Chemistry in Dentistry: Extrinsic Stain Lightening
Research Question: How does increasing concentration of hydrogen peroxide solution (0.0 mol dm-3,
1.0 mol dm-3, 2.0 mol dm-3, 3.0 mol dm-3, 4.0 mol dm-3) affect the initial rate of oxidation of tannin stains on
teeth over 20 minutes (AU s-1), monitored by measurement of absorbance of light at 468 nm using colorimeter?
1. Introduction
During my high school years, the increased amount of work has caused me to sleep later than usual to finish my
assignments. However, my inability to stay awake for prolonged periods of time has caused me to become
dependent on black tea during all-nighters. Yet, I have realised that my teeth are getting increasingly stained
despite brushing my teeth properly, twice a day. Upon further research, I realised that tannin, a substance
present in black tea, was the culprit to my new teeth stains. With a mass percentage of 11.76-15.14% of tannin,
black tea has the highest tannin concentration in all types of teas (Boyers, 2019). On a routine visit to my
dentist, I asked him about common techniques to lighten teeth. He told me that hydrogen peroxide is often used
by dentists to conduct both in-clinic and at-home treatments. Becoming more intrigued, I decided to focus my
Chemistry Internal Assessment on teeth stain lightening by hydrogen peroxide. I will be investigating the
effects of an increasing concentration of hydrogen peroxide solution (0.0 mol dm-3, 1.0 mol dm-3, 2.0 mol dm-3,
3.0 mol dm-3, 4.0 mol dm-3) on the initial rate of oxidation of tannins in tea, with aid of a colourimeter.
2. Background
2.1. Tannins in daily life
Tannins are a class of dark-coloured polyphenols that have the ability to bind to, precipitate or shrink proteins.
Naturally occurring in nearly all plants, the astringency of it gives a puckering feeling in the mouth when
consumed. This compound is a vital component in beverages such as black tea. (Ashok & Upadhyaya, 2020)
For tannins, the monomer is called catechins. Catechins are able to react to form widely popular antioxidants
such as epigallocatechin (EGC) and epigallocatechin gallate (EGCG). These molecules, which are colourless in
nature, react with each other during the fermentation of black tea via enzymatic oxidation and condensation
(Robertson & Bendall, 1983). This forms theaflavins and thearubigins, which are red in colour and primarily
responsible for the staining of teeth by tea (Frey, 2018). In theaflavins and thearubigins, the structural feature
responsible for their characteristic colour is called the chromophore, which are 6-member rings with alternating
carbon-carbon single and double bond that give resonance to the molecule. The promotion of an electron in pi (
π) bond from ground state to a higher energy level, i.e., the pi-star (π *) antibonding orbital requires the
absorption of a certain wavelength of light from the surroundings, causing its complementary colour to be
observed (Soderberg, 2016). Referring to figure 2, the π * orbitals all have a higher energy than the π orbitals.
In theaflavins and thearubigins, the wavelength of light absorbed is about 468 nm, which is a blue colour,
resulting in a complementary reddish-brown colour observed.
With a more complex polymer of catechins, the colour of the molecule deepens (Schwitters, 1995). This is due
to the difference in network of alternating carbon-carbon single and double bond present. With a larger network,
electrons can be more extensively delocalised, which means that the wavelength of light is generally longer and
lower in energy, thus exhibiting coloured properties to the human eye, as the energy of the π * orbital is
decreased when the extent of delocalisation is increased. With reference to figure 1, theaflavins and thearubigins
are larger in molecular size with more alternating carbon-carbon single and double bond than one molecule of
catechin, EGC and EGCG. Thus, theaflavins and thearubigins exhibit a red colour, while catechins, EGC and
EGCG appears to be colourless as the energy difference between π and π *orbital is larger so that light with
wavelength beyond visible light region, i.e. UV light is absorbed.
2.2. The chemistry in teeth stain formation and whitening
My dentist told me that there are two types of teeth stains, namely extrinsic (outward) and intrinsic (inward)
teeth stains. He also told me that teeth staining caused by consumption of black tea is an extrinsic stain. Upon
further research, extrinsic discolouration is usually superficial and a result of consuming tannin-rich food or
beverages, where the tannins may easily slip into the cervices of teeth (“Types of tooth discoloration”, 2017).
Due to the presence of delocalised electrons in the resonance structures of theaflavins and thearubigins, these
stains are regarded as oxidisable due to the fact that these electrons can be easily removed. Chemical
compounds called bleaches often act as oxidising agents to, as the name suggests, ‘bleach’ or decolourise
chromogens. When the theaflavins and thearubigins are oxidised, some double bonds in the chromophore are
broken, producing smaller fragments of organic molecules like EGC and EGCG that do not absorb light from
the visible wavelengths of light (Benckiser, 2016). The complementary colour of the molecules is not observed
by the human eye. As these smaller molecules can be more easily washed away from the crevices of teeth, the
processes outlined above give a whitening effect on the teeth.
