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1) Aims and explanation of the experiment and Chemical Theories

The aim of this investigation is to find the best way of extracting blue plant pigment from a plant by
chromatography, and finding RF Values in order to deduce the best method and chemicals to use.
The next aim is to determine the pH of the mid-point colour of the blue pigment; this will be done
using a pH meter and titration. Readings will be taken at intervals to measure when the mid-point
colour change occurs.
Background
There are 6 buffer solutions;
1) Pentan-1-ol is an alcohol with five carbon atoms and the molecular formula C5H12O. It is a
colourless liquid.
2) Ethanoic Acid is a weak acid with two carbon atoms and the molecular formula
CH3COOH. It is a colourless liquid.
3) Butan-1-ol is an alcohol with a four carbon structure and the molecular formula C4H10O.
4) Ethyl Acetate is an organic compound with four carbon atoms and the molecular formula
CH3COOCH2CH3. It is a colourless solution.
5) Methanoic Acid is a weak acid with a one carbon structure and the molecular formula
CH2O2. It is a colourless solution.
6) Sodium Methanoate is a compound with a one carbon atom structure and the molecular
formula HCOONa. It is usually a colourless deliquescent solid.

These solutions have a definite pH so when things are added the pH is not changed.

The variety of plant colours is produced by the natural dyes of anthocyanins, carotenoids and
chlorophyll. 1 Each of these absorbs light at different parts of the visible light spectrum; the light
that is reflected gives the plant its colour.
Anthocyanins are responsible for the blue colour of plants. The anthocyanin is bound to an alkali in
the plant3, therefore it is blue.
The structure for an anthocyanin that is blue/purple, Delphinidin, is

