Water and Citric Acid

A. Introduction
Chelating agent is a compound that combines with metal ions to form stable ring structures. It is used to reduce the concentration of free metal ion in solution by complexing it. From the Greek term “chela” that means “the great claw” of the lobster or other crustaceans, chelate, root word for “chelating”, suggests the way in which an organic compound “clamps” onto the cationic element, which it chelates.

In order for a compound to be called a true chelating agent, it must have certain chemical characteristics. This chelating compound must consist of at least two sites capable of donating electrons (coordinate covalent bond) to the metal it chelates.

For true chelation to occur the donating atom/s must also be in a position within the chelating molecule so that a formation of a ring with the metal ion can occur. The term sequestered deals more with the action of chelation or complexing, not the actual chemical arrangement or definition. The term “complexed” originates from combinations of minerals and organic compounds that do not meet the guidelines of a true chelate.

Chelators are used in producing nutritional supplements, fertilizers, chemical analysis, as water softeners, commercial products such as shampoos and food preservatives, medicine, heavy metal detox, and industrial applications. Citric Acid is one of the organic acids commonly used as a chelating agent. It is considered an excellent chelating agent that binds metals. It is used to remove lime scale from boilers and evaporators. It can be used to soften water, which makes it useful in soaps and laundry detergents. By chelating the metals in hard water, it lets these cleaners produce foam and work better without need for water softening devices. Citric acid is the active ingredient in some bathroom and kitchen cleaning solutions. A solution with a 6% concentration of citric acid will remove hard water stains from glass without scrubbing. In industry, it is used to dissolve rust from steel. Citric acid can be used in shampoo to wash out wax and coloring from the hair.

One of the many sources of natural citric acid is citrus fruits. The calamansi, a citrus fruit, is believed native to China and thought to have been taken in early times to Indonesia and the Philippines. It became the most important Citrus juice source in the Philippine Islands. In the Philippines, the extracted juice, with the addition of gum tragacanth as an emulsifier, is pasteurized and bottled commercially. The fruit juice is used in the Philippines to bleach ink stains from fabrics.

Because of such may uses and benefits to Filipinos and also because of the likely warm climate of the Philippines, the fruit is indigenous and widely cultivated in the Philippines. Calamansi is available year round in the Philippines and is usually seen in its unripened state as a dark green fruit, but if left to ripen it turns a tangerine orange color. It was introduced to the U.S. as an “acid orange” about 1900.

In 2010, the Asia-Pacific region was the largest outlet, generating about 45% of worldwide demand for chelating agents. The region was then followed by Western Europe and North America. The global chelating agents market is expected to reach more than 5 million tones in 2018.

In year 2006, global production of citric acid has already reached 1.4 million tones. The actual price of citric acid is about $1 or 41 pesos per kilo. As a result, there is an average of ₱57,400,000,000 for year 2006. Furthermore, there is an annual growth of 3.5–4.0 % in demand/consumption of citric acid. Due to the aforementioned annual growth of 3.5–4.0 % in demand and consumption of citric acid, there is an obvious need for increase in citric acid productivity. This is early recommended for scarcity prevention and wise management of citric acid production.

One of the main reasons why the researchers decided on this study is to contribute to this scarcity prevention and wise management of citric acid production. The Philippines is one of the many countries that significantly benefits from citric acid mainly as a chelating agent in products like shampoos, laundry detergents and soaps. In 1917, the American food chemist James Currie discovered certain strains of the mold Aspergillusniger could be efficient citric acid producers, and the pharmaceutical company Pfizer began industrial-level production using this technique two years later, followed by CitriqueBelge in 1929.In this production technique, which is still the major industrial route to citric acid used today, cultures of A. niger are fed on a sucrose or glucose-containing medium to produce citric acid. The source of sugar is corn steep liquor, molasses, hydrolyzed corn starch or other inexpensive sugary solutions. After the mold is filtered out of the resulting solution, citric acid is isolated by precipitating it with lime (calcium hydroxide) to yield calcium citrate salt, from which citric acid is regenerated by treatment with sulfuric acid.(Lotfy, Walid A. et al., 2007)

Prior to the fermentative process, citric acid was isolated from citrus fruits. The juice was treated with lime to precipitate calcium citrate, which was isolated and converted back to the acid. Citric acid exists in greater than trace amounts in a variety of fruits and vegetables, most notably citrus fruits. Lemons and limes have particularly high concentrations of the acid; it can constitute as much as 8% of the dry weight of these fruits (about 47 g/L in the juices). The concentrations of citric acid in citrus fruits range from 0.005 mol/L for oranges and grapefruits to 0.30 mol/L in lemons and limes. Within species, these values vary depending on the cultivar and the circumstances in which the fruit was grown.(Penniston KL et al., 2008) Usually produced in powder form, citric acid is naturally found in citrus fruits. It easily mixes into liquids, making it a valuable acid. Lemons and limes have high concentrations of citric acid, accounting for their bitter taste. (Ellis-Christensen, Tricia 2012) Based on information gathered by the researchers, the country’s very own fruit Calamansi can be possible source of Citric Acid. From this, the researchers have ascertained that abundance of Calamansi in the country can be of great use in a wise major production of Citric acid.

