INVES. on the light emission produced in the oxidation reaction and chemiluminescence of Luminol

Updated: Nov 19, 2019


Introduction

Luminescence on it’s own is the spontaneous emission of light in the form of cold body radiation from a substance. Such emission of light can occur due to stress of a crystal, electrical energy, subatomic motions as well as chemical reactions (5). Due to the light being emitted by all these factors except heat, is exactly what separates luminescence from Incandescence, being light emitted as a result of heat. Other luminescence exist such as the bioluminescence, electroluminescence, mechanoluminescence, phosphorescence, thermoluminescence, fluorescence and photoluminescence.

(9&10)The substance I would like to focus on that emits light, is a chemical one, which in turn is a chemiluminescent. Chemiluminescence is the emission of light from a chemical reaction between two chemicals, forming a high-energy excited intermediate. The chemical will in turn want to go back to ground state, resulting in the release of its energy in the form of photons.

A + B ⇒ AB* ⇒ Products + Light

There are many fields that use chemiluminescence and its light emission to advantage and the one I am mostly interested in, is in the Forensic field. In Forensic chemistry, Luminol is used to detect blood at crime scenes (1). Luminol is then diluted with a bit of Hydrogen Peroxide to then spray on where blood is assumed to be. Iron, found in the Hemoglobin acts as a catalyst to the reaction and in a dark room glows blue for about half a minute. Considering Iron acts as a catalyst, not much of it is needed for a reaction to occur and so even non-visible blood can be detected, giving a good response. The reaction and its use in such sensitive situations are what really excites me and in generally how science helps solve mysteries.

Another field I am also interested in is the Bioluminescence (8), being production of chemiluminescence in living organisms. The most famous example being the organism “Firefly” with the biochemical reaction being the action of an enzyme with the substrate “Luciferin”, which in turn emits energy rich photons. Other organisms that produce bioluminescence are some algae and some bacterias as well.

Background information

The main chemical I am to investigate is Luminol otherwise known as (IUPAC name: 5-amino-2,3-dihydro-1,4-phthalazinedione). Its cyclic aminophthalhydrazide form is due to 3-nitrophthalic acid and hydrazine. (6) When Luminol is given to react with an oxidizing agent, the phthalhydrazide oxidizes from the molecule and, in turn the cyclic shape breaks as Hydrogen gas, Nitrogen gas as well as Phthalic acid are produced as products and depending on the oxidative agent used, other products could also be produces.

In this case, (2) it is partly the gaseous oxygen O2 and partly Hydrogen Peroxide H2O2 that are the oxidizing agents. Once the shape of the cyclic breaks due to it getting oxidized, an energy rich and unstable state which is returned back to the ground state through multiple steps, with the last one being the emission of energy in the form of photons. Luminol powder is mixed with liquid Hydrogen Peroxide, a Hydroxide which in my case is Sodium Hydroxide and potassium ferricyanide as a catalyst. Instead of Iron as a catalyst in forensic investigations, Potassium Ferricyanide acts as the catalyst, accelerating the chemiluminescence of the reaction. In this investigation, Hydrogen Peroxide oxidizes the luminol molecules, being the cause of an energy rich and unstable state which is returned back to the ground state through multiple steps, with the last one being the emission of energy in the form of photons.

In a alkaline solution (4) luminol is found in equilibrium with its anions charged -2. These anions exist in two tautomers, with the electrons delocalised on either side of oxygens being the enol-form or on the nitrogen being the ketol-form. O2, once combined with the enol-form of the luminol anion, oxidises it, creating a cyclic peroxide. (3) Essentially, oxygen is produced in this redox reaction with reactants Hydrogen Peroxide, Sodium Hydroxide and Potassium Hexacyanoferrate (K3[Fe(CN)6). In this reaction, the Hexacyanoferrate(III) ion ([Fe(CN)6]3-) reduces to Hexacyanoferrate(II) ion, making a Potassium Ferrocyanide K4[Fe(CN)6]) and the two oxygen atoms from Hydrogen Peroxide oxidise from -1 to 0.

From the reaction mechanism above and to the side, the reaction is presented and can be broken down into three steps. As mentioned to exhibit a blue-like luminescence from luminol, the luminol must be activated by an oxidizing agent such as H2O2 as well as Hydroxide ions in the water present due to Sodium Hydroxide breaking down as the activators. (7) NaOH as well as Hydrogen Peroxide added to


the reaction, cause luminol to reach an excited stage with product N2 and H2O, and the total reaction rate increased using a catalyst such as Potassium Ferricyanide (C6N6FeK3).

