Effect of Copper Ion Chelation On Curcumin’s Antioxidant Capacity

1. Effect of Copper Ion Chelation On Curcumin’s Antioxidant Capacity Chelsea Massaro, 11th grade Christian Home & Bible School A.P. Environmental Science Instructor: Mrs. McElligott I have participated in science fair for the past six years. I have attended and placed at the state science fair for all six years. I have attended the International Science and Engineering fair for two years. I have been working with Tandy Grubbs at Stetson University for three years. I have taken Chemistry, Biology, Anatomy and Physiology, and Physical Science. I am currently take A.P. Environmental Science. I plan on attending college and majoring in Chemical Research.

2. Introduction Curcumin is a curcuminoid found in the spice turmeric, which is often used in curries. Many experiments have showed that curcumin may have numerous medical qualities, although few have been confirmed. Like most herbal remedies, it was first used as a spice, and it was later discovered to have healing properties. Curcumin interests medical researchers because it shows many properties that may fight specific forms of cancer and Alzheimer’s disease. For my science fair project I chose to test if curcumin work as an effective antioxidant, and, if so, what effect will copper (II) have on curcumin’s antioxidant capacity. This subject interests me because I have an interest in chemistry. In addition, I have been researching uses of curcumin for two years and have become very interested in its numerous abilities. I have previously researched the use of curcumin as a chelating agent and discovered that the spice does have the ability to bind with certain transition metal ions. I have also researched curcumin’s ability to reduce the amount of oxidation that occurs to N-acetyl-tyrosine in an in vitro Cu(II)/H2O2 model of Alzheimer’s disease. I contacted Dr. Tandy Grubbs, professor of chemistry at Stetson University, and asked if he would work with me on this project. This research paper contains information on curcumin, copper (II) nitrate, DPPH, the electron dot diagram, free radicals, the free radical theory of aging, antioxidants, ascorbic acid, phenols, and chelating agents.

3. Problem Will curcumin work as an effective antioxidant, and, if so, what effect will copper (II) have on curcumin’s antioxidant capacity? Hypothesis If curcumin is added to a solution containing DPPH, then curcumin will work as an effective antioxidant and scavenge the radicals. If copper is added to said solution, then it will have no effect curcumin’s on antioxidant capacity. Initial Research Curcumin is a yellow spice extracted from the dried roots of the Curcuma longa plant, which is part of the ginger family of herbs. Curcuma longa is native to southern and southeastern Asia. The roots of the Curcuma longa plant are ground into a deep yellow powder called curcumin, which has a very bitter and peppery taste. Curcumin is the principle curcuminoid in the Indian curry spice turmeric. A curcuminoid is curcumin or a byproduct of curcumin. Curcumin is used in spicy dishes as well as curries in Asia, India, and the Middle East. Curry gets its yellow color and strong flavor from curcumin. Curcumin is a polyphenol, or a group of chemical substances found in plants. These polyphenols are responsible for the yellow color of curcumin. Copper(II) nitrate , also referred to has Cupric nitrate , is a chemical compound with the formula Cu(NO3)2. A chemical compound is a substance containing two or more elements that is formed by the chemical-bonding of those elements. The Roman numeral in the name is used to specify the oxidation number of copper. Copper(II) has the oxidation number of positive two. An oxidation number is the ionic charge of the

4. element. Copper(II) physical form is dark blue crystals. It is soluble in water and ethyl alcohol. Copper(II) is stable under normal conditions. In addition, it is an oxidizer. An oxidizer is a substance that oxidizes, either adds oxygen to or removes electrons from, another substance. Copper is one element in a somewhat small group of metallic elements that are essential to human health. It is a trace element, which is necessary for a human’s overall health. The metallic elements, including copper, are required for ordinary metabolic processes. Because the body cannot synthesize, or create, copper, the diet of humans must supply regular amounts. Copper combines with certain proteins to produce enzymes. About 20 enzymes in the human body contain copper and at least ten depend on copper to function. An enzyme is a protein that accelerates chemical reactions. The enzymes appear in most tissues and affect the functions of the cardiovascular, nervous, and immune systems. Diphenylpicrylhydrazyl, commonly referred to as DPPH, is an organic chemical compound composed of stable free radical molecules. Its two major applications in laboratory research are monitoring chemical reactions involving radicals and studying the position and intensity of electron paramagnetic resonance signals. DPPH is a well known radical and scavenger of other radicals. Therefore, the reduction rate of a chemical reaction upon the addition of DPPH into a solution can be used as an indicator of the antioxidant ability of the other reactants. DPPH has a strong absorbance at around 520 nm, which gives the radical a deep violet color when in a solution. However, when it is neutralized it becomes colorless or pale yellow. This allows the visual monitoring of the reaction. Many scientists have conducted studies using the specific absorbance properties of DPPH. A study documented in Sensors Scientific Journal revels that “the absorbance decreases when the radical is reduced by antioxidant”. In a study of DPPH involving oxygen active species, P. Ionita states “because DPPH can be kept indefinitely with little decomposition and because it does not react with oxygen, it proved to be quite useful in a variety of investigations, such as the determination of antioxidant properties of amines, phenols, or natural compounds…”. Free radicals are formed when a weak bond is spilt leaving an atom with an outer shell that contains an odd number of electrons. They react quickly with other compounds and are very unstable. Free radicals try to capture the needed electron from the nearest stable atom to gain stability. When the stable atom loses its electron, it becomes a free radical. This starts a chain reaction. After the process starts it has the ability to result in the disruption of a living cell. Free radicals are often responsible for aging, tissue damage, and some diseases. The body’s immune system can sometimes purposefully create free radicals to neutralize viruses and bacteria. The body can normally handle free radicals. However, when antioxidants are unavailable or

5. free radical production becomes excessive damage can occur. Free radical damage often occurs with age. The free radical concept of aging was first proposed by Dr. Denham Harmon. The theory implies that antioxidants such as vitamin C and E, which prevent free radicals from removing electrons from sensitive biological molecules, will slow the process of aging. Antioxidants are molecules capable of inhibiting the oxidation of other molecules. Oxidation is a chemical reaction that occurs when an electron is transferred from a substance to an oxidizing agent. This process can produce free radicals. Many oxidants contain an unpaired electron, which can be highly dangerous to humans due to the possible reaction to nucleic acids, proteins, and lipids. These reactions are dangerous because they can result in a whole chain of free radical reactions. The interaction of the initial free radical with another molecule changes that molecule into a free radical, which proceeds to react with other molecules turning them also into free radicals. Antioxidants donate an electron to the unstable free radical to stop the reaction. Although oxidation reactions are essential for life, they can also be damaging. To protect against these harmful affects plants and animals maintain a complex system of antioxidants, including vitamins C and E. Low levels of antioxidants can cause oxidative stress and may damage or kill many cells. Ascorbic acid is water-soluble sugar acid with antioxidant properties. It is considered an enol because of the high acidity of its hydroxyl groups. One form of the acid, L-ascorbic acid, is commonly known as vitamin C. It is found in plants, animals, and single-cell organisms. Ascorbic acid works as an antioxidant by acting as a stable electron donor. Phenolic groups are typically thought to be responsible for the antioxidant abilities of substances. A phenol consists of a benzene ring and a hydroxyl group. Although it is considered an alcohol, phenols are much more acidic than typical alcohols. The ability of phenolic compounds to stop free radicals comes from both their acidity, which allows them to donate protons, and their delocalized electrons, which gives them the ability to transfer electrons while remaining relatively stable. Electron delocalization and easy ionizations account for the bright colors of most substances containing phenols. A chelating agent is an ion or compound that can form various bonds to a single metal ion. The compound formed by the metal ion and the chelating agent is called a chelate. Chelating agents are often used in food manufacturing to remove metal ions from the food. Some herbs and supplements are used as chelating agents. Chelation is the process of the binding of the chelating agent’s molecules and the metal ion’s molecules and removing the metal ions from the body. This process has been scientifically proven to remove excess or toxic metals before they can cause damage to the human body. Chelation can treat

6. circulatory disorders and lead or other heavy metal poisoning. The ability of curcumin to work as an antioxidant could have numerous medical benefits. In addition, studying the affect of copper on this ability could confirm that the phenol group is responsible for the antioxidant abilities. Work Cited Antioxidants and Free Radicals (1996). Free Radicals. Retrieved June 8, 2009, from http://www.rice.edu/~jenky/sports/antiox.html Ara, N. (2009). In Vitro Antioxidant Activity of Methanolic Leaves and Flowers Extracts of Lippia Alba. Research Journal of Medicine and Medical Sciences, 4, 107-110. Ascorbic Acid. (2010). Retrieved January 11, 2010 from PubHealth: http://www.ncbi.nlm.nih.gov/​pubmedhealth/​pmh0000720. Copper in Human Health (2008). Copper and Health. Retrieved June 8, 2009,from http://www.copperinfo.co.uk/health/homepage.shtml Curcumin (2007). Real Age. Retrieved June 8, 2009, from http://www.realage. com/researchlibrary/searchResults.aspx? link=crsfiles/ma/ma_curcumin_ma.htm Free Radical Biology & Medicine (1997). Oxidative stress hypothesis in Alzheimer's disease. Retrieved June 8, 2009, from http://d.wanfangdata.com.cn/NSTLQK_NSTL_QK9841268.aspx Free Radicals and Your Health (n.d.). Radicals. Retrieved June 8, 2009, from http://www.healingdaily.com/conditions/free- radicals.htm Ionita, P. (2005). Is DPPH Stable Free Radical a Good Scavenger for Oxygen Active Species? Chem Papers, 59, 11-16. Leh, F. (1972). Organic Free Radical and Metal Complexes.2,2-Diphenyl-1-picrylhydrazyl with Cu(1). Canadian Journal of Chemistry, 50, 999-1004. Masuda, T. (2001). Chemical Studies on Antioxidant Mechanism of Curcumin: Analysis of Oxidative Coupling Products from Curcumin and Linoleate. Journal of Agricultural and Food Chemistry, 49, 2539-2547. Phenol. (2009). Retrieved January 11, 2010 from Greener Industry: http://www.greenerindustry.org.uk/​pages/​phenol/​2phenoluses.htm. Smart, J. (2010). Curcumin: A Powerful Brain Protection Supplement. Retrieved January 11, 2010 from Acceleration Studies Foundation: http://www.accelerating.org/​articles/​curcumin.html. Szabo, M. R. (2009). Improved DPPH determination for antioxidant activity spectrophotometric assay . Chemical Papers, 61, 214- 216. Understanding Free Radicals and Antioxidants (2009). What are Free Radicals. Retrieved June 8, 2009, from http://www.encyclopedia.com/doc/1E1-chelatin.html What is Curcumin (2008). Wise Geek. Retrieved November 10, 2008, from http://www.wisegeek.com/what-is-curcumin.htm

7. Materials • Rubber gloves, lab coat, eye protection • Pen or pencil and paper • Plastic garbage bag • Sodium hydroxide solution • Copper (II) sulfate pentahydrate • L-ascorbic acid reagent ACS • Sodium phosphate monobasic monohydrate • 1 mL volumetric pipette • Distilled water • Curcumin • Ethyl alcohol • DPPH • Bulb pipette • Spatula • Kimtech kimwipes • Weight boats • One 1,000 mL flask • Two 250 mL flasks • Two 100 mL flasks • Eight 20 mL flasks • Cuvette • pH meter and probe • Analytic balance Independent Variables • Copper (II)’s chelation to curcumin • Curcumin added to a solution containing DPPH Dependent Variables • Curcumin’s antioxidant ability • Copper (II)’s effect on curcumin’s antioxidant capacity Constants • Concentrations of stock solutions • Total volume in each sample • Amount of DPPH in each experimental solution

8. Control Group • Solution containing .5 mL ethyl alcohol, 1 mL curcumin stock solution, and 1 mL sodium • phosphate monobasic monohydrate stock solution • Solution containing .5 mL DPPH stock solution, l mL ethyl alcohol, and 1 mL sodium • phosphate monobasic monohydrate stock solution Experimental Group • Solution containing .5 mL DPPH stock solution, 1 mL curcumin stock solution, .5 mL • copper (II) stock solution, and .5 mL sodium phosphate monobasic monohydrate • stock solution • Solution containing .5 mL DPPH stock solution, 1 mL copper (II) stock solution, and 1 • mL curcumin stock solution • Solution containing .5 mL DPPH stock solution, 1 mL copper (II) solution, and 1 mL ethyl • alcohol • Solution containing .5 mL DPPH stock solution, 1 mL ethyl alcohol, and 1 mL ascorbic acid • stock solution • Solution containing .5 mL DPPH stock solution, 1 mL curcumin stock solution, and 1 mL • sodium phosphate monobasic monohydrate stock solution

9. Procedure Perform all steps under the supervision of the Qualified Scientist in University laboratory. Gather Materials. Put on disposable rubber gloves, lab coat, and eye protection. Have the designated supervisor do so as well. Using an analytic balance, spatula, and weight boat, measure 20.8 grams of sodium phosphate monobasic monohydrate into a 1,000 mL flask. Add distilled water into the flask containing the sodium phosphate monobasic monohydrate to obtain a total volume of 800 mL. Using a pH meter and probe, add aliquots of one molar sodium hydroxide solution until reaching a pH of 7.4. Dilute up to one liter to create sodium phosphate monobasic monohydrate buffer stock solution. Mix well. Clean surface analytic balance using Kimtech Kimwipes. Clean spatula with distilled water and dispose of weight boat in trash. Using an analytic balance, spatula, and weight boat, measure .002 grams of ascorbic acid into a 250 mL flask. Add sodium phosphate monobasic monohydrate stock solution into flask containing ascorbic acid to obtain a total volume of 250 mL to create ascorbic acid stock solution. Mix well. Clean surface analytic balance using Kimtech Kimwipes. Clean spatula with distilled water and dispose of weight boat in trash. Using an analytic balance, spatula, and weight boat, measure .0018 grams of Cu(II) into a 250 mL flask. Add sodium phosphate monobasic monohydrate stock solution into flask containing Cu(II) to obtain a total volume of 250 mL to create Cu(II) stock solution. Mix well. Clean surface analytic balance using Kimtech Kimwipes. Clean spatula with distilled water and dispose of weight boat in trash. Using an analytic balance, spatula, and weight boat, measure .002 grams of DPPH into a 100 mL flask. Add ethyl alcohol to flask containing DPPH to obtain a total volume of 100 mL to create DPPH stock solution. Mix well. Clean surface analytic balance using Kimtech Kimwipes. Clean spatula with distilled water and dispose of weight boat in trash.

10. Using an analytic balance, spatula, and weight boat, measure .001 grams of curcumin into a 100 mL flask. Add ethyl alcohol to flask containing curcumin to obtain a total volume of 100 mL to create curcumin stock solution. Mix well. Clean surface analytic balance using Kimtech Kimwipes. Clean spatula with distilled water and dispose of weight boat in trash. Using a one mL volumetric pipette, add 1.5 mL ethyl alcohol and 1 mL sodium phosphate monobasic monohydrate buffer stock solution to a 20 mL flask. Mix well. This is the baseline. Using a bulb pipette, insert sample of baseline solution in cuvette. Insert cuvette into UV Visible Spectrometer. Run UV Visible Spectrometer for a baseline. Properly dispose of solution as indicated in Clean up & waste disposal. Wash cuvette with warm soapy water. Using a one mL volumetric pipette, add .5 mL ethyl alcohol, one mL curcumin stock solution, and one mL sodium phosphate monobasic monohydrate buffer stock solution to a 20 mL flask. Mix well. Using a bulb pipette, insert sample of solution in cuvette. Insert cuvette into UV Visible Spectrometer. Run UV Visible Spectrometer. Record results. Properly dispose of solution as indicated in Clean up & waste disposal. Wash cuvette with warm soapy water. Using a one mL volumetric pipette, add .5 mL DPPH stock solution, one mL ethyl alcohol, and one mL sodium phosphate monobasic monohydrate buffer stock solution to a 20 mL flask. Mix well. Using a bulb pipette, insert sample of solution in cuvette. Insert cuvette into UV Visible Spectrometer. Run UV Visible Spectrometer. Record results. Properly dispose of solution as indicated in Clean up & waste disposal. Wash cuvette with warm soapy water. Using a one mL volumetric pipette, add .5 mL DPPH stock solution, one mL ethyl alcohol, and one mL ascorbic acid stock solution to a 20 mL flask. Mix well. Using a bulb pipette, insert sample of solution in cuvette. Insert cuvette into UV Visible Spectrometer.

