Chromatographic Analysis

Ilagan, Mhariel Anne Denisse, Lacsamana, Marco Sebastian, Lanuza, Reggiana Mae, Latonio, Juan Paulo*
Group 6, 2Bio5
Department of Biology, University of Santo Tomas, Manila, Philippines




Abstract

Chromatography is used to identify different components of certain samples and on
plants, their different pigments. In this experiment, samples of common over-the-counter drugs
and the sili were used as samples. They had different preparations. The analgesics were
subjected to capillary-type of chromatography were the solvent travels up the TLC plate and
the sili extract was subjected to column chromatography. The results for the Rf values of SX,
Asp, Mef, Para, and Mef are 0.3538, 0.5538, 0.6462, 0.5077, and 0.6615, respectively. This
highly suggests that all are of different components. The column chromatography also yielded
different pigments such as yellow, orange, light orange, and dark orange, each coming up after
the sample was subjected to hexane, hexane-DCM, DCM, and DCM-MeOH, respectively.

Introduction
There are a lot of drugs and medications for different types of diseases, some may look
and smell the same but are totally different. One may be for diarrhea while the other one for
migraine. The label on the package of the drug usually tells us what they are and are made of.
One can identify the difference of the drugs using chromatography; the collective term for a set
of laboratory techniques for the separation of mixtures. The various components of the mixture
travel at different speeds, causing them to separate. [1]
Chromatography was first employed by Russian scientist Mikhail Tsvet in 1900. He was
famous for the actual separation of plant pigments such as chlorophyll, carotenes,
and xanthophylls. Since these components have different colors. Further work done by
researchers found that the main principles of Tsvet's chromatography could be applied in many
different ways. [3]
Chromatography also allows for the separation and identification of the different
pigments of plants, which were mentioned above. Different pigments tend to move together
with different reagents, and within the intervals, the change in color can be observed.
This experiment is separated into two: chromatography for commercial analgesics and
for plant pigments. This experiment aims to extract those pigments or colors and determine
the rate in which they diffuse and to what type of reagent they react to.

Experimental

Drugs namely aspirin, ibuprofen, paracetamol, and mefanamic acid, along with a sample X
were crushed and placed on points 1 centimeter apart at the bottom of a TLC plate (as shown
in figure 1), marked and labeled. The TLC plate was then placed in a sample solution and was
let alone for capillary action to happen. The results were then observed under UV light and
recorded.


Figure 1 showing how the samples were placed

The second part of the experiment was for the plant pigments (setup shown at figure 2) . Sili
was crushed together with sand and dichloromethane. The extract was then gathered and set
for hours. The dropper was also prepared. A piece of cotton was placed to stop the silica from
falling along with the liquids and to filter as well. The silica was then added. A few drops of the
extract were then added and was followed by these reagents:
Hexane
Hexane-dichloromethane (1:1)
CH2Cl2
CH2Cl2-MeOH (1:1)
each were added until the presence of another color. The colored extracts are then collected
and recorded.

Figure 2 the setup and the differentiation of colors
(not actual colors; retrieved from http://www.chemguide.co.uk/analysis/chromatography/column3.gif)




Results and Discussion

Sample Rf Value
SX 0.3538
Asp 0.5538
Mef 0.6462
Para 0.5077
Mef 0.6615
Table 1 Table of Rf Values



Rf is computed by


.
The difference of Rf value clearly suggests that these samples are of different
components. The rate of which they diffuse confirms this. Their components tend to bind to the
paper and the solvent diffuses them up. The more bound the sample is, the less likely it would
travel up, resulting into a lower Rf Value (figure 3 roughly shows the outlining of the diffused
samples).[2]


Figure 3


Compound Solvent Color Volume (ml)
1 Hexane Yellow 0.1
2 Hexane-DCM Orange 0.6
3 DCM Light orange 0.5
4 DCM-MeOH Dark orange 0.09
Table 2 Data on experiment 4B: plant pigmentations


Different colors reacted to different solvents. The first color extracted using hexane was
a bright yellow, which yielded 0.1ml. Then it changed to an orange coloration upon addition of
Hexane-DCM, yielding about 0.6ml. Light orange was extracted using DCM only and yielded
0.5ml and finally DCM-MeOH had dark orange coloration of the extract. This means to say that
different pigments dissolve in different solvents, and that there are more than one pigment in a
plant even though only one is visible most of the time.

Conclusion

The different sample drugs all responded to the solution used, meaning to say that they
are reactive to it but at different rates of reaction, hence the different Rf values and distances
theyve reached. This means that SX was the least to react to the reagent and mefanamic acid
to be the most reactive. This is one way to identify drugs and used also in forensics [4].

As for the presence of different colors of extracts from the sili sample, it is proof that
plants do not carry only one pigment, but rather many. Some of this pigments are dormant and
some are only a variation of the main pigment. This is evident in the sili plant, as the common
person sees pepper as red and the extracted colors are within the color range and along the
spectrum and shade of red.

References

[1] Chromatography. Retrieved from http://en.wikipedia.com/wiki/Chromatography#cite
_note-OnlineEtDict-1

[2] Chromatography. Retrieved from
http://www.chemguide.co.uk/analysis/chromatography

[3] Ettre, L.S., Chapters in the Evolution of Chromatography pp. 412-413, 2001

[4] Forensic Toxicology. Retrieved from
http://www.trutv.com/library/crime/criminal_mind/forensics/toxicology/10.html