Luminol will in turn react with the Hydroxide ions, forming a dianon and the oxygen produced from H2O2 will in turn also react with the luminol dianion. The unstable and excited organic peroxide formed is due to the loss of a nitrogen molecule, change of its electrons from excited state to ground state leads to the emission of energy as photons. It is due to the oxidizing agent that breaks the very weak bond between the two nitrogen atoms in the Luminol compound creating N2 (g) as product and leaving an energy rich molecule that in turn emits blue glow.

In conclusion, it is the presence of the catalyst that causes H2O2 to decompose, forming O2 and H2O, It is NaOH inclusively with H2O2 that create an excited 3-aminophthalic acid *, unstable organic peroxide which by time goes back to its ground state releasing photons in a bright blue colour ranging in around ~ 490–450 nm of wavelength.

Research question

A further and more explanatory research question for this investigation would be;

  • Investigation of a variety of hydrogen peroxide and sodium hydroxide concentrations on the intensity and duration of the light emissions produced in the oxidation reaction and chemiluminescence of Luminol using Hydrogen peroxide and Sodium Hydroxide.

Hypothesis

I assume that by increasing the concentration of Hydrogen peroxide, a trend of higher luminosity will be shown. Meaning, when the concentration of hydrogen peroxide is the least in the reaction, the luminosity will be the least and when the concentration is the highest, the luminosity will be the highest.

When it comes to the concentration of Sodium Hydroxide, I predict that since the Hydroxide ions needed to activate luminol (together with Hydrogen Peroxide) comes from this molecule, that as the concentration increases, so will the luminosity and might reach a plateau at one point.

As mentioned in the background information, these two molecules work together to activate luminol to reach an excited state, thus I also believe both of them might show the same pattern and effect on the overall reaction.

  1. The surface of the table was cleaned and all the materials needed placed on it. Two solutions were prepared named A and B in separate beakers with a marker.

  2. Solution A was prepared by measuring 0,5384 g ± 0.0001 of NaOH and 0,1289 g ± 0.0001 of C8H7N3O2 on a measuring scale using a cupcake form and then dissolving it into 50 ml ± 0.6 of water using a measuring cylinder.

  3. Solution B was prepared by measuring 0,3249 g ± 0.0001 of K₃[Fe(CN)₆] on a measuring scale using a cupcake form and dissolving it into 97 ml ± 0.6 of water.

  4. 10 ml ± 0.6 of solution A was later diluted into 40 ml ± 0.6 of water and the solution was named “Solution C”.

  5. 10 ml ± 0.6 of solution B was also diluted in 40 ml ± 0.6 of water in a separate beaker and 0,3 ml ± 0.2 of H2O2 was added named “Solution D”.

  6. Solution D and C were carried on a tray to a very dark room with a computer and sparkvue (which is a software that collects the data from the spectrometer).

  7. Lights were turned off and solution D was mixed with solution C and at the same moment the Sparkvue was turned on and measured Luminescence, % of green, blue and red light.

  8. When the blue light was no longer detectable by the naked eye, the last two trials were performed and then the tray was carried back into the lab, cleaned and a new solution D was made with a different Hydrogen Peroxide volume used.

  9. 3 trials with different volumes of H2O2 were tested, being (0,3 ml - 0,6 ml - 0,9 ml - 1,1 ml) and all of them were measured using a measuring pipette.

  10. When all different Hydrogen Peroxide volumes were tested, then the H2O2 was kept constant and instead the volume of NaOH was changed and tested in three trials. Volumes of NaOH (0,2 ml - 0,4 ml - 0,6 ml - 0,8 ml) were tested.

  11. Once all the data was collected, all the materials waste was poured into a big beaker and the rest were cleaned to be reused.

  12. As too many tables with data were obtained, an average of all the illuminecence and % of green, blue and red light was taken and several graphs and tables were made as processed data.

  13. A slope of the illuminance over time was taken to make the different volumes over illuminance tables and graphs.

Safety

  • Safety glasses and robe is necessary!

Considering many chemical substances used are corrosive/poisonous to the touch, safety glasses are needed as well as robe to protect clothes from getting contaminated. Sensitivity towards the chemicals, danger from breathing in the chemicals too much and allergic reactions can occur. Thus all the materials need to be handled with high care and if skin is contaminated, it has to get rinsed thoroughly and quick.

  • Hydrogen peroxide is a strong oxidizing agent and if it comes in contact with the skin, it will leave burn marks. Thus it is important to handle with safety glasses and if necessary, use gloves.

  • Sodium Hydroxide is corrosive, thus safety glasses are essential. This chemical can be disposed off down the sink, but the others must be poured into the waste beaker and handing it to the supervisor to be disposed off in an environmentally friendly way.