As aforesaid, hydrogen peroxide is commonly used for at-home teeth whitening treatments. Thus, hydrogen
peroxide was chosen as the chemical to be studied in this experiment. Hydrogen peroxide is an unstable
molecule, which easily dissociates and gives free radicals in the following equations (Torres et al., 2014):
Initiation of free radical substitution:
H2O2 → H+ + HO2HO2- + H2O2 → HO2⋅ + HO⋅ + OHPropagation of free radical substitution:
HO⋅ + H2O2 → HO2⋅ + H2O
The free radicals of hydroxyl (HO⋅) and perhydroxyl (HO2⋅) are highly reactive as they contain an unpaired
electron. These are the molecules that are responsible for breaking down the double bonds in the chromophore.
Tannins are considered as antioxidants, i.e., they donate electrons to free radicals without becoming free radicals
themselves (Gülçin et al., 2010). Thus, when encountering hydroxyl and perhydroxyl free radicals, tannins are
able to donate some π electrons such that some double bond breaks, resulting in the formation of smaller
molecules. When tannins break down to the extent where they become catechin oligomers or monomers, the
network of alternating carbon-carbon single and double bond in each molecule becomes smaller. The extent of
delocalisation decreases in each molecule, which means that the wavelength of light absorbed to promote
remaining π electrons is generally shorter and higher in energy. When the complementary wavelength of the
absorbed wavelength falls out of range of the visible region of light, the molecules have a colourless
appearance, giving the bleaching effect.
2.3. Rate of reaction
The collision theory states that, for a successful reaction, molecules must collide in correct geometry with
energy equal to or higher than the activation energy. For this investigation, concentration of reactant is studied.
By an increase of concentration of reactant, the likelihood of molecules colliding increases as well. Thus, the
number of successful collisions per unit time increases with concentration of reactants.
2.4. Methodology: colourimetry
A colourimeter is a device that is used to determine colour intensity through measuring the absorbance of light
passing through a sample of solution. It is based on the Beer-Lambert law, which states that the absorption of
light transmitted is directly proportional to the concentration in the sample.
𝐴 = ε𝑙𝐶
Here, 𝐴 is absorbance (in AU (absorbance unit)), ε is the absorptivity (in cm-1 mol-1 dm3), 𝑙 is the optical path
length in cm (in this case, length of cuvette), and 𝐶 is the concentration of the species (in mol dm-3).
In a colourimeter, light of a specific wavelength is passed
through the sample via lenses, which guides the light to the
detector. Then, the colour is analysed by comparison to a
standard, which usually is distilled water, i.e., a colourless
solution. A detector measures the transmittance and a
microprocessor calculates the absorbance with the formula
1
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 = 𝑙𝑜𝑔10 𝑇𝑟𝑎𝑛𝑠𝑚𝑖𝑡𝑡𝑎𝑛𝑐𝑒 . This process is repeated
every second such that change in colour can be observed.
The absorbance of blue light at 468 nm was measured in this
experiment. This is because black tea has a reddish orange to brown colour. As higher absorption of light at its
complementary colour results in higher sensitivity when recording data, the wavelength of the complementary
colour was used.
After all the data points were collected, they were plotted on a graph and a tangent was drawn at 𝑡 = 0 to
visualise the effects of varying concentration of hydrogen peroxide on the rate of oxidation of tannins in tea.
3. Variables
Independent variable: Concentration of hydrogen peroxide solution (0.0 mol dm-3, 1.0 mol dm-3, 2.0 mol dm-3,
3.0 mol dm-3, 4.0 mol dm-3). At-home treatments using hydrogen peroxide are often effective at 3.0 mol dm-3 of
hydrogen peroxide (Frank, 2019). To reflect reality, the above concentrations were used. The concentrations
were achieved from diluting 4.0 mol dm-3 of hydrogen peroxide. Method is provided in procedures.
Dependent variable: Colour absorbance (in AU) of the reaction mixture containing hydrogen peroxide and
English Breakfast Tea at wavelength of 468 nm (blue region in the visible colour spectrum). This is measured
every 2 minutes starting from t = 10 s. This is because, during preliminary trials, I had found out it was
impossible to record the data starting from when t = 0: I had to screw the lid on the cuvette, place the cuvette
into the appropriate slot, close the lid of the colorimeter and press “record” after the transferral of solutions.
This process usually took about 8-10 seconds. Yet, the reaction will already have begun to occur in the 10
seconds where I was unable to record the absorbance due to these physical limitations. Therefore, I am only
taking the data starting from t = 10 s into account, and the data will be recorded in 120-second intervals for the
next 20 minutes. A digital stopwatch will be used to ensure that this process is within 10 seconds.
