The chemiluminescence of luminol induced by bleaches. A study of glows over periods of time.

Summary

The luminol solution is an effective reagent for detecting blood. However, it has been shown to give false positive results by reacting with some household cleaners. To solve this problem, I investigated the reaction of luminol with 5 commonly accessible bleaches, which contained different concentrations of sodium hypochlorite (NaClO), the reactant capable of oxidising luminol.

In the first part of my study, I tested the hypothesis: “The intensity of emitted light depends on the concentration of sodium hypochlorite in a given cleaning substance”. To investigate the influence of time on the samples, I collected the data at 3 time periods – 1, 48 and 96 hours after preparing the sample. There was no significant correlation between the concentration of NaClO and the intensity of the glow. However, the chemiluminescence significantly changes in time and the closest false positive reaction for blood was given by the bleach containing the highest concentration of NaClO.

The second part of the experiment investigated the problem of detecting blood traces washed by bleach. The hypotheis: "Bleaches with higher concentrations of NaClO mask blood traces with greater efficiency" was supported by the data, as the bleaches with the highest concentrations masked the blood trace most efficiently. The heme group (in heamoglobin) got destroyed by the hypochlorite, and therefore, less oxidation of luminol could be possible.

The results of this study provide crime investigators with useful knowledge about the chemiluminescence  of luminol induced by commonly accessible bleaches and how they can be differed from a blood trace.

Question / Proposal

In this study I wanted to investigate the problems and limitations of luminol, one of the most popular chemicals used to detect blood traces. As luminol gets in contact with blood, it goes through a series of oxidations and reductions and emits blue light, making the place covered by blood visible. However, there are some chemicals, such as sodium hypochlorite (NaClO), which is present in bleaches, which can also induce this reaction. This can mistake and confuse crime investigators. I was very interested to investigate to what extent can bleach "resemble" a blood trace and what happens, when a blood trace is washed by bleach! I divided my experiment into two parts . For each of them, I posed a hypothesis. In the first part i tested the following one: "The intensity of emitted light depends on the concentration of sodium hypochlorite in a given cleaning substance". Here I expected, a positive correlation (the higher the NaClO % of a bleach, the more intensive the luminol glow). In the second part of this research I posed a hypothesis: "Bleaches with higher concentrations of sodium hypochlorite mask blood traces with greater efficiency". In this part of experiment, I also expected a positive correlation, as from what I already knew hydrogen peroxide (one of the components of luminol) reacts with blood. This made me believe, that the NaClO, which is also a strong oxidant, will have a similar effect on the haemoglobin.

Research

Much of the attention of the current world of forensic science is focused on the flaws and limitations of detecing blood traces. Before I started my project, I wanted to read more about the subject of luminol and the interference that bleaches can cause. All of the articles, which I was inspired by, can be found in the bibliography section of this Google Science Fair document. Although there were serveral papers about "bleach induced luminol chemiluminescence", I could not find any materials, which actualy used commerial and commonly accesible bleaches, because in all of the papers, the blach was made artificially (mixing NaClO with water). What was even more interesting for me is that in most of these papers, the scientists used only one or two sodium hypochlorite concentrations. I wanted to extend both the range of bleaches which I useed, their concentrations and availability on the market. I also found four papers, which to certain extetns resemble and verify the results of my experiment:

1. Antonini, James M., 1995 et al. Attenuation of acute inflammatory effects of silica in rat lung by 21-aminosteroid, U74389G. Inflammation 19.1: 9-21.

2. Lahav, N., 1973. Chemiluminescence of Luminol in the Presence of Bentonite and Other Clays. Clays and Clay Minerals 21: 257-259.

3. Maitra, Dhiman, et al. "Reaction of hemoglobin with HOCl: mechanism of heme destruction and free iron release." Free Radical Biology and Medicine 51.2 (2011): 374-386.

4. Yamaguchi, H., et al. "The influence of two concentrations of sodium hypochlorite on human blood: changes in haemolysis, pH and protein." International endodontic journal 34.3 (2001): 231-236.

To make the conclusions of my experiment more valid I send them to Dr. Michał Adam Dobrowolski from the University of Warsaw (Chemistry faculty), who is an expert in the field of forensic science chemistry. He verified them and agreed, that they are not "outliers" and can be trusted by the scientific community. 