  • Potassium Ferricyanide is not generally poisonous but could release poisonous cyanide if placed near heat. It is also not easily cleaned as spillage on skin and or textile can be difficult to wash out.

  • Luminol is not a compound known to be poisonous but could be rather irritating when inhaled or in contact with the skin.

Results and analysis

Visual Observation

Before the reaction:

  • Solution C was appeared clear and solution D had a tint of strong yellow colour because of the Potassium Ferricyanide and none of them glew in the dark room.

During the reaction:

  • Once one solution was poured into the other in the dark room, the solution immediately glew blue and became more intense for some time before slowly losing brightness and luminosity back to transparent.

After the reaction:

  • The waste was fully transparent and their was no residue of other substances or colours. What made this experiment a bit difficult and can be considered as a limitation is knowing when to stop collecting data using only the naked eye, considering the solution even when it had lost all blue brightness still glowed in the dark. Thus I just depended on the Sparkvue program and once it could not detect anymore data, I stopped the trial and began with a new one.

Conclusion

In my hypothesis, I predicted that as the concentration of H2O2 increases, so will the luminosity and as for Sodium Hydroxide, I believed that as the concentration of NaOH increases, so will the overall luminosity of the reaction and also believed that at one point the luminosity would reach a plateau.

My hypothesis in regards to what effect the change of concentration of Hydrogen Peroxide has on the intensity and duration of the reaction was half wrong. I predicted that as the concentration increases, the intensity would increase and duration would decrease, but the experiment showed the opposite. The intensity and the duration both decreased as the concentration increased. When it comes to the concentration of Sodium Hydroxide, I predict that since the Hydroxide ions needed to activate luminol (together with Hydrogen Peroxide) come from this molecule, that as the concentration increases, so will the luminosity and might reach a plateau at one point.

In regards to my prediction of Sodium Hydroxides effect on the reaction, I was right on the luminosity increasing with increased concentration. However, I did not reach a plateau and thus cannot confirm that part of my hypothesis. A solution to that could be using a higher concentration of Sodium Hydroxide in order to investigate whether a plateau is reached or not.

Thus, I believe by analyzing the data as well as the stepwise chemical pathway provided in the background information, the results can be explained. Sodium Hydroxide is the first step in the chemical pathway, acting as a base and “Modifies” the luminol molecule to lose its hydrogen molecules as well as leaving Hydroxide ions in the solution which are activating agents that work with Hydrogen Peroxide to oxidise and activate the chemiluminescent. If Sodium Hydroxide is increased, creating ready and modified luminol molecules to react and glow, then at one point Sodium Hydroxide will become excess as well as all the modified luminol molecules and so leave a lot more for Hydrogen Peroxide to react with and make the solution glow. Thus as Sodium Hydroxide increases, it is understanding for the luminosity as well as the time duration to increase, since so much more is prepared for Hydrogen Peroxide to react with to glow blue.

Change in Hydrogen Peroxide showed an increase in concentration leading to a decrease in time duration as well as luminosity. This can also be explained through the stepwise chemical pathway showing how it is mainly Hydrogen Peroxide that oxidizes the modified Luminol to an excited 3-aminophthalic acid that emits photons to return to ground state with water and Nitrogen gas as products. Since it is mainly Hydrogen Peroxide that is the last step added to the solution before any glow occurs, if its concentration increases it will decrease both luminosity and time duration since it immediately uses up all the modified luminol. Also, a catalyst is also playing a role here, increasing the reaction rate by lowering the activation energy of the reaction and thus making the reaction to reach completion even faster together with Hydrogen Peroxide.

My average tables (found in section; Appendix B) showed a clear visual effect of Hydrogen Peroxide having a decreased duration and luminosity as the concentration increased and the Sodium Hydroxide showing an increase in duration and luminosity as concentration increased.

Table 1&2 are processed data I derived of the first slope of each of my “Luminosity over Time” graphs with their equivalent concentrations that can be seen in a graph form in graph 1&2. The graphs are meant to show my point on the effect of change in concentration of Sodium Hydroxide and Hydrogen Peroxide on the chemiluminescence of luminol. Graph 1. presents the concentration of Sodium Hydroxide over illuminance, which shows that as the concentration increases, so does the luminosity as well. Graph 2. Presents the concentration of Hydrogen Peroxide, which shows that as the concentration increases, the luminosity decreases. The highest achieved luminosity is with using 0.3 ml of Hydrogen Peroxide.