11. Run UV Visible Spectrometer. Record results. Properly dispose of solution as indicated in Clean up & waste disposal. Wash cuvette with warm soapy water. Using a one mL volumetric pipette, add .5 mL DPPH stock solution, one mL curcumin stock solution, and one mL sodium phosphate monobasic monohydrate buffer stock solution to a 20 mL flask. Mix well. Using a bulb pipette, insert sample of solution in cuvette. Insert cuvette into UV Visible Spectrometer. Run UV Visible Spectrometer. Record results. Properly dispose of solution as indicated in Clean up & waste disposal. Wash cuvette with warm soapy water. Using a one mL volumetric pipette, add .5 mL DPPH stock solution, one mL curcumin stock solution, .5 mL Cu(II) stock solution, and .5 mL sodium phosphate monobasic monohydrate buffer stock solution to a 20 mL flask. Mix well. Using a bulb pipette, insert sample of solution in cuvette. Insert cuvette into UV Visible Spectrometer. Run UV Visible Spectrometer. Record results. Properly dispose of solution as indicated in Clean up & waste disposal. Wash cuvette with warm soapy water. Using a one mL volumetric pipette, add .5 mL DPPH stock solution, one mL curcumin stock solution, and one mL Cu(II) stock solution to a 20 mL flask. Mix well. Using a bulb pipette, insert sample of solution in cuvette. Insert cuvette into UV Visible Spectrometer. Run UV Visible Spectrometer. Record results. Properly dispose of solution as indicated in Clean up & waste disposal. Wash cuvette with warm soapy water. Using a one mL volumetric pipette, add .5 mL DPPH stock solution, one mL Cu(II), and one mL ethyl alcohol stock solution to a 20 mL flask. Mix well. Using a bulb pipette, insert sample of solution in cuvette. Insert cuvette into UV Visible Spectrometer. Run UV Visible Spectrometer. Record results. Properly dispose of solution as indicated in Clean up & waste disposal. Wash cuvette with warm soapy water. Dispose all paper waste in a sealed plastic garbage bag and place in a garbage can for pick up by disposal service. Return all other lab equipment to laboratory storage Return all used chemicals to chemical storage at University Repeat all steps above for each trial.

12. Safety Measures • Work in a University Chemistry Laboratory • Work under the supervision of qualified scientist. • Wear a lab coat, disposable gloves, face mask, and eye protection. • Avoid inhalation, eye contact, skin contact, and ingestion of the nitrates and curcumin • Keep chemicals and solutions away from heat, sparks, and flame • Wash hands thoroughly after experimentation Clean up and Waste Disposal • Paper waste will be sealed in a plastic garbage bag and placed in garbage can for pick up by disposal service. • Return all other lab equipment to laboratory storage. • Return all unused chemicals to chemical storage at University. • Using hot water, rinse the curcumin stock solution down the drain of a sink. • All other solutions will be disposed of by the lab manager of Stetson • University using the following procedure: • Pour the transition metal ion solutions collected in the waste jar into a large dish. • Allow the water to slowly evaporate out of the dish. • After the water has fully evaporated, dissolved components are left behind as a solid. Stetson University accumulates the components and combines them with a large amount of mixed transition metal salts gathered over the course of a year. • After a year, Stetson University sends the salts to a waste disposal company which properly disposes of the waste.

13. Data Analysis • Using the UV visible spectrometer I will measure the absorbance of the solutions listed above. The following equations will be used to determine the solutions’ reactions to DPPH: • Cu+2 = .5 mL DPPH, 1 mL Cu(II), 1 mL Ethanol absorbance - .5 mL DPPH, 1 mL • Ethanol,& 1 mL Phosphate Buffer absorbance • Curcumin = .5 mL DPPH, 1 mL Curcumin, 1 mL Phosphate Buffer absorbance- • .5 mL Ethanol, 1 mL Curcumin, 1 mL Phosphate Buffer absorbance • Curcumin and Cu+2 = .5 mL DPPH, 1 mL Curcumin, .5 mL Cu(II), .5 mL • Phosphate Buffer absorbance - .5 mL Ethanol, 1 mL Curcumin, 1 mL • Phosphate Buffer absorbance • Ascorbic = .5 mL DPPH, 1 mL Ethanol, 1 mL Ascorbic Acid absorbance - .5 • mL DPPH, 1 mL Ethanol,& 1 mL Phosphate Buffer absorbance • Curcumin and Cu (II) 2= .5 mL DPPH, 1 mL Curcumin, 1 mL Cu(II) absorbance - • .5 mL Ethanol, 1 mL Curcumin, 1 mL Phosphate Buffer absorbance • Compare the spectra results of these equations to the control groups to determine the components’ reactions to DPPH.

14. Curcumin Bonded to Copper Ion Phenolic Group Red: Oxygen White: Hydrogen Grey: Carbon Red: Oxygen White: Hydrogen Blue: Carbon Curcumin Molecule

17. Discussion A possible error made was that a chemical reaction that took place when copper was added to the curcumin solution. This chemical reaction caused the curcumin to precipitate, or become insoluble in ethyl alcohol. If more ethyl alcohol was used to dissolve the curcumin, this problem could possibly be avoided. In addition, if the ascorbic acid solution was allowed to set for more than fifteen minutes then it would no longer react with DPPH. My data varied in a few of my trails. I believe this is do to the ascorbic acid solution being allowed to set for a long period of time. This altered some of the results. However, it did not affect the outcome of the experiment significantly. If this experiment were to be continued, the recommendations would be to test iron’s affect on curcumin’s antioxidant abilities as well as varying concentrations of DPPH and curcumin.

18. Conclusion The key results for this experiment were the UV Visible Spectrometer results that show curcumin is effectively scavenging the radicals in the solution and that copper (II) only moderately reduces curcumin’s antioxidant capacity. This is indicated by the rise and falls of the peaks in absorbance of the different samples shown on the spectra. My results supported my hypothesis by showing curcumin’s ability to work as an antioxidant. However, my hypothesis was disproved by copper (II)’s slight negative on curcumin’s antioxidant capacity. The measurements also indicated that curcumin’s antioxidant capabilities are a result of its phenolic groups.

19. Value of Experiment This experiment has great value. It has shown that curcumin can be used as an effective antioxidant. Alzheimer’s disease is thought to be caused by the oxidation of copper ions. The oxidized copper ions react with the amyloid beta protein in the brain, creating plaques. Curcumin, which is able to cross the blood brain barrier and enter the area around the brain, could stop the oxidation of copper ions by working as an antioxidant. This would stop the free radical reaction and, therefore, the formation of plaques. Thus, curcumin’s antioxidant capabilities could slow the progression of Alzheimer’s disease. Acknowledgements I would like to thank Dr. Tandy Grubbs, Professor of Chemistry at Stetson University for helping me with my project. He assisted me in determining which metals to test and the most accurate method of testing. In addition, Dr. Grubbs showed me how to conduct the experiment. Without his advice and assistance this project would have never happened. I would also like to thank Stetson University for allowing me to conduct my experiments in one of their labs and to use their materials.

20. Bibliography Ascorbic Acid MSDS(2010). Ascorbic Acid. Retrieved 2010, from http://www.sciencelab.com/xMSDS-Ascorbic_acid-9922972 Copper Nitrate MSDS (2008). Copper Nitrate. Retrieved June 8, 2009, from http://www.palomar.edu/ehs/Chemistry%20MSDS/COPPER%20NITRATE.pdf Curcumin MSDS (2008). Curcumin. Retrieved June 8, 2009, from http://www.palomar.edu/ehs/Chemistry%20MSDS/TUMERIC.pdf DPPH MSDS(2010). DPPH. Retrieved 2010, from http://www.sigmaaldrich.com/catalog/ProductDetail.do?D7=0&N 5=SEARCH_CONCAT_PNO%7CBRAND_KEY&N4=43180%7CFLUKA&N25=0&QS=ON&F=SPEC Ethyl Alcohol MSDS (2008). Ethanol. Retrieved June 8, 2009, from http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en &N4=459836|SIAL&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F =SPEC International Rules-For Precollege Science Research: Guidelines for Science and Engineering Fairs (2011). ISEF. Retrieved June 8, 2010, from http://www.societyforscience.org/document.doc?id=9 Phosphate Buffer MSDS (2009). Phosphate Buffer. Retrieved June 8, 2009, from http://www.sigmaaldrich.com/catalog/Product Detail.do?lang= en&N4=P3288|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Sodium Hydroxsol MSDS(2010). Sodium Hydroxsol. Retrieved 2010, from http://www.fishersci.com/msds?productName=SS2761&productDescription=SODIUM+HYDROX+SOL+N%2F10+CR+L &catNo=SS276-1&vendorId=VN00033897&storeId=10652

21. Lab at Stetson University Experiment Performed In Mixing Phosphate Buffer Solution All Photos by T. Massaro