The researchers have also considered the fact that if this study will make it, a lot of Filipinos who are unemployed can be potential workers and farmers for the said manufacture. The study specifies at the utilization of citric acid from Calamansi as chelating agent. On the assumption that the study will be a success, Calamansi chelator can be a major breakout for the country’s advancement in economy in terms of producing chemical analysis, as water softeners, commercial products such as shampoos and industrial applications. Aside from the personal use the country can get from this study, the researchers have also realized the chance of being exported to other lands; all depends to the study’s success. Using this study, the need for citric acid as a chelating agent in the country will be aided and exportation of Citric Acid from other lands will be disregarded. Through this research, the researchers will create citric acid mainly using Calamansi and determine if it is an effective chelator in terms of hardness of water, pH level and suds produced. Therefore, the title of this study is “THE UTILIZATION OF CALAMANSI (Citrus microcarpa) IN MAKING POWDERED FORM CHELATING AGENT” B. Statement of the Problem

The following are the objectives of the study:
B.1 General Objective:
Generally, the study aims to utilize Calamansi as water softener by its Citric Acid property as chelating agent. B.2 Specific Objectives:
Specifically, the study also aims to answer the following questions: 1.) Was the Calamansi citric acid effective as a chelator in terms of: a. pH level?  b. Suds produced?

C. Hypothesis
Null Hypothesis: There is no significant difference on the effectiveness of Calamansi as a chelator in terms of: a. pH level?
b. Suds produced?
D. Significance of the Study
This study will greatly benefit the following:
1. Small Households. Every household and every factory uses water, and none of it is pure. The cure to these water impurities is too much in price for most of the Filipino households. In addition, it is not available locally and might have to order via online in order to consume. This will no longer be assuming that the success of the study will establish a future steady industry of the product which will make the accessibility of product easier to Filipinos in need for the product’s usage. 2. Country’s Economy. First, it will serve as a big support in the country’s personal use. Thus, exportation of Citric Acid from other lands will be disregarded. Citric acid as a chelating agent alone is a great assistance to a lot of products here in the country. Other uses of Citric acid has been concisely explained and can also be considered and experimented for more immense use for the betterment of the nation. It can also be a potential product for export if it receives more financial support and much focused in the chemistry industry.

The Calamansi Processing Facility in Barangay San Miguel, Roxas was successfully launched last September 8, 2012 through the collective efforts of the Oriental Mindoro Calamansi Producers Association (OMCPA), the Department of Science and Technology – MIMAROPA (DOST-MIMAROPA), the Department of Labor and Employment Region IV-B (DOLE IV-B), and the Office of Oriental Mindoro 2nd District Rep. Reynaldo V. Umali. The project aims to develop new and competitive products from calamansi as well as address the problems long besetting the calamansi producers: low prices during peak seasons through value-adding which would guarantee steady income for the farmers and offer employment opportunities for the women in the area. This occurrence serves as the first step for the exportation. With even more support from the government, exportation is possible. 3. Future Researchers. This study is beneficial to them because this will serve as a spring board or background for their future studies. E. Scope and Limitations

The study entitled “THE UTILIZATION OF CALAMANSI (Citrus microcarpa) IN MAKING POWDERED FORM CHELATING AGENT” was scheduled by the researchers to finish within a time span of nine months as allotted by their research teacher, Mrs. Luzviminda M. Bago.

The only raw material used is Calamansi which was purchased in the local market at Baliuag, Bulacan since the study was conducted in the laboratory Montessori de Sagrada Familia. The chemicals used which is Calcium Hydroxide and Sulfuric Acid was purchased in a medical supplies store in Bambang, Rizal Avenue, Manila.

The researchers were after the utilization of Calamansi as water softener by its Citric Acid property as chelating agent, And not in the massive production of the product for market use. F. Definition of Terms

The study included words that may need further understanding in order to fully interpret the concept and idea of this research. The following terms served as keywords to the study that may sound unfamiliar and needs explanations to totally relate suchlike the research is about. Calmansi. Citrofortunella microcarpa, the Calamansi or Calamondin, is a citrus fruit in the family Rutaceae native to the Philippine Islands. It is used as a variable in the study. Calcium Hydroxide. Ca (OH)2, colorless crystal or white powder. It can be generated as a part of the fermentation process that makes citric acid. Compounds such as this cause the reaction that generates the mineral. Chelation. Chelation describes a particular way that ions and molecules bind metal ions. According to IUPAC, the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central atom. Usually these ligands are organic compounds. Chelating Agent/Chelator.