Graph 3&4&5&6 are all presenting the different percentages of green, blue and red light emitted from different averaged concentrations of Hydrogen Peroxide. Following Graph 7&8&9&10 are all presenting the different percentages of green, blue and red light emitted from different averaged concentrations of Sodium Hydroxide. Hydrogen Peroxide showed a lower percentage of blue light detected in all its graphs in contrast to Sodium Hydroxide which showed a higher percentage of blue light detected as the concentration increased.

To analyze more on the duration of the reaction depending on different concentrations, Graph 11&12&13&14 all show the averaged “Illuminance over time graphs” of different Hydrogen Peroxide concentrations. The duration of the experiment proves yet again my hypothesis wrong, since as the concentration increased for e.g. from 0.3 to 0.6, the duration decreased with a difference of 15 seconds.

Graph 15&16&17&18 all show the averaged “Illuminance over time graphs” of different Sodium Hydroxide concentrations. Here my hypothesis is visually proven since a clear best fit of all graphs shows an increase in time duration with increased concentration.

Evaluation and limitations

One of my main limitations in this investigation was knowing when the luminance is over, thus effecting the time duration.It was difficult to know the real duration of the chemiluminescence of luminol, considering even after it had lost all blue light, it still glew and was detectable with the naked eye in the dark room. Thus I depended on the spectrometer I was using and when it could no longer measure any luminosity, I stopped collecting data. Getting a stronger device that can detect the light better than Sparkvue could be a good improvement since I could still see the clear solution for a short period of time even after the device could not measure anymore data.

To aid and improve collecting time duration data of this investigation if a good spectrometer is not possible to get a hold of, is by using a spiral and pouring Solution C&D through that. Then it helps to make the luminance last longer and give a better visual confirmation as to when the reaction stops glowing with less uncertainty.

One part of my Hypothesis was also not proven, being Sodium Hydroxide reaching a Plateau at one point and left in excess in the solution. An obvious improvement for this can be to investigate higher concentrations of Sodium Hydroxide for the next time, but the overall Hypothesis was proven that the as the concentration increased, so did the luminosity as well as the time duration collected by the spectrometer.

In regards to the measurements of Sodium Hydroxide as well as Hydrogen Peroxide, they were done using a measuring pipette that measures up to 1 ml. It is a very precise measuring apparatus, but considering it was so difficult sometimes to keep the long pipette vertically straight, the human factor and a small angle of tilt, could be a limitation to an extent. An improvement here would be to connect the pipette through a clutch to a stand, stand on a chair and measure. It is not a very efficient method as well as time consuming, but it is still an improvement.

Other improvements such as sterilizing/cleaning all equipments thoroughly before the start of the experiment to avoid even the slightest contamination as well as make sure to dry after cleaning each beaker to avoid water droplets to contaminate the experiment are both minor but still to a certain extent significant improvements to the investigation and getting more accurate results.

Bibliography

  1. How Luminol Works. Howstuffwork.https://science.howstuffworks.com/luminol.htm (Accessed; 2018-12-10)

  2. Luminol. PubChem.https://pubchem.ncbi.nlm.nih.gov/compound/luminol (Accessed; 2018-12-10)

  3. Luminol-hydrogen peroxide chemiluminescence produced by sweet potato peroxidase. NCBI.https://www.ncbi.nlm.nih.gov/pubmed/17089355 (Accessed; 2019-01-05)

  4. Chemistry, Standard Level, for the IB Diploma (Student Book with eText Access Code) (Pearson Baccalaureate) (2nd Edition) (Pearson International Baccalaureate Diploma: International E) 2nd Edition (Accessed; 2018-12-10)

  5. Luminescence. Wikipedia.https://en.wikipedia.org/wiki/Luminescence (Accessed; 2019-01-06)

  6. http://chem-www4.ad.umu.se:8081/Skolkemi/Experiment/experimentfordjupning.jsp?id=144 (Accessed; 2019-02-07)

  7. Chemical equation of Potassium Ferricyanide, Luminol and Hydrogen Peroxide. Quora.https://www.quora.com/What-is-the-chemical-equation-of-a-reaction-between-potassium-Ferricyanide-C6N6FeK3-sodium-hydroxide-NaOH-Luminol-C8H7N3O2-and-Hydrogen-Peroxide-H2O2 (Accessed; 2019-02-10)

  8. Bioluminescence. Ocean.https://ocean.si.edu/ocean-life/fish/bioluminescence (Accessed; 2019-02-10)

  9. Luminol. NCBI.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760923/ (Accessed; 2019-02-10)

  10. Luminol oxidation reaction with Potassium. Reddit. https://www.reddit.com/r/chemicalreactiongifs/comments/4g2gwx/luminol_oxidation_reaction_with_potassium/ (Accessed; 2019-02-10)

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