Separate to identify
Introduction:
A short while ago I was assigned to a large chemical plant called Chemcalequip. The plant was
often asked to identify substances and as a part of my test, I had to write a detailed report. In
this report I had to demonstrate my abilities, show the extent of my experience and last but not
least prove that I am qualified for this job. The report will be about identifying components in
mixtures and other solvents by using different chromatographic techniques. It consists of a part
where I am explaining the techniques, analysing and evaluating the results, and lastly
suggestions for some improvement.
Chromatography is everywhere. You may not know that it exists everywhere in our daily life. A
very modern example of chromatography is the covid pandemic. People are busy trying to find
new vaccines and improve them. Chromatography is a helpful and useful method for this case.
It helps to determine which antibodies fight various viruses. This further helps scientists to find
the most effective antibodies to kill the wanted virus. Chromatography was also used to find out
what the food contained. According to the peak page1beef was suspected to contain
manufactured materials. In 2013, chromatography was a traditional method used to discover the
beef’s truth. The people were able to find out if the meat was beef, horse, or mixed meat, which
generally reduced the number of adulterated foods.
When it comes to drug testing, gas chromatography is a commonly used method. A very small
breath can, by using a very big machine, reveal the whole truth. This big machine takes
advantage of the various boiling points of the different compounds in the sample.
Chromatography is also used in forensics and can easily identify the samples from murders,
terrorist incidents, and robberies. It can also be used to know if the dead person died naturally
or was poisoned. The machine separates e.g. blood particles from drug particles and further
drug-like particles from drug particles. This leads to easy visual analysis, as Reginald Davey
mentioned in his article 2.
TLC and paper chromatography works using a mobile phase e.g. solvent and a stationary
phase( paper or plate). The stationary phase allows the mobile phase to carry the molecules
through the paper depending on their size, attraction, and solubility with the mobile phase. This
means that the results we will get are going to be separated dyes in different places in the
stationary phase. Paper chromatography is a separation method of mixtures of coloured
substances. It can be used to identify fingerprints, additives, and purify chemicals. etc.
1 bmcbride (2007). 5 Everyday uses for Chromatography [online]. Available from:
<https://www.peakscientific.com/discover/news/5-everyday-uses-for-chromatography/>.
[Accessed 22/10/21].
2 Reginald Davey (2020). Chromatography and Forensics [online]. Available from:
<https://www.news-medical.net/life-sciences/Chromatography-and-Forensics.aspx>.
[Accessed 22/10/21].
,HPLC is high-performance liquid chromatography. It has a column that contains silica, alumina,
or cellulose powder. The mobile phase is a solvent that moves through the column by high-
pressure pumps. Before the column, there is a sample injector that injects the samples with the
solvent. The column is then attached with a detector that is attached to a computer. When the
molecules are separated data appears on the computer that then will be compared to actual
data to identify the compounds 3. Gas chromatography takes advantage of the different boiling
points that the compounds have. The mobile phase here is the gas, which is kept in a cylinder.
The cylinder is connected with a molecular sieve that removes the unwanted compounds. The
sieve is connected to a column that has the stationary phase packed in the wall of it. The
stationary phase is silicone grease or wax, which withstands the high temperature. The column
is connected with a detector that shows the data. This method works according to the volatility.
The science behind
Thin layer chromatography and paper chromatography are separating methods used to
separate mixtures of substances or coloured chemicals into their components. The stationary
phase in paper chromatography is solid. It can be a paper or plate. These two methods are very
similar and can be used to identify additives in foods, purify chemicals, and know the
components for an unknown substance. A gas or liquid is the mobile phase in paper
chromatography. However, TLC has a thin layer of silica gel or alumina as the stationary phase
and a suitable liquid ( solvent or a mixture ) as the mobile phase.
Jim Clark (2007) named that paper chromatography and TLC work according to size, solubility,
and partition 4. Paper chromatography works because of different solubilities. The more soluble
substance travels further up than the less soluble substance. Paper is made of cellulose fibres
attracted to water molecules that are polar from the atmosphere. Against that background, polar
solvents will have a high attraction for water molecules and will dissolve near the baseline. That
entails a smaller Rf value than paper with non-polar solvents. Non-polar solvents do not have
the same attraction and will travel a long way up the paper leading to a larger Rf value.