Chelating agents are chemical substances that contain molecules capable of bonding securely to minute particles of metal called ions. Citric Acid. Citric acid is a natural acid that is found in many fruits. It is very bitter, and it gives fruits like lemons and limes their characteristic sour flavor. Citric acid is an excellent chelating agent, binding metals. Hard Water. Hard water is water that has high mineral content (in contrast with “soft water”). Hard drinking water is generally not harmful to one’s health, but can pose serious problems in industrial settings, where water hardness is monitored to avoid costly breakdowns in boilers, cooling towers, and other equipment that handles water. In domestic settings, hard water is often indicated by a lack of suds formation when soap is agitated in water. Wherever water hardness is a concern, water softening is commonly used to reduce hard water’s adverse effects. Sediments. They are the material that settles to the bottom of a liquid; lees. Also, the solid fragments of inorganic or organic material that come from the weathering of rock and are carried and deposited by wind, water, or ice. These tend to precipitate out as adherent solids on the surfaces of pipes and especially on the hot heat exchanger surfaces of boilers.

They act as thermal insulation that impedes the flow of heat into the water; this not only reduces heating efficiency, but allows the metal to overheat, which in pressurized systems can lead to catastrophic failure. Organic Acid. An organic acid is an organic compound with acidic properties. pH. In chemistry, pH is a measure of the activity of the (solvated) hydrogen ion. pH, which measures the hydrogen ion concentration, is closely related to, and is often written as, pH. Pure water has a pH very close to 7 at 25°C. Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline Sulfuric Acid. Sulfuric acid is an oily, colorless liquid with no odor. It is a key component in batteries, wastewater treatment, ore production, and fertilizer creation. Sulfuric acid (alternative spelling sulphuric acid) is a highly corrosive strong mineral acid with the molecular formula H2SO4. It is a colorless to slightly yellow viscous liquid which is soluble in water at all concentrations

G. Conceptual Framework

Paradigm
The paradigm shows the input, process and output of the experimental study. Citric Acid is naturally found in Citrus Fruits like Calamansi. To get the citric acid in Calamansi, the fruit has undergone different processes like extracting, heating, cooling, filtering, pounding and adding of chemicals such as Calcium Hydroxide and Sulfuric Acid. After processing, the product is subjected to different tests to measure its effectiveness as a chelating agent.

Chapter II
Review of Related Literature and Related Studies
This section indicates the related studies and the preliminary data gathered. It also includes research findings, published or unpublished theories and principles formulated by experts or authorities in some field of discipline; and ideas or opinions of experts contained in books, pamphlets, magazines and periodicals. Related Literature

Calamansi
According to the Germplasm Resources Information Network, accessed on 2011, Citrofortunella microcarpa, the Calamondin or Calamansi, is a fruit tree in the family Rutaceae native to the Philippine Islands and has been dubbed the calamondin, golden lime, panama orange, chinese orange, acid orange, calamonding, or calamandarin in English.] It is believed to originate from China and has spread throughout Southeast Asia, India, Hawaii, the West Indies, Central and North America. The plant is characterized by wing-like appendages on the leaf stalks and white or purplish flowers. Its fruit has either a spongy or leathery rind with a juicy pulp that is divided into sections. The fruit is indigenous and widely cultivated in the Philippines. Calamansi is available year round in the Philippines and is usually seen in its unripened state as a dark green fruit, but if left to ripen it turns a tangerine orange color. According to the Food and Nutrition Research Institute on 2007, it is said that calamansi is an acid citrus, a group that includes lemons and limes. This is because the flesh is orange, juicy and acidic and with a fine lime-orange flavor. One bite of this fruit can pucker your mouth.

Calamansi fruit, when ripe, is very sour when first tasted but subsequent tasted fruits can make your mouth sweet. If the fruit is picked too soon, calamansi is bitter.The many uses of calamansi make this fruit a wonder fruit. Calamansi halves or quarters may be squeezed on iced tea, seafoods and meat, to enhance iron absorption. It can also be preserved whole in sugar syrup or made into sweet pickles or marmalade. The calamansi juice is primarily valued for making acid beverages. It is often incorporated like lime or lemon juice to make gelatin salads and custard pie. Calamansi juice is also used as a meat tenderizer and adds flavor to the dishes. According to the Nutritional Guidelines for Filipinos 2000 developed by the Technical Working Group headed by the Food and Nutrition Research Institute of the Department of Science and Technology (FNRI-DOST), one should consume two servings of fruit daily. Said serving ranges from 45 to 300 grams, depending on the size and variety of fruit, one of which is a vitamin C-rich food . Thirty-four medium sized pieces of calamansi are needed to meet the daily requirement of vitamin

C. The calamansi fruit also has many medicinal uses. The fruit juice is also applied to the scalp after shampooing to eliminate itching and promote hair growth. Rubbing calamansