Unit 2C: Undertake chromatographic techniques to identify compounds in mixtures
Unit 19C: Investigate chromatographic techniques to identify components and determine the amount present in samples
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Unit 2: Practical Scientific Procedures and Techniques
Unit 19: Practical Chemical Analysis
U2C: Undertake chromatographic techniques to identify compounds in
mixtures
U19C: Investigate chromatographic techniques to identify components
and determine the amount present in samples
Separation of components of spinach leaves by paper chromatography
Section One -
Scenario
I work for a multinational pharmaceutical business as a junior technician.
I employ Gas Chromatography (GC) and High-Performance Liquid
Chromatography (HPLC) techniques at the company’s UK research centre
to routinely identify chemicals created by the research chemists so that
they, may track their progress toward their research goals.
Every year, a sixth-form student receives a two-week experience
internship from my department. To prepare for this, I will write an
instructional handbook that will provide students with an outline of the
work they will be doing on placement. I will also show you how to make
simple chromatograms for routine analysis.
Real life applications of paper and thin-layer chromatography
Paper
This technique is normally used the analysis in the food industry,
identifying any artificial colours in the food that is produced; this
can range from ice cream to jelly.
Only real colouring agents should be used, this technique is used to
identify the fakes.
TLC
This technique is mainly used in the forensics industry to identify
the presence of drugs in fluids such as blood and urine that is
collected from the scene of a crime.
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Substances such as octadecyl and white silica are used for the
stationary phase to identify certain drugs. [6]
Reference – 6
What factors affect the separation?
Paper
The quality of the paper used.
The concentration and quality of the solvent used.
The temperature of the atmosphere the apparatus is set in.
TLC
The concentration and size of the sample being used.
Size of the TLC plate.
The climate of the room that the experiment is being performed in.
How to show stains of a chromatogram?
TLC
One method to clearly visualise the stains upon the TLC plate is the
process of using iodine vaporisation, iodine is used because it has
the property of high affinity for some unsaturated compounds. [7]
Another way is to use a high intensity UV lamp, with assorted
colour filters, this machine would allow stains to appear that are
difficult to see with the human eye.
Reference – 7
Hypothesis
I should see multiple colours being produced this being traveling upwards
from the origin line. Colours should be bright and visible enough to
calculate Rf values, a full set of Rf values for each visible colour should be
calculated.
Introduction
In this experiment I will be using a process called Paper Chromatography
to separate the different pigments found in spinach, these may include,
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,Louis Stanford
Carotene, Xanthophyll, Chlorophyll a, and Chlorophyll b. These different
pigments should be shown on the chromatography paper, traveling
upwards from the origin line.
Aim
By the end of this experiment, I am expected to use the process of Paper
Chromatography correctly with the equipment provided, and produce a
set of dependable calculated Rf values from the colours that I can identify
from the Chromatogram
Before the experiment begins, I am asked to produce a risk assessment
labelling the risks and methods put forward to reduce risks. For example,
this could be any spillages that occurred along the way, or broken
glassware.
Figure 2 - A photo showing the chromatography apparatus I used for the experiment – Photo taken at 13:26 on 01/02/2022
– From Louis Stanford’s Android
Apparatus and Equipment
Safety –
Goggles
Laboratory coat (specific size, buttoned up)
Gloves – To reduce the chance of amino acids being applied on the
chromatography paper.
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Experiment –
Spinach leaves (stork cut off)
Approximately 3cm3 of the extraction solvent – Propanone
Approximately 3cm3 of the chromatography solvent
Boiling tube apparatus complete with bung and a drawing pin
1 x 5cm3 measuring cylinder (to extract the chromatography
solvent)
Pestle and Mortar
Capillary tube
Chromatography paper (pre-cut for fit into a boiling tube)
Scissors
Sharpened pencil and a ruler.
Boiling tube rack
Stopwatch / timer
Risk Assessment
Hazard Risk Level of Precautionary Emergency
Hazard Measures
Extraction Irritation High Wear a lab Wash eye
solvent could occur. coat. out at
Serious skin Wear eye emergency
and eye protection. eye wash
irritation. Avoid station.
touching Wash skin
liquid. under
water for a
couple of
minutes.
If
swallowed,
swill out
mouth with
water –
seek
4
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