I hope that in the future, the findings of this study will strenghten the knowledge of crime investigators and help them with differentiating between a blood trace and bleach. Not only do they help to avoid the risk of possible misinterpretations of the luminol chemiluminescence glow spectra , but also provide much detailed knowledge about using bleach to clean blood. By comparing the glow intensities of an examined trace to the results of this study, one can determine which bleach may be altering the reaction of blood with luminol, or which bleach is producing a false - positive reaction. Also, to the best of my knowledge, this is one of the first experiments in the field of forensic sceince to examine so many sodium hypochlorite concentrations in bleach and to actually test real, not artificial solutions!

 

Method / Testing and Redesign

The following method and procedures were used in my experiment:

1. Luminol Solution Preparation – chemicals and procedure.

The luminol solution was prepared by dissolving 11.5 mg of luminol (C8H7N3O2) in 40 ml of water. An alkaline environment was then created by adding 0.8 g of NaOH. Just before the experiment was conducted, 1.2 ml of H2O2 was added to the solution. The luminol solution was made each day before conducting the experiments, so the regent was fresh and not affected by time. Samples were taken from this stock solution.

2. Bloodstain Preparation – chemicals and procedure.

Real blood was not used in this study for ethical reasons, so in order to prepare a haemoglobin solution, a fixed amount of dry haemoglobin was dissolved in enough water to achieve a approximate 10% concentration. The samples were taken from this stock solution.

3. Preparation of the control and experimental samples. Luminol application.

The study used 96 well 300 microliter plates. Each of the bleaches - Home Sense® Bleach Original (8.25% of NaClO), Clorox® Regular Bleach 2 (6% of NaClO), Tilex® Mold & Mildew Remover (2.4% of NaClO), Lysol® Brand II Bleach Toilet Bowl Cleaner (2% of NaClO), Clorox® Clean-Up® Cleaner with Bleach 1 (1.84% of NaClO) - were tested in triplicate (i.e. the mean of  three measurements form each of the samples was calculated). Each well contained 125 ml of the imitated blood or a given bleaching substance and just before reading the result, 95 ml of luminol was added. One additional well for each of the investigated substances was prepared to serve as a control sample (luminol was not added before reading the measurements). The figure below illustrates the procedure of the experiment:

4. Measurement of the luminol response

After adding 95 ml of luminol to a well containing 125 ml of a given bleaching agent or the imitated blood, the intensity of chemiluminescence in Relative Luminescence Units (RLU) was measured using the BioTek Synergy H1 Hybrid Multi-Mode Reader, with the gain set to 100 (used at Dr. Amy Palmer's lab at the University of Colorado, Boulder).

Safety

In order to perform this experiment in a profesional laboratory, at each time I had to wear saftety googles, a lab coat and gloves. Also, I had to dispose all the waste products to special containers, not poure them to the sink.

Statistical Analysis

To test the hypotheses and find the solutions to the problems I posed in the introduction part of this document, I had to learn how to use several statistical tests. I finnaly used a one - way analysis of variance test (ANOVA) and a regression analysis. Using statistics, makes my conclusions more valid, as they have a mathematical background and can be critiaclally analysed.

 

Results

Results from the first part of experimentation:     

A one way analysis of variance test (ANOVA) was performed to analyse the data from each measurement. The P value smaller than 0.05 and the F value < F critical value indicates that the differences between the means are statistically significant. To test  the correlation between the concentration of NaClO in a bleaching agent and the intensity of chemiluminescence a regression analysis was performed. 

ANOVA RESULTS:

Time period F F critincal P value
1 hour 68.44 3.48 < 0.01
48 hours 7.5 3.48 <0.01
96 hours 8.38 3.48 <0.01

REGRESSION ANALYSIS:

 

Time period r - value r2 P value
1 hour 0.42 0.18 0.48
48 hours 0.78 0.62 0.12
96 hours 0.2 0.04 0.75

Conclusions:

As shown in the ANOVA table the chemiluminescences are significantly different from each other. The regression analysis supports the hypothesis that the intensity of emitted light depends on the concentration of NaClO in a given cleaning substance, but only in the second time period (48 hours), when the r2 was the highest in all of the trials. However, due to the small sample size (concentrations of NaClO in bleaches on the market fit within the range of 1.5% - 9%) a firm relationship can't defined. This is because the intensity of the glow also depends on other ingreients - such as montmorillonite (found in the Lysol® Cleaner) or silica (found in Clorox® Clean-Up Bleach). Thus, the amount of NaClO in bleach in most cases does not have an effect on the intensity of the glow, which can be altered by other chemicals present in the cleaner. However, the cleaning substance which gave the closest false positive reaction for blood was identified (Home Sense Bleach® with 8.25% concentration of NaClO).