Controlled variables:
1. Initial temperature of solutions
A higher temperature of hydrogen peroxide solution increases the rate of reaction between tannins and itself by
increasing the kinetic energy of hydrogen peroxide particles such that there is an increased number of successful
collisions. A lower temperature reverses this effect. On deciding the controlled temperature of the experiment, I
had run several preliminary trials to test out the decolourisation effect of hydrogen peroxide of 4.0 mol dm-3
with different temperatures (25.0 °C, 40.0 °C, 60.0 °C, 80.0 °C). In a limited timeframe of 20 minutes, the 80.0
°C set up showed most significant visible difference in decolourisation. By a larger change in colour
absorbance, the percentage uncertainty of the data will be smaller. Hence, the temperature of 80.0 °C was
chosen for the experiment, and the solutions were kept in a 80 °C water bath.
2. Volume of hydrogen peroxide solution used in each trial; Volume of tea used in each trial
Increasing the amount of hydrogen peroxide solution or tea while keeping the overall volume constant (as the
cuvette has a fixed volume) may change the rate of reaction. For example, an increase in the concentration of
hydrogen peroxide will increase the rate of reaction, but the resultant decrease in the concentration of tannins
may decrease the rate of reaction. As the extent is unknown, these variables must be kept constant. As the
cuvette is around 5 cm3 in volume, 2.5 cm3 of hydrogen peroxide solution and 2.5 cm3 of tea was used,
measured and deposited with separate pipettes to avoid contamination.
3. Type of tea
Different types of tea may contain different levels of tannins due to varying tea oxidation conditions. For
example, green tea has the smallest mass of tannins while black tea has the most (Jaime et al., 2014). In
determining which type of black tea should be used, I had selected two black tea brews that are commonly
found in supermarkets, i.e., English breakfast tea and Earl Grey tea. After soaking one tea bag of each type in
200 cm3 100.0 °C water, I had found out that English breakfast tea showed a darker colour. Hence, by selecting
English breakfast tea, the colour difference may be more obvious when conducting the experiment. I had also
chosen to use tea bags instead of tea leaves as the material of the bag may more easily keep the tea leaves
separate from the tea. Thus, type of tea is controlled.
4. Source and batch of tea
Different sources of the same type of tea may lead to a different quantity of tannins due to varying growth and
tea oxidation conditions. Using a tea of higher tannin concentration may cause an overestimation on rate of
oxidation (as higher concentration of tannin may lead to a higher rate of reaction), which affects experimental
results. Thus, tea bags obtained from the same box tea were chosen, controlling type and batch of tea.
5. Wavelength of light used for colourimetry
The value for absorbance of light is different for each wavelength. Changing the wavelength would change the
absorbance value such that correct comparisons cannot be drawn. Thus, the absorbance of blue light at 468nm
(complementary colour of black tea) was measured in this experiment.
Uncontrolled variables:
1. Temperature of sample after it had been put into the colourimeter
As the sample was no longer being actively heated, there would be heat loss to surroundings through
conduction. To minimise this disparity between measurements and the resulting systematic error, the
colourimeter was allowed 5 minutes to cool down in between each measurement such that heat loss in each trial
is more similar.
2. Evaporation of solutions
During heating at a relatively high temperature of 80.0 °C, water molecules could easily evaporate into the air.
For the tea, this may cause an increase in concentration of tannins, as tannins are not evaporated. This may
affect the experimental results by an increased tannin content in the same volume of tea for the set ups done at a
later time. For the hydrogen peroxide solution, this supplied heat may accelerate the rate of decomposition of
hydrogen peroxide into water and oxygen (2H2O2 → 2H2O + O2) in addition to the evaporation of water
(Benckiser, 2016). This may change the concentration of hydrogen peroxide, depending on whether the rate of
water evaporation or the rate of hydrogen peroxide decomposition is quicker. A random error would occur, as
evaporation and decomposition affect all solutions to a similar extent. However, evaporation and decomposition
are natural processes that cannot be stopped. Thus, this is considered a limitation of this investigation. If the
extent of evaporation and decomposition is small, its effect on concentration can be deemed negligible.
4. Apparatus
-
6 beakers (500 cm3)
1 colorimeter (±0.001 AU)
3 cuvettes
1 digital stopwatch (±0.01 s)
1 measuring cylinder (100.0 ± 0.5 cm3)
-
1 pipette (10.0 ± 0.1 cm3)
7 thermometers (±0.05 °C)
1 vacuum-insulated bottle
1 water bath
-
225 cm3 hydrogen peroxide solution
(4.0 mol dm-3)
Tap water (to fill water bath)
575 cm3 distilled water
5. Materials
-
2 Twinning’s English Breakfast Tea tea
bags
- 100 cm3 hydrogen peroxide solution
(1.0 mol dm-3)
6. Procedures
-
For the preparation of tea, according to the instructions on the tea bag packet, it is recommended that 200 cm3 of
water is used for per tea bag, hence giving the ratio of teabag to water as 1:200 cm3. Distilled water was used to
minimise the chance for random impurities in water that may affect the experiment. A vacuum-insulated bottle
was used as keeping the tea at a higher temperature may raise the kinetic energy of tea particles, leading to a
more efficient diffusion of tea into the water.