4

Anthocyanins are soluble in water and are easily extracted by a weakly acidic solution. The colour
of the plant depends upon the uptake of soil aluminium, but, it is also pH dependent because it
involves the release and capture of hydrogen ions, i.e. HIn H+ + In- 2
Theory
All materials absorb photons (quanta’s of light). Molecules are very selective about what photon
energies they will and will not absorb, they can only exist in certain energy sates. A photon will
only be absorbed if they carry the exact energy the material requires to absorb it, if they absorb
visible light, they will have a colour. It is suggested that the electron confinement may determine
the colour of a plant, for example, the more confinement the more blue light is absorbed,2 therefore
it will look more orange. Electron confinement is when electrons are confined into a smaller space,
this increases the size of the energy spacing levels as part of the atom, this shows that only certain
standing wave frequencies can exist which correspond to certain energy states, therefore when an
atom or molecule absorbs visible light/radiation the electrons become excited and move up one of
these energy states, moving into an electronically excited state.5 But the electron does not stay in
this excited state for long, the energy needed to promote the electrons is re-emitted as visible light
which enables the human eye to see the colour.
Chromatography is used to separate the different components of a mixture. It involves passing a
mixture dissolved in a mobile phase over a stationary phase. Paper chromatography works by
dotting the solution on a pencil line near the bottom of the paper (stationary phase), using a
capillary tube for maximum precision; it is then placed in a solvent (mobile phase) and covered with
a lid. The solution moves up the paper by capillary action, this is the tendency of a liquid to rise up.
It rises up as the attraction of the solvent to the paper (adhesion force) is greater than the attraction
of the solvent to itself (cohesion force). As the mobile phase continues to rise, it takes the compound
solution with it.6 When a component of the solution is absorbed it stops moving with the mobile
phase, and this is why we get the separated colours.
Thin-layer chromatography is a more widely used method for the separation of a compound, as it
is quicker and provides better separations. It uses the same methods as paper chromatography,
except the stationary phase in a thin layer of silica gel; the mobile phase is still the solution the plate
is put in. The solvent rises due to capillary action. Separation is achieved by the space occupied and
bound by either the mobile phase or compound on the stationary phase. Silica gel is polar, so the
more polar solution will be more attracted to the silica, and is therefore more likely to occupy the
space on the stationary phase, this means the less polar solution will move further up the plate,
resulting in a higher Rf value.7
A buffer solution is a solution which can resist changes in pH despite adding an acid or alkali to
it; they have a constant pH. These buffer solutions are made by mixing a weak acid with its
conjugate base, or a weak base with its conjugate acid, this way it is possible to make a buffer
solution with any pH desired. A buffer works due to an equilibrium reaction between the weak acid
and its conjugate base, i.e. HA (aq) H+ (aq) + A- (aq). If more H+ were added to the solution it
would disturb the equilibrium and push it further to the right. A- ions from the salt would react with
these extra H+ ions, forming HA and water, preventing a dramatic fall in pH. In the same way that if
A- ions were added, H+ ions are removed from the solution because the acid produces more H+
which once again prevents a dramatic rise in pH. Both the weak acid and its conjugant bases need to
be present in a buffer solution, this is because there must be plenty of HA to act as a source of H+
ions and A- ions when necessary, these act as a sink if anything extra is added.8
The donation of these H+ ions can be measured using the pH scale. The scale measures how much
acid or alkali is present in a given solution. If the ratio of hydrogen ions is greater than the hydroxyl
ions, the solution is acidic, having a pH that is less than 7. If the hydroxyl ions have a higher ratio to
hydrogen ions, the solution is basic, having a pH of more than 7. If there are equal parts hydrogen
to hydroxyl ions, the solution is neutral with a pH of 7. The ‘p’ in
pH stands for power and the ‘H’ stands for number of H+ ions,
therefore pH means the amount of H+ ions in a solution, and can be
defined as pH= -lg[H+(aq)] 9, where H+ is the concentration.
Techniques
Titration is a method usually used to find an unknown
concentration of a known reactant. However, in order to find the
pH of the mid-point colour change, titration can be used in
conjunction with a pH meter. An indicator is added in order to
visibly see the mid-point colour change or, the end point in an acid- Picture of Titration Practical
base titration. The end-point when finding the mid-point colour
change is when Kin = [H+(aq)] × [In-(aq)] / [HIn(aq)] and pKin =
-log10 Kin . This means that the mid-point colour change occurs when
pH=pKin10. Indicators are weak organic acids that appear different
colours in their dissociated and undissociated forms. It works by
the shifting of the equation, applying Le Chatelier’s principle, for example, if the indicator was
bromothymol blue the excess H+ ions in the solution would shift the equilibrium to the left, making
it appear yellow. When an alkali solution is added the OH- ions will react with H+ ions to form
water, which removes the H+ ions. This shifts the equilibrium to the right in order to restore the
balance, resulting in a colour change to yellow.11
In order to find the mid-point colour change using the titration technique, the equipment should
be set up the same as if doing an acid-base titration. The indicator should be added to the NaOH or
in the conical, it is not important which solution is in the flask or the burette. Draw a table to record
how much is HCl added to the NaOH and to record the pH at those specific amounts. Titrate the
HCl into the NaOH and measure the pH at the amounts decided using the pH meter (see below).
A pH meter measures the acidity or alkalinity of a solution. A pH meter is a combination
electrode and has two electrodes within it; the internal electrode and the reference electrode, which
are made of the same thing. The internal electrode is the one that interacts with the environment, the
reference electrode acts as a
stabiliser. The sensing part of the
electrode is typically a specific glass
bulb at the bottom; this is what goes
into the solution. This is coated both
inside and out with hydrated gel, but
separated by a layer of dry glass.
There is sometimes a small amount Picture of pH meter
of participate sitting in the bottom of
the bulb. It is shaped as a bulb to
encourage the mobility of Na+ ions,
meaning they can diffuse into the
solution being measured, and then the
H+ ions, which indicate pH, can
diffuse into the hydrated gel. The H+
does not pass the gel of the electrode, it is the Na+ ions that do, allowing a change in free energy.
When an ion diffuses from one region of activity to a different region of activity, it changes free
energy, and this is what the pH meter measures. The abundance of H+ is ‘relayed’ to the inside of
the probe by the Na+ ions.12
To begin practically measuring pH using a pH meter, you must first calibrate the meter. This is
done by rinsing the electrode with distilled water. It should then be put into a buffer solution; the
scale on the meter should then be adjusted to match the pH of the solution. The electrode must
always be kept wet to avoid it drying out. When the unknown solution is ready to be measured
simply place the electrode into it, and read off the display on the meter, record on a pre-prepared
table. Rinse the electrode and place back into the storage container. Repeat as necessary.
With the results from the mid-point titration and the pH readings it is possible to calculate Ka
values and a more accurate and precise pH. This will hopefully highlight the best method for
finding an accurate pH. The equation to find the pH of a strong acid is pH= -log[H+]. Hydrochloric
acid is a strong acid because H2O + HCl H+(aq) + Cl-(aq), it fully dissociates in the water. So for
a 0.1M solution the equation would become pH= -log[0.1], pH= 1. The equation for a weak acid is
slightly more complex, Ka = [H+(aq)][A-(aq)] , but it is also necessary to know that pKa = -log(Ka)
[HA(aq)]
Consider that the pH found with the titration and pH meter is 4 and concentration 0.1M.
[H+]= 10-pH, 10-4= 0.0001, [H+] = [A-]
Therefore (0.0001)2/ 0.1 = 0.0000001 –or– 1x10-7 13

With the results from the titration a graph can be drawn of the results to show the trend of pH
values.
2) Methods Used
Equipment used:
• Mortar and Pestle
• Funnel
• 100ml Glass Beaker
• 200ml Glass Beaker
• 25ml Glass Beaker
• Chromatography Paper
• TLC Plates
• Capillary Tube
• Pipette
• 50ml Bulb Pipette
• Conical Flask
• Burette
• White Tile
• pH Meter
• Glass probe

Chemicals used:
• Ethanoic Acid
• Pentan-1-ol
• Butan-1-ol
• Ethyl Acetate
• Methanoic Acid
• Sodium Methanoate
• pH Buffer Solutions ranging from pH 2-12
• Hydrochloric Acid 4M
• Hydrochloric Acid 0.1M
• Sodium Hydroxide 4M
• Sodium Hydroxide 0.1M
• Propanone

Risk Assessment

Hazardous chemical/procedure/equipment Nature of the Control measures to reduce the Measures to be taken in the event of
being used Hazard risks spillage or accident

Ethanol Flammable Wear eye protection Eye contact: Rinse immediately
Remove sources of ignition Skin contact: Rinse immediately
Swallowed: Seek medical attention
Spillage: Apply mineral absorbent to
area and scoop into a bucket and add
water.
Hydrochloric Acid 4M Corrosive Wear eye protection Eye contact: Rinse immediately
Toxic Wear gloves Skin contact: Rinse immediately
Good ventilation Swallowed: Drink plenty of water or
milk. Seek medical attention
Spillage: Evacuate. Apply sand and
neutralise with sodium bicarbonate and
dispose in a sealed bag.
Sodium Hydroxide 4M Corrosive Wear eye protection Eye contact: Rinse immediately
Toxic Wear gloves Skin contact: Rinse immediately
Good ventilation Swallowed: Give 1-2 cups of water.
Seek medical attention.
Spillage: Evacuate. Apply sand and
dispose in a sealed bag.
Propanone Flammable Wear eye protection Eye contact: Rinse immediately. Seek
Harmful Good ventilation medical attention
Remove sources of ignition Skin contact: Rinse immediately.
Remove clothing
Swallowed: Rinse mouth. Seek medical
attention.
Spillage: Ventilate area. Apply sand and
mineral absorbent, dispose of in a sealed
bag.
Buffer Solutions Irritant Wear eye protection Eye contact: Rinse immediately
Skin contact: Rinse continuously
Swallowed: Drink large amounts of
water.
Spillage: Apply sand and dispose of in a
sealed bag
Hydrochloric Acid 0.1M Corrosive Wear eye protection Eye contact: Rinse immediately
Toxic Skin contact: rinse immediately.
Swallowed: Drink plenty of water. Seek
medical attention
Sodium Hydroxide 0.1M Corrosive Wear eye protection Eye contact: Rinse immediately
Toxic Skin contact: rinse immediately.
Swallowed: Drink plenty of water. Seek
medical attention

Experimental Method
The petals of an African violet were crushed with a pestle and mortar. Propanone was added to the
mixture along with sand in order to extract the colour. The mixture was then filtered through a
funnel with filter paper to filter out the debris.
6 Glass beakers of Pentan-1-ol, Ethanoic Acid, Butan-1-ol, Ethyl Acetate, Methanoic Acid and
Sodium Methanoate were set up with the top covered up. A pencil line was drawn along the bottom
of six sheets of chromatography paper; a capillary tube was used to spot the dye on the paper. They
were then placed in the buffer solutions in the beakers. The solutions were observed as they soaked
up the paper, just before or when the solution stopped, the paper was removed and a pencil line was
drawn where the solution ended. Notes were taken on the results.
Another 6 beakers of Pentan-1-ol, Ethanoic Acid, Butan-1-ol, Ethyl Acetate, Methanoic Acid and
Sodium Methanoate were set up with the top covered up. A capillary tube was used to spot the dye
onto the plate, but this time, TLC plates were set in the solutions. When the solutions had soaked up
the plate and stopped they were taken out and a pencil line was drawn at the ending line. They were
then covered in sticky-back plastic to preserve the plates.
Eighteen test tubes were collected and set up in two rows of nine in separate test tube racks, with
an extra large one in the end. One rack was labelled ‘X’ and one was ‘Y’. They were positioned so
that it was possible to see through both sets when looking through the front. Each test tube was
initially filled with 10cm3 of distilled water. One drop of 4M hydrochloric acid was added to the
larger test tube in rack ‘X’, this had the dye in it; the tube was then shaken up. A note of its colour
was taken. After, one drop of sodium hydroxide was added to the larger tube in rack ‘Y’, this had
the dye in it, the tube was then shaken up and a note made of the colour. A pipette was then used to
place an increasing amount of drops of each solution into its corresponding rack, for example, one
drop of HCl was added to test tube ‘A’ in rack ‘X’, two drops in tube ‘B’ in rack ‘X’ etc, until nine
drops were added to the last test tube in the row. The same occurred through rack ‘Y’. The test
tubes were then viewed form the front to see which ratio looked most like the mid-point colour. A
note of the colour and ratio was taken.
Buffer solutions of varying pHs were then brought out, the range was between pH 2 and pH 12.
The buffer solutions were placed in test tubes with a pipette and drops from the larger test tubes ‘X’
and ‘Y’ were added to them. A note of the colour changes was taken. The buffer solution that
produced the closest match to the visible mid-point colour was then used as a reference.
A titration procedure was then set up for a simple acid-base titration between hydrochloric acid
and sodium hydroxide. A pH meter was also set up in order to accurately take pH measurements
throughout the titration. The hydrochloric acid was titrated into the sodium hydroxide in 5ml
intervals and the pH recorded after each addition. This would now give and accurate pH for the
mid-point colour change.
Recording
The first recording taken was the chromatograms from the twelve chromatography procedures set
up. Many of the chromatograms from the paper did not show any colour separation; however,
sodium methanoate separated the colours to a degree as did methanoic acid. The TLC plates were
more successful as many of them produced a colour separation, the best colour separations occurred
with pentan-1-ol and butan-1-ol.
The Rf values were calculated as follows:
The formula is A= a
x
Where ‘A’ is the position of the first spot, ‘a’ is the distance between the first line and ‘A’
and ‘x’ is the total distance the solution travelled up the paper or plate.

Paper Chromatography:

Sodium Methanoate
a= 0.4 A= 0.4 = 0.083
x= 4.8 4.8
B= 0.344
C= 0.813

Methanoic Acid
a= 0.9 A= 0.9 = 0.021
x= 4.3 4.3

TLC Plates:
Pentan-1-ol
a= 1.4 A= 1.4 = 0.322
x= 4.35 4.35
B= 0.46
C= 0.851

Butan-1-ol
a= 1 A= 1 = 0.196
x= 5.1 5.1
B= 0.657

The next recording was of the colour of the solutions in the two larger test tubes when an acid and
an alkali were added to them. It was found that in the acidic form, the dye changed from a mid
purple to a bright red, and in the alkali form it went from the same mid purple to a murky green.

Picture of test tube colours

The next observation was the identification of the mid-point colour using pH solutions. The first pH
used was pH 2, this found that there was too acidic as it was very pink. The next pH used was pH 9,
this had too little acid in it as it was turquoise. The next pH to be trialled was pH 3, this was found
to be quite peachy, the correct range of colour. To be certain pH 5 was also trialled, this found to be
the most suitable pH as it was the closest to the mid-point when compared to looking through the
pairs of test tubes. The colour was 30% green and 70% red.
The last observation was when the mid-point colour change occurred during the titration. The pH
5 solution and the test tubes forming that colour were kept and referred to. This colour change
occurred around the pH range of 8.9 and 1.6, after 18-20cm3 of hydrochloric acid was added to the
sodium hydroxide. This is indicated in the table of records taken below:
Amount of 0 2 4 6 8 10 12 14 16 18 20 22 24
HCl added
pH 11.9 11.6 11.6 11.5 11.6 11.5 11 11.1 10.4 8.9 1.6 2 1.9

A graph was also produced to show the trend of pH more graphically, and in order to help predict
and show the pH of the mid-point colour change.

Graph for pH trend

4) Analysing Evidence and Drawing Conclusions/Interpretation
The above graph shows that the pH of the mid-point colour change is 6.5.
In order to prove that this number makes sense the following calculations can be solved:
For a strong acid: pH= -log[H+], H+ is the concentration of hydrogen ions which is the same as the
pH, 0.1. pH= -log[6.5] = 8.13
For a weak acid: Ka = [H+(aq)][A-(aq)]
[HA(aq)]
pH= 6.5 and concentration= 0.1
[H+]= 10-6.5, = X, [H+] = [A-]
Therefore (X2)/0.1=
Conclusions
The experiment and results have shown that the best way to extract blue plant pigment is to firstly
to grind it with a mortar and pestle with a mixture of white sand and propanone, and then to use
chromatography with TLC plates with the mobile phase being either pentan-1-ol or butan-1-ol. This
is because the bonds in the pentan-1-ol and butan-1-ol have weak intermolecular forces, so they are
more easily and willingly going to bond with the silica gel. Pentan-1-ol and butan-1-ol both belong
to the alcohol group which means they are polar, as they so readily bond with the silica gel this
must make them less polar than the gel as they move further up the plate. The chemicals must
therefore occupy less space on the stationary phase.
The investigation shows that the pH of the mid-point colour change of the blue plant pigment is
6.5. This means that the ratio of hydrogen ions to hydroxyl ions is greater which makes the dye
slightly acidic as it has a pH that is less than 7.

5) Evaluating Evidence and Procedures
The experimental procedure was very accurate as the data could be collected to 2 decimal places
and the materials enabled good data collection as they were from broad areas of chemicals, there
was a lot of variety and room for experimentation within the experiment.
Procedural Errors
The determination of the mid-point colour change.
The calculation of Rf values.
Precision Errors
The standard error (SE) for a burette is 0.05cm3, this would make the percentage error (PE) 0.10%
SE of a bulb pipette is 0.06cm3, PE= 0.12%
The error of the pH meter used is 0.5, PE= 4.20%
15cm ruler is 0.1, PE= 2.08%
The percentage error for both the burette and the bulb pipette are very small which makes them very
accurate, and negligible so they did not contribute to a significant amount of error in any
calculations. However the pH meter has a very high percentage error and therefore may have caused
a mistake in reading of results which could result in inaccuracies when calculating and collecting.
The main sources of error were the data that was open to interpretation; the mid-point colour.
This is dependent on who conducted the experiment as some people may see colours differently
from another. The results were not scientific quantities that could be tested properly, it was notary
accurate and it could have been read differently by another experimenter. this may have been
negligible as the same person decided the colour each time, however it is undeterminable as there
were day lapses between colour investigations, therefore there could have been extraneous variables
that were unknown and effected the perception of colour. Another source of error would be the
calculation of Rf values. This is also open to interpretation as it is difficult for another person to see
the lines and spots once the paper and plates have dried. The percentage error for a 15cm ruler is
relatively high as well, so the interpretable measurements are less accurate. One of the main sources
of error is the pH meter as it has a high percentage error. Considering the meter was a vital part in
the experiment and which gave the most significant results, it is surprisingly inaccurate. The results
could be 4% either way, which is a large amount for such small numbers.
The burette and the bulb pipette have very low percentage errors and therefore are negligible,
they do not present an accuracy problem.