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Assignment 3 Unit 2 - Practical Scientific Procedures and Techniques
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Unit 2 Assignment CAimee Baish FSC year 1
Chromatography
Introduction
Chromatography is the separation of mixtures or samples, and when analysed we can see which
substances are in the mixture. Chromatography has many different types, but the most common
ones are thin layer chromatography (TLC), paper chromatography and gas chromatography. Thin
layer chromatography and paper chromatography are very similar but have some slight differences.
TLC is where you have a small strip of paper which is coated in a substance like silica, and then you
put a drop of the mixture you are analysing on a dot about 1cm from the bottom of the paper. Once
this has been done, you pour your solvent (the mobile phase) into a McCartney bottle, make sure
that the amount of solvent isn’t going to go above the pencil line at 1cm from the bottom of the
paper. After around 10-20 minutes, the solvent should have moved all the way up the paper and
carried the substances with it, now you have a chromatogram. You can now use this to work out the
Rf values. Which can tell you what the substances are. Paper chromatography is where you draw a
pencil line on the paper around 2.5cm from the bottom. (make sure the paper is horizontal) Then
draw around 4-5 dots about 2.5cm apart. When you have put your 4-5 mixtures/ samples and your
unknown sample on the paper, then make a cylinder (without the bottom overlapping) and
paperclip it at the top. Then pour your solvent in the 250ml beaker, again make sure that the
amount of solvent is not going above the pencil line on the paper. When putting the paper in the
beaker make sure its not touching the sides of the beaker. After around 20-30 minutes take the
paper out, take off the paperclip and leave it to dry. When dried then spray the ninhydrin on the
paper (if needed). After that, find your Rf values and then find out which substances you have. Gas
chromatography works by putting your sample/mixture into a column, which is coated in an aqueous
solution on the inside, and then when put into the machine, the sample will eventually separate and
stick to the column wherever it travels to. This is what gives us the reading and tells us what
substances they are.
How does chromatography work?
Chromatography works by separating samples into the different substances that make it up. For
example, separating a colour into the different pigments that make up that colour, you can also use
chromatography to show which substances are in a substance by putting the unknown and known
samples on a paper and then comparing them. Chromatography is carried out by using a stationery
phase, which is what you put the sample on, and a mobile phase, for example a solvent, which will
carry the substances along the stationary phase. In the case of Thin Layer Chromatography (TLC), the
stationary phase is the TLC paper and the mobile phase is the solvent, which in our case is a
cyclohexane, propanol and ethyl-ethanoate solvent mixture. The cyclohexane, propanol and ethyl-
ethanoate solvent mixture is used in TLC rather than another solvent because it is a mixture of polar
and non-polar solutions, and the TLC paper is usually coated in a non-polar substance like silica,
which means that the mobile phase can move along the TLC paper quite quickly, especially if it’s a
polar solvent and it can separate the substance fully. However, TLC can work both ways, where the
stationary phase is polar and the mobile phase is non-polar however, this is rarer. In Paper
chromatography the stationary phase is the paper, and the mobile phase is Propan-1-ol. However,
when using paper chromatography for amino acids, ninhydrin is also used. Ninhydrin is used to make
the clear amino acids, visible by reacting with the amino acids and making a pigment. Propan-1-ol is
, used as the solvent because as it is a polar solution the mixture will be separated, it also evaporates
quickly meaning that it will dry quickly, which also means that ninhydrin can be applied shortly after.
In gas chromatography the stationary phase is the column which you put the sample into, which is
covered in an aqueous solution on the inside, which is the mobile phase, when the column has the
sample inside, it goes into the machine and then similar to paper chromatography and TLC, the
different substances stick to different parts of the inner column. This is what tells us what substances
make up the sample. However in gas chromatography, if the column is filled with a solid stationary
phase instead, then, as the mobile phase with the mixture in it, passes through the column, the
stationary phase will grab onto the different substances at different parts of the column, this shows
us what each substance is. However, another way to describe this is that the molecules in the
stationery phase grip onto the substances in the test sample, as the sample is travelling through the
column, this is called absorption.
Safe working practices
When performing the experiments, we must follow safety precautions. We must wear lab coats to
protect our skin and our clothes, this prevents our clothes being stained or discoloured. However, it
also prevents our skin from getting burnt, cut or irritated. Wearing goggles protects our eyes from
getting irritated if a chemical splashed into your eye or you have chemicals on your hands and forget
to wash them then rub your eyes. Wearing gloves meant that if the solvent spilled it wouldn’t
encounter your hands as that could cause chemical burns. We also kept the windows open to make
sure the room was well ventilated to prevent inhaling too much of the solvent because it can make
you lightheaded or drowsy. We also had the lab technicians add the ninhydrin to the paper
chromatography for us, because the ninhydrin is more potent than the solvents and we had limited
ventilation, so we made sure it was done safely by having the lab technicians do it.
Rf values
Thin Layer Chromatography (TLC)
Length of TLC paper- 5.25cm
1st dot- 0.7cm
2nd dot- 2cm
3rd dot- 3.8cm
1st dot- Rf= 0.7/5.25= 0.13
2nd dot- Rf= 2/5.25=0.38
3rd dot- Rf= 3.8/5.25= 0.72
When comparing my values to this table (figure 1). I can see that my 1 st dot was most like
anthocyanin as the Rf value for anthocyanin was 0 and the Rf value for my 1 st dot was 0.13. I can also
see that my 2nd dot was closest to Xanthophyll as the Rf value for my 2 nd dot was 0.38 and the Rf
value for xanthophyll was 0.35. I also was able to notice that my 3 rd dot was closest to chlorophyll A,
I came to this conclusion as the Rf value for my 3 rd dot was 0.72 and the Rf value for chlorophyll A
was 0.52.
Paper chromatography
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