For the dilution of hydrogen peroxide, different measuring cylinders for water and hydrogen peroxide were used
to avoid contamination. The measuring cylinders were labelled as the two solutions are both colourless, making
it easy to mix up.
The set up with 0.0 mol dm-3 hydrogen peroxide (i.e., distilled water) acted as a controlled set up as it does not
undergo any chemical reaction related to hydrogen peroxide. This enables me to understand that the variation
observed in the dependent variable is due to the change in independent variable (concentration of hydrogen
peroxide) and not other factors (presence of water).
Stage 1: Pre-experiment preparation
1. Put 2 English breakfast tea bags and 400 cm3 of 100.0 °C distilled water, measured with a 100.0 ± 0.5 cm3
measuring cylinder, into a vacuum-insulated bottle and let the tea brew overnight.
Stage 2: Main experiment
2. Calibrating the colorimeter:
2.1. Set the wavelength of light detected to 468 nm.
2.2. Fill cuvette with distilled water.
2.3. Put cuvette into colorimeter and press the calibration button.
3. Preparing a water bath
3.1. Obtain a water bath of 80.0 °C from the laboratory technicians.
3.2. Place a thermometer inside the water bath to ensure the temperature does not undergo major
fluctuations.
4. Pour the tea into a 500 cm3 beaker and place it in the 80.0 °C water bath. Monitor the temperature of the
solutions using a thermometer until it reaches 80.0 °C.
5. Prepare hydrogen peroxide solution (1.0 mol dm-3, 2.0 mol dm-3, 3.0 mol dm-3, 4.0 mol dm-3, 100 cm3 each).
4.1. 4.0 mol dm-3 hydrogen peroxide solution: obtained from laboratory technicians.
4.2. 3.0 mol dm-3 hydrogen peroxide solution: mixing 75 cm3 4.0 mol dm-3 hydrogen peroxide and
25 cm3 water
4.3. 2.0 mol dm-3 hydrogen peroxide solution: mixing 50 cm3 4.0 mol dm-3 hydrogen peroxide and
50 cm3 water
4.4. 1.0 mol dm-3 hydrogen peroxide solution: obtained from laboratory technicians.
4.5. For the dilutions:
4.5.1. Use a 100.0 ± 0.5 cm3 measuring cylinder to obtain a suitable amount of 4.0 mol dm-3
hydrogen peroxide solution.
4.5.2. Use a different 100.0 ± 0.5 cm3 measuring cylinder to obtain an appropriate amount of
distilled water.
4.5.3. Pour the solutions into a 500 cm3 beaker for storage. Each concentration should have a
separate beaker.
6. Place these beakers in an 80.0 °C water bath to control the initial temperature of solutions. Monitor the
temperature of the solutions using a thermometer until it reaches 80.0 °C.
7. Prepare 100 cm3 of distilled water with a measuring cylinder. Pour this into a 500 cm3 beaker and place it in
the 80.0 °C water bath. Monitor the temperature of the solutions using a thermometer until it reaches 80.0
°C.
8. Using a pipette to more precisely control the volume of reactants used, put 2.5 cm3 of tea and 2.5 cm3 of
distilled water at 80.0 °C into a cuvette. Insert the cuvette into the colourimeter and press “record” to
measure the absorption of blue light for 20 minutes. This process should take only 10 seconds (measured by
a digital stopwatch), as the reaction occurs immediately upon contact of solutions.
9. After the 20 minutes, remove the cuvette from the colourimeter. Allow the colourimeter to cool down for 5
minutes.
10. Repeat steps 8 and 9 for 2 more times.
11. Wash and dry cuvettes.
12. Replace distilled water with 1.0 mol dm-3, 2.0 mol dm-3, 3.0 mol dm-3, 4.0 mol dm-3 hydrogen peroxide, and
repeat steps 8 to 11.
7. Photographs
Figure 4: The set-up of the experiment.
8. Ethical, environmental and safety issues
There were no ethical issues related to this experiment.
According to US regulation of chemicals, from the Safety Data Sheet (SDS, or previously MSDS) for hydrogen
peroxide, the chemical poses a threat to aquatic life with a 25% chronic hazard (Hydrogen Peroxide Safety Data
Sheet, 2018). Thus, the hydrogen peroxide leftovers were handed over to laboratory technicians for further
treatment before disposal.
For safety issues, hydrogen peroxide is a substance that is corrosive according to the aforementioned SDS.
Thus, protective gloves, laboratory gown and safety goggles were worn when handling it to protect the skin and
eyes against splashes. Special notice was taken to not swallow or inhale the vapour. However, in such case, it
was noted that the mouth should be rinsed and induce vomiting was not encouraged, while breathing fresh air
until comfortable should be done if discomfort were experienced after inhaling hydrogen peroxide.
In addition, hydrogen peroxide is known to have the ability to intensify a fire with its properties as an oxidising
agent. Thus, it was kept away from hot surfaces and open flames in the laboratory. In the case of a fire, water
spray, foam, dry powder or carbon dioxide was used for extinction.
Also, hot solutions at 80.0 °C were handled during the experiment. Thus, after the experiments, the beakers
were taken out of the water bath and left to cool down before cleaning up. In the case of being burnt, the area
affected would be washed under running tap water for 10 minutes.
9. Raw data
9.1.Qualitative data
To visualise the colour change, 1:1 ratio of hydrogen peroxide solutions of different
concentrations to tea solution, measured and deposited with different pipettes to
avoid contamination, were put in separate test tubes and left for two hours. The
results are as shown in Figure 5.
There is no visible change in the solutions used for the real set-ups, as
the difference in colour is too small to be detected by human eye.
9.2.Quantitative data
In table 1, the data recorded is the absorbance of blue light, which it is a
relative number. The unit for absorbance is denoted as AU (absorbance units).
Concentration
of H2O2 (mol
dm-3)
0.0 (±0.0)
10
130
250
0.965
0.963
0.966
0.963
0.966
0.963
Table 1: Experimental results
Time (seconds ±1)
370
490
610
730
Absorbance (AU±0.001)
0.966
0.967
0.967
0.966
0.962
0.961
0.961
0.963
850
970
1090
1210
0.966
0.962
0.966
0.963
0.966
0.963
0.966
0.963
0.966
0.965
0.968
0.963
0.960
0.963
0.966
0.954
0.964
0.958
0.959
0.957
0.955
1.0 (±0.0)
2.0 (±0.02)
3.0 (±0.02)
4.0 (±0.0)
0.966
0.953
0.954
0.950
0.929
0.932
0.935
0.913
0.914
0.905
0.911
0.909
0.908
0.966
0.939
0.940
0.937
0.907
0.909
0.913
0.884
0.887
0.879
0.879
0.876
0.874
0.966
0.930
0.933
0.927
0.890
0.893
0.910
0.863
0.866
0.861
0.850
0.849
0.845
0.966
0.924
0.927
0.920
0.878
0.881
0.890
0.842
0.845
0.838
0.822
0.820
0.816
0.966
0.919
0.922
0.913
0.864
0.869
0.875
0.823
0.827
0.819
0.799
0.794
0.791
0.967
0.912
0.916
0.909
0.851
0.855
0.841
0.804
0.811
0.801
0.775
0.772
0.769
0.967
0.905
0.907
0.901
0.839
0.841
0.835
0.786
0.792
0.781
0.749
0.746
0.740
0.966
0.900
0.901
0.899
0.828
0.832
0.825
0.769
0.771
0.756
0.729
0.724
0.719
0.966
0.895
0.898
0.898
0.821
0.822
0.818
0.754
0.759
0.751
0.711
0.706
0.700
0.966
0.893
0.895
0.896
0.814
0.815
0.810
0.742
0.748
0.736
0.680
0.691
0.683
For uncertainties related to concentration of hydrogen peroxide of 4.0 mol dm-3 and 1.0 mol dm-3, it is assumed
that the solutions have no uncertainties as they were prepared by the laboratory technicians. There is no way to
know the uncertainties of the solutions without knowing the method of preparation.
For uncertainties related to the concentration of hydrogen peroxide solution of 3.0 mol dm-3 and 2.0 mol dm-3,
the dilution was done by this formula:
)× 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 4.0 𝑚𝑜𝑙 𝑑𝑚−3 𝐻2𝑂2 𝑢𝑠𝑒𝑑 𝑓𝑜𝑟 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 (±0.5𝑐𝑚3)
= 𝐷𝑒𝑠𝑖𝑟𝑒𝑑 𝑐𝑜
−3
3
3
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 4.0 𝑚𝑜𝑙 𝑑𝑚 𝐻2𝑂2 𝑢𝑠𝑒𝑑 𝑓𝑜𝑟 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 (±0.5𝑐𝑚 ) + 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑑𝑖𝑠𝑡𝑖𝑙𝑙𝑒𝑑 𝑤𝑎𝑡𝑒𝑟 𝑎𝑑𝑑𝑒𝑑 (±0.5𝑐𝑚 )
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 4.0 𝑚𝑜𝑙 𝑑𝑚
−3
(
𝐻2𝑂2 𝑢𝑠𝑒𝑑 𝑓𝑜𝑟 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 ±0.0 𝑚𝑜𝑙 𝑑𝑚
−3
.
