveries of low levels of semi-volatiles and often
gives cleaner extracts. Other than that, hexane also is a popular solvent for extraction as its
unique properties make it efficient and effective to use. The main reason behind hexane
being the preferred chemical for extractions is because of its high evaporation rate and
low boiling point.