Regarding TLC, it works in the same way as paper chromatography. Silicon molecules in the
stationary phase are attached to -OH groups. The stationary phase, therefore, is polar. That
means that TLC and paper chromatography have hydrogen bonds in them. Different
compounds travel at different rates because of their different solubility in the solvent. Non-polar
solvents will travel further up than polar solvents because polar solvents have attraction
between the compounds and the solvent adsorption 5. High adsorption that exists in polar
3 Quick Biochemistry Basics (2019). HPLC | High performance liquid chromatography.
[online]. Available from: <https://www.youtube.com/watch?v=ZN7euA1fS4Y>. [Accessed
22/10/21].
4 Jim Clark (2007). THIN LAYER CHROMATOGRAPHY [online]. Available from:
<https://www.chemguide.co.uk/analysis/chromatography/thinlayer.html#top>. [Accessed
22/10/21]
5
Jim Clark (2016). PAPER CHROMATOGRAPHY [online]. Available from:
<https://www.chemguide.co.uk/analysis/chromatography/paper.html>. [Accessed 22/10/21].
,solvents leads to a low Rf value, but with non-polar solvents, the same attraction that gives a
larger Rf value does not exist.
The main advantage of paper chromatography is its cheapness. It’s easier to perform with lower
costs than TLC. It also requires fewer samples and less space for the setup. Paper
chromatography can separate water-soluble compounds, e.g. ink. A very good thing is that the
result can easily be detected by the naked eye, not as in TLC where you can not see the result
with the naked eye without using something that reacts with the compounds on the stationary
phase. Paper chromatography also offers some disadvantages, namely that the data you get is
not as accurate as it is in TLC.
Paper chromatography can not accommodate a large number of samples and separate complex
substances. Similar to TLC there are limited types of mobile phases that can be used. When it
comes to TLC advantages, it provides accurate results in less time. It’s a faster method to use
and where ninhydrin is used to separate the colourless substances. A disadvantage is that only
nonvolatile compounds like sugar are applicable. Volatile compounds can not be used in this
technique.
Experiment 1: Comparison of paper chromatography and TLC of extracted plant
pigments- ivy leaves.
Today’s experiment aims to look at the pigments that different solvents will provide. Additionally,
learn about Rf values and how to identify unknown compounds. The main point for today’s
experiment is to identify which method of TLC or paper chromatography is better to use to
separate different compounds from a solvent.
Hypothesis: What is expected to happen is seeing separated pigments of more than one
colour. The reason for this prediction is that most of the solvents are often not pure, but have
mixed compounds. It’s rare to have a pure solvent for the experiment and propanone is not one
of them, which its formula clearly proves. I know that the TLC plate pigments will have higher rf
values because it’s a quicker separating method than PC.
Variables: In this experiment, we have three main variables: independent, control, and
dependent variables. The dependent variable in this experiment is the Rf values of the pigments
that will be seen separated. The type of solvent used is the independent variable because the
results differ while using different solvents. The control variables are variables that have to be
constant. The temperature of the room, type of solvent, type of the leaves, and the separating
material are some examples of control variables.
, Hazard Risk Precaution Action
The scissor is sharp. There is a risk of Be very careful and If you cut your hand
cutting your hand have all your or finger, the action
when you are cutting concentration when depends on the
the leaves into small you are using the injury. If the cut is big
pieces. scissor. Don’t let you have to cover it
anything distract you. and go to the
hospital. If it's small
you have to clean it
and put a bandaid on
it.
There is glass in this If it e.g. accidentally You have to be If you get a wound,
experiment and they falls from you, it will careful and use the action depends
are hard. break into small goggles to protect on the injury. If the
sharp parts that can your eyes if any part wound is big you
cause deep wounds. flies away. have to cover it and
go to the hospital. If
it's small you have to
clean it and put a
bandaid on it.
Propanone is If the propanone gets You have to use eye If the propanone
flammable and very hot it can cause protection or goggles comes into the eye,
irritant. a fire and if it comes to protect your eyes, flood your eye with
on your skin the first of all. Wear a lab water for 10 min, if
propanone can coat and gloves to you swallow it wash
irritate it. reduce the risk if the your mouth and if you
chemical comes on breathe a high
your skin. Ventilate concentration of its
the room very well to vapour, ventilate the
reduce the room and breathe
temperature. fresh air. For all
these, you have to
contact a medic. If
propanone comes on
your skin, wash it
immediately.
To find the source used in the risk assessment. Search Hazcards for your chemical and use
Cleapss website6.
6
CLEAPSS (2019). Student safety sheets 61 Propanone also known as Acetone [online].
Available from: <https://science.cleapss.org.uk/resource/sss061-propanone.pdf>.
[Accessed 22/10/21].