The uncertainty of the concentration of 4.0 mol dm-3 hydrogen peroxide used for dilution was, as aforesaid,
assumed to be negligible. The volume of 4.0 mol dm-3 hydrogen peroxide used for dilution and volume of
distilled water added has an uncertainty of ±0.5 cm3 as it was measured with the 100.0 ± 0.5 cm3 measuring
cylinder. Thus, the absolute uncertainty of the final concentration of hydrogen peroxide solution should be
calculated with the following formula:
𝐴𝑏𝑠𝑜𝑙𝑢𝑡𝑒 𝑢𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 = 𝐶𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 × (𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑢𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 𝑜𝑓 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 4. 0 𝑚𝑜𝑙
So, for 3.0 mol dm-3, the absolute uncertainty in concentration would be:
0.5
(
)
(
)
−3
0.0
0.5
0.5
1000
3. 0 × ⎡⎢ 4.0 × 100% + 0.075
× 100% + 1000 + 1000 × 0. 1 × 100%⎤⎥ = 0. 02 𝑚𝑜𝑙 𝑑𝑚 (1𝑠. 𝑓.)
⎣
⎦
Similarly, for 2.0 mol dm-3, the absolute uncertainty in concentration would be:
0.5
−3
0.0
0.5
0.5
1000
2. 0 × ⎡⎢ 4.0 × 100% + 0.050
× 100% + 1000 + 1000 × 0. 1 × 100%⎤⎥ = 0. 02 𝑚𝑜𝑙 𝑑𝑚 (1𝑠. 𝑓.)
⎣
⎦
For uncertainties related to time, the colourimeter records time by the second. Thus, the uncertainty is ±1
second. For uncertainties related to absorbance, colourimeter records absorbance corrected to 3 decimal places.
Thus, the uncertainty is ±0.001 AU.
From table 1, the absorbance values for the set ups with concentration of hydrogen peroxide at 0.0 mol dm-3
(distilled water) do not undergo major changes over the 20 minutes in each trial. Thus, it can be assumed that
these set ups do not undergo any chemical reaction related to tannin oxidation. The variation in absorbance
observed in other set ups is due to the change in concentration of hydrogen peroxide and not other factors like
the presence of water.
10. Data processing and analysis
First, using the =AVERAGE function in Excel, the averages of each trial (excluding 0.0 mol dm-3 set up) were
taken. The results are shown in Table 2.
Concentration
of H2O2 (mol
dm-3)
10
130
Table 2: Average values of experimental results
Time (seconds “1)
250
370
490
610
730
850
970
1090
1210
1.0 (±0.0)
2.0 (±1.0)
3.0 (±1.0)
4.0 (±0.0)
0.965
0.933
0.894
0.837
0.952
0.902
0.846
0.789
0.939
0.880
0.818
0.756
0.930
0.862
0.798
0.728
Absorbance (AU“0.001)
0.924
0.918
0.912
0.850
0.839
0.823
0.777
0.758
0.740
0.699
0.675
0.652
0.904
0.808
0.721
0.625
0.900
0.798
0.705
0.604
0.897
0.790
0.690
0.586
0.895
0.783
0.677
0.565
Then, the data were plotted into graphs of absorbance against time. The graph of 0.0 mol dm-3 hydrogen
peroxide, i.e., distilled water, was not plotted in this manner as the data shows no significant change, i.e., it had
not undergone any chemical reaction. Then, these averages were plotted on 4 separate graphs with a best-fit line
plotted. When choosing whether to plot a linear best-fit line or exponential best-fit line, I referred to Panizza
and Cerisola’s literature study done on removal of tannins by oxidation their data indicates that tannin oxidation
“follows a first-order rate”. So, I chose to plot a curved, exponentially decreasing best-fit line over a linear
best-fit line. , meaning there would not be a data point at t = 0. (Sth about not wanting to rely on extrapolation)
The R2 value along with the equation of best fit were shown as well. The R2 value is a statistical figure that
shows correlation between the independent and dependent variables. If the value is closer to 1, it shows a
positive correlation.
Here, all best-fit lines show an R2 value of over 0.9600, meaning that the independent and dependent variables
show a very strong correlation.
Although the exact concentrations of the tannin in the sample is not known, the initial rate of the reaction can be
derived from the graphs above by plotting an exponential best-fit line, then looking for the equation of the
tangent at 𝑡 = 0. The slope of the tangent, i.e., 𝑚 in the equation 𝑦 = 𝑚𝑥 + 𝑐, gives the initial rate of
oxidation of tannin. As rate is always positive, the rate of oxidation of tannin is taken as |𝑚|. The unit for
absorbance is denoted as AU, while the unit for time is seconds. Thus, the unit for rate in this experiment is AU
s-1.
As Excel does not have a tangent-plotting function, the equations were taken to GeoGebra for tangent-plotting.
For
example,
for
Graph
1
(𝑦 = 0. 959𝑒
−6Ε−5𝑥
),
the
equation
of
tangent
−5
-1
𝑦 =− 0. 0000575𝑥 + 0. 959. Thus, the rate is |− 0. 0000575| = 5. 75×10
at
𝑡= 0
is
AU s , meaning for every
−5
second, the 5. 75×10 . Hence, the oxidation rates for each concentration of hydrogen peroxide are:
−5
5. 75×10
−5
AU s-1 for 1.0 mol dm-3, 9. 51×10
−4
and 2. 844×10
−4
AU s-1 for 2.0 mol dm-3, 1. 888×10
AU s-1 for 3.0 mol dm-3
AU s-1 for 4.0 mol dm-3.
11. Uncertainties
The uncertainty in this experiment follows this equation:
𝑀𝑎𝑥𝑖𝑚𝑢𝑚 𝑟𝑎𝑡𝑒−𝑚𝑖𝑛𝑖𝑚𝑢𝑚 𝑟𝑎𝑡𝑒
𝐴𝑏𝑠𝑜𝑙𝑢𝑡𝑒 𝑢𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 =
2
The maximum rate and minimum rate need to be found by plotting all three trial data sets from each
concentration of hydrogen peroxide from Table 1 into graphs of absorbance against time. Then, the equations of
best-fit are plotted, and the slopes of the equations of tangent at 𝑡 = 0 are found.
For example, for 1 mol dm-3 hydrogen peroxide:
After calculation in GeoGebra, the initial rate (absolute slopes of equation of tangent at 𝑡 = 0) from Graphs 5,
−5
−5
6 and 7 are 6. 71×10 , 5. 76×10
−5
and 5. 73×10
respectively. Thus, the uncertainty is:
−5
𝐴𝑏𝑠𝑜𝑙𝑢𝑡𝑒 𝑢𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 =
−5
6.71×10 −5.73×10
2
−6
= 2×10
−5
−6
The initial tannin oxidation rate in 1.0 mol dm-3 hydrogen peroxide is reported as 5. 8×10 ± 2×10 AU s-1.
Thus, the initial oxidation rates of each individual set-up (AU s-1), reported initial oxidation rate (AU s-1), and
their uncertainties (mol dm-3) are as follows:
Table 3: The initial oxidation rates of each individual set-up (AU s-1), reported initial oxidation rate (AU s-1),
and their uncertainties under various concentrations (mol dm-3)
Concentration (mol
dm-3)
Trial 1
1.0
6. 71×10
2.0
9. 48×10
3.0
4.0
Trial 2
−5
−5
−4
1. 89×10
−4
2. 85×10
Reported initial
oxidation rate (AU s-1)
Trial 3
−5
−5
5. 76×10
5. 73×10
−5
−5
9. 51×10
9. 58×10
−4
−4
1. 89×10
1. 88×10
−4
−4
2. 84×10
2. 84×10
−5
5. 8×10
−5
9. 51×10
−4
1. 888×10
−4
2. 844×10
Absolute
uncertainty
(AU s-1)
−6
Percentage
uncertainty
(%)
3%
−7
0.5%
−7
0.3%
−7
0.2%
2×10
5×10
5×10
5×10
With the information at hand, graph 8 can be plotted.
Here, the R2 value is at 0.969, which shows a strong positive correlation between initial oxidation rate and
concentration of hydrogen peroxide, i.e., there is an increasing initial rate of oxidation of tannins with an
increase in concentration of hydrogen peroxide in mol dm-3; for every 1 mol dm-3 increase in concentration of
−5
hydrogen peroxide, the rate increases by 7. 00×10
AU s-1.
Due to random errors from uncontrolled variables, fluctuations in data between each trial can be observed.
Thus, the error bars in graph 5 acts as visual representations of the calculated uncertainties as presented in table
3 and are indicators of the precision of data. Overlapping error bars signify that the difference between the data
points is insignificant. From graph 5, there are no overlapping error bars. Therefore, it can be concluded that the
data collected are precise and have a high reliability.
12. Conclusion
The data collected shows that the oxidation rate of tannins increase with an increase in concentration of
−5
hydrogen peroxide, from 5. 8×10
−4
−6
± 2×10
AU s-1 for 1 mol dm-3 of hydrogen peroxide to
−7
2. 844×10 ±5×10 AU s-1 for 4 mol dm-3 of hydrogen peroxide. Due to an increase of concentration of
hydrogen peroxide, the likelihood of hydroxyl and perhydroxyl free radicals from hydrogen peroxide colliding
with the double bonds in tannin increases as well. This gives an increase of successful collisions per unit time.
As antioxidants, tannins can donate some π electrons to the free radicals due to the presence of delocalised
electrons in the resonance structures of tannins that are easily removable. The breaking of some double bonds
causes the tannin to eventually become catechin oligomers or monomers. The network of alternating
carbon-carbon single and double bond in each molecule of catechin oligomers or monomers is smaller than that
in tannins, thus the extent of delocalisation decreases in each molecule, which means that the wavelength of
light absorbed to promote remaining π electrons to the π * orbital is generally shorter and higher in energy.
