Results from the paper chromatography and TLC of extracted plant pigments from paper chromatography of amino acids. An explanation of the principles behind the chromatographic separations. Suggestions for improvements to the chromatographic procedures carried out and full justification of these sug...
Unit 2C - practical scientific procedures and techniques
Paper Chromatography:
The analytical technique known as paper chromatography is used to separate pigments of
substances. Paper chromatography has been replaced as an inexpensive and easily
accessible teaching tool which has replaced Thin Layer Chromatography. To carry out TLC,
it involves using two solvents and rotating the paper in between. It separates complex
substances with similar polarity, commonly, amino acids. Any form of chromatography
consists of a mobile phase and a stationary phase. Mobile phases are the solution that
travels up the stationary phase. A mixture of polar organic solvent with water is the mobile
phase. In addition, paper is used to help the stationary phase. TLC and paper
chromatography are different, the stationary phase of TLC is a layer of silica gel or
adsorbent whereas for paper chromatography it is water.
All polar substances will adhere to the silica showing the travel of the colour up the paper.
Less polar and nonpolar substances won’t adhere to the silica and will travel up the paper
with the solvent being used.
Introduction:
Paper chromatography uses filter paper to identify different substances in a mixture, the
substances dissolve in the alcohol (solvent) and travel up the paper. Heavier substances will
move slower and lighter substances move quicker up the paper.
Aim of the experiment: To find the pigments present in spinach leaves using paper
chromatography
Equipment:
Acetone 1 part water : 1 part acetone
Spinach leaves
Filter paper
Pestle and mortar
Beaker
Ruler
Scissors
Pencil
Coplin jar
Sand
Spatula
Capillary tube
Hypothesis: The filter paper can be used to separate the pigments in spinach leaves.
Risk assessment:
Hazards: Acetone is an irritant and is a highly flammable substance
,Risks: Acetone is flammable, may be harmful if inhaled, ingested and can act as an irritant.
Scissors can cause wounds.
Glassware can be smashed causing injuries.
All of these risks and hazards can affect students, technicians and teachers.
Control measures: Ensure there are no open flames or other ignition sources in the lab.
Ensure full PPE is worn including goggles.
Keep windows open to avoid inhalation of acetone.
Method:
1) Place 3-5 leaves into the pestle and mortar, add one spatula of sand and then add
10 ml of acetone to the pestle and mortar.
2) Grind the mixture together until a liquid solution is made containing the spinach
leaves.
3) Measure the filter paper at both ends 1 cm using a pencil and ruler.
4) Pour some of the spinach mixture into a watch glass, using the capillary tube,
transfer one drop of pigment solution to the start line centre. Wait till the drop has
dried and repeat 5 times.
5) Pour enough acetone into the base of the Coplin jar for it to cover the base.
6) Place the paper into the Coplin jar and do not let the acetone touch the start line, it
must be below. Allow the solvent to rise until it reaches the solvent line (top of the
paper).
7) When the line reaches 1 cm before the finish line remove it from the Coplin jar. Wait
for the chromatography paper to dry completely, record the colours and then
calculate the Rf value for the paper using the formula:
As calculated above, I measured the distance of the solvent travelled and the distance of the
spinach pigment. I then calculated the Rf value for each spot.
I calculated the Rf value of the paper using the formula:
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