 

Results from the second part of experimentation:  

Similary to the first part of experimentation, I performed an ANOVA to examine whether the chemiluminescecne intesities are significantly different from eachother. In order to test the hypothesis for this part of experimentation (higher concentartions of NaClO mask haemoglobin traces more efficiently) I did a regression analysis:

ANOVA:

Time Period F value F critical P value
1 hour 1.53 3.48 0.27
48 hours 131,8 3.48 <0.01
96 hours 277.26 3.48 <0.01

RESULTS:

Time Period r - value r P value
1 hour 0.68 0.46 0.2
48 hours 0.74 0.56 0.14
96 hours 0.89 0.8 0.04

The results of the second set of experiments allow for some additional conclusions. Most importantly, the data supports the hypothesis that higher % of NaClO mask the haemoglobin traces more efficiently, especially in the second and third time intervals of the experiment. The highest concentrations of NaClO (8,25% and 6%) gave less intensive glows than the bleaches with the lowest concentration of NaClO (1,84%). These results support the data from previous studies, indicating that the increased concentration of NaClO produces more oxidation in haemoglobin, leading to the destruction of heme. Consequently, the smaller amount of heme in the mixture, the weaker and slower the oxidation of luminol. 

Conclusion

The results of this experiment have the potential to provide crime investigators with useful and helpful knowledge about the interference of bleaches on crime scenes and the false–positive chemiluminescence outcomes they can produce. Indeed, the first part of the experimentation presented here shows how various concentrations of sodium hypochlorite in bleach react with luminol and how they compare to a haemoglobin solution (HS) - luminol reaction. However, the hypothesis (the intensity of emitted light depends on the concentration of sodium hypochlorite in a given cleaning substance) was not supported by the data obtained in this part of my experiment. This may be due to other chemicals present in bleaches, which can also influence the oxidation of luminol  Therefore, in order to identify the cleaner which induces a false positive reaction, its components must first be identified and researchers cannot rely on the glow spectrum only. Thus, it would be necessary to test how solutions with sodium hypochlorite being the only component would react with luminol and compare these data to the results of this experiment. This would allow seeing how other components of bleach influence the luminol chemiluminescence and which part of the glow spectrum the sodium hypochlorite is responsible for.

The outcomes of the second part of the experimentation carry an even more important lesson for forensic scientists about identifying the bleaching agent that may have been used to clean bloodstains. The hypothesis (bleaches with higher concentrations of sodium hypochlorite mask blood traces with greater efficiency) was supported by the data obtained in this part of the study. The bleaches with higher concentrations of sodium hypochlorite (6%, 8.25%) were able to mask haemoglobin traces more efficiently because of the greater amount of oxidation they induced. This may be helpful for crime investigators, who by knowing the mean chemiluminescence intensity after adding the luminol solution to a mixture of bleach and HS will be able to approximate the NaClO % in the bleach used to clean a blood trace. It would be interesting to use real blood samples – this would make the results of this study more reliable for the scientific community, and other components of blood would also be included.

Overall, the outcomes of this study evoke some interesting conclusions. These results may strengthen the knowledge of crime investigators and enable them to differentiate between the oxidation of luminol induced by a blood trace or by bleach. This decreases the risk of possible misinterpretations of the luminol chemiluminescence glow spectra and helps to avoid unjustifiable accusations.

About me

For me, science and technology are one of the most interesting and fascinating areas of knowledge which are currently explored by people. STEM subjects have permeated into our lives to the extent that sometimes we can't even imagine living without them! Growing up in the era, where each day is abundant in new discoveries and technological breakthroughs, I decided to become a scientists one day. One of the most important and inspiring people for me is my grandfather, Leon Gradoń, who despite coming from a very poor Silesian family became one of the greatest polish chemical engineers. I often think about his unbelievable story, and the great courage he had to "think outside of the box" about his projects during the communist times in Poland. His ambition, hard work and deep love for science will always inspire me and motivate to be engaged in the scientific community. Following my grandfather’s steps, in the future, I aim to pursue an academic career. Although the project I am submitting to Google Science Fair is from the field of chemistry, I want to become a biochemist in order to explore the fascinating field of stem cells and regenerative medicine. Winning a prize in this competition would definitely help me to get closer to my dreams and plans. With no doubt, I would use this money and other benefits to develop a complex scientific project, on which I would work with young people just as excited and curious about the scientific world as me!

Health & Safety

In order to perform an experiment at Dr. Amy Palmer's lab, I had to go through a series of safety tests, learn how to use all of the necessary equipment, handle chemicals and how to dispose them. I also had to go through a serie of tutorials, during which I learnt useful lab techniques.

Contact to Dr. Amy Palmer:

E-mail: amy.palmer@colorado.edu
Office phone: (303)-492-1945
Office location: JSCBB C317
Laboratory location: JSCBB C381
Laboratory phone: (303)-492-8536

Bibliography, references, and acknowledgements

I would like to acknowledge Dr. P. Strode, Dr. M. A. Dobrowolski and Mrs. Katarzyna Zaremba for their help in deciding what to focus the subject of my research on. I would also like to thank Dr. A. Palmer and the members of her lab at the University of Colorado at Boulder for their helpful advice in the laboratory and teachnig me all the neccessary lab techniques to use in the experiment. 

Works and sources which I used throughout my experiment:

1. Castelló, A., Francés, F. and Verdú, F., 2009. Bleach interference in forensic luminol tests on porous surfaces: More about the drying time effect. Talanta, 77(4), pp.1555-1557.

2. Barni, F., Lewis, S.W., Berti, A., Miskelly, G.M. and Lago, G., 2007. Forensic application of the luminol reaction as a presumptive test for latent blood detection. Talanta, 72(3), pp.896-913

3. Cassidy, B.M., Lu, Z., Martin, J.P., Tazik, S.K., Kellogg, K.W., DeJong, S.A., Belliveau, E.O., Kilgore, K.E., Ervin, S.M., Meece-Rayle, M. and Abraham, A.M., 2017. A quantitative method for determining a representative detection limit of the forensic luminol test for latent bloodstains. Forensic science international, 278, pp.396-403.

4. Stoica, B.A., Bunescu, S., Neamtu, A., Bulgaru‐Iliescu, D., Foia, L. and Botnariu, E.G., 2016. Improving Luminol Blood Detection in Forensics. Journal of forensic sciences, 61(5), pp.1331-1336.

5. https://weirdscience.eu/Chemiluminescencja luminolu aktywowana żelazicyjankiem.html

6. http://mriquestions.com/types-of-hemoglobin.html

7. Oldfield, C., Morgan, R.M., Miles, H.F. and French, J.C., 2017. The efficacy of luminol in detecting bloodstains that have been washed with sodium percarbonate and exposed to environmental conditions. Australian Journal of Forensic Sciences, pp.1-10.

8. Creamer, J.I., Quickenden, T.I., Apanah, M.V., Kerr, K.A. and Robertson, P., 2003. A comprehensive experimental study of industrial, domestic and environmental interferences with the forensic luminol test for blood. Luminescence, 18(4), pp.193-198.

9. Kent, E.J., Elliot, D.A. and Miskelly, G.M., 2003. Inhibition of bleach-induced luminol chemiluminescence. Journal of forensic science, 48(1), pp.1-4.

10. Lahav, N., 1973. Chemiluminescence of Luminol in the Presence of Bentonite and Other Clays. Clays and Clay Minerals 21: 257-259.

11. Antonini, James M., 1995 et al. Attenuation of acute inflammatory effects of silica in rat lung by 21-aminosteroid, U74389G. Inflammation 19.1: 9-21.

12. Maitra, Dhiman, et al. "Reaction of hemoglobin with HOCl: mechanism of heme destruction and free iron release." Free Radical Biology and Medicine 51.2 (2011): 374-386.

13. Yamaguchi, H., et al. "The influence of two concentrations of sodium hypochlorite on human blood: changes in haemolysis, pH and protein." International endodontic journal 34.3 (2001): 231-236.

 

I performed this experiment at the University of Colorado at  Boulder in Dr. Amy Palmer's lab (Jennie Smoly Caruthers Biotechnology Building). Beside of the regular lab equipment which I used in the experiment, Dr. Palmer permitted me to use the BioTek Synergy H1 Hybrid Multi-Mode Reader in order to measure the chemiluminescence. I also recieved some help from the University of Warsaw with the understanding of how luminol-oxidation reaction works. Beside that, the project was designed and developed entirely by myslef, I did not join any research group and did this project independently, occasionaly seeking adive from experienced scientists to gain a better understanding of some chemical processes.