When the complementary wavelength of the absorbed wavelength falls out of range of the visible region of
light, the molecules become colourless in appearance, giving the bleaching effect that the data collected
suggests.
Based on high R2 value in graph 8 from the line of best-fit, it can be said with confidence that there is strong
positive correlation between concentration of hydrogen peroxide and initial rate of oxidation rate of tannin.
13. Evaluation
Strengths
1. Choosing to measure absorbance at 468 nm
The absorbance of blue light at 468 nm was measured in this experiment. This is because black tea has a reddish
orange to brown colour. As higher absorption of light at its complementary colour results in higher sensitivity
when recording data, this decreases the errors in experiment.
2. Repeated trials
3 trials were done under each temperature set to minimise random errors. This shows high precision of the data
collected, as reflected by the low percentage uncertainty of 0.2%-3%.
Weaknesses
Methodological issues
1. Heat loss after it had been put into the colourimeter
Without constant supply of heat to the solutions in the cuvette, heat was undesirably lost to the surroundings.
This systematic error caused an underestimation of the initial rate as the reactants lose kinetic energy to the
surroundings in the reaction process, leading to lower number of successful collisions per unit time. To improve
this in a situation where time was sufficient, solutions of lower temperature can be used to minimise this effect.
Another possible improvement is to use more advanced colourimeters with heating elements such as the
PFXi-950/P model from Lovibond.
2. Uncontrolled evaporation of water
Water molecules could easily evaporate into the air during heating at a relatively high temperature of 80.0 °C,
which may change the concentration of tannin in tea and concentration of hydrogen peroxide, causing a random
error in my data. However, evaporation is a natural process, and it cannot be stopped. As no apparent changes in
volume of solutions were observed, it was assumed that the effect of evaporation on concentration was
negligible.
3. Unrealistic temperature
Although the experiment aimed to investigate the removal of tea stains on teeth, at-home treatments rarely use
solutions of 80.0 °C, as it would cause damage to pulp vessels in the mouth (Mondelli et al., 2016). 80.0 °C was
only used due to the time limitation. Besides, there was no teeth-like surface for the tannin to adhere to. Thus,
the investigation was limited to the lightening of tannin as a chromogen and may not be completely applicable
to real life situations. To improve on this, eggshells can be used. Having 97% calcium carbonate, eggshells are
similar to the human enamel in terms of chemical composition (Hunton, 2005). Eggshells can be soaked in tea
and a video can be recorded of the decolourisation occurring on the surface of the eggshell. Then, digital
analysis of the colour change on eggshell can be done using software that can analyse colour objectively using
the RBG (Red, Blue, Green) system.
4. Uncertainty of exact components and concentrations of tannin in tea
As the exact components and concentrations of tannin in tea was unknown, it was uncertain if the measured
results were solely due to the oxidation of tannins and not other chromogens such as anthocyanins, which are
red or purple in colour (Kerio et al., 2011). However, it was assumed that the change in colour was due to the
loss of the two major chromogens in tea, i.e., theaflavins and thearubigins, which many research papers agree
with (Boyers, 2019; Jaime et al., 2014). To further improve, solutions of pure tannic acid can be used instead of
a mixture like tea to help assess my hypothesis. Without complications from different substances in the tea, the
change in oxidation rate of tannic acid with change in concentration of hydrogen peroxide can be tested.
Procedural issues
1. Exposure of tea to UV light and air causing undesirable oxidation
Undesirable free radicals from UV light and air may react with the tannins in the tea, causing an
underestimation of the results of the experiment as the reactants in the tea had already been oxidised, leaving a
lower concentration of tannins in the tea for reaction. This systematic error was minimised by completing the
experiment as fast as possible. Also, the tea was stored in a light-proof bottle until the conduction of the
experiment to prevent oxidation. To further improve, the experiment can be done in a room without sunlight to
minimise the chances of oxidation of tea via free radicals generated by UV light.
14. Further exploration
First, instead of just English Breakfast Tea, other types of teas can also be tested. By comparing the time
required for the colour absorbance to decrease to a certain value with a fixed concentration of hydrogen
peroxide, the relative abundance of chromogens in each beverage can be derived and assessed. Other common
teeth staining beverages such as coffee and red wine can also be tested.
Also, this investigation only touched upon the aspect of colour change of tannins that shows its antioxidant
abilities. Other methods of testing for the antioxidant properties of tannins can also be done, such as the FRAP
(ferric reducing ability of plasma) test. While it requires materials that the school does not provide, the test can
potentially provide a more technical, accurate and precise finding (Benzie & Strain, 1996).
Lastly, according to recent findings, casein in milk can bind to tannins to prevent stains, which are proven to be
as effective as whitening treatments (Davies, 2015). Thus, the staining power of pure tannic acid solution and
tannic acid solution with milk on a surface can be compared to affirm this theory, which assists me in drinking
tea daily with minimal staining to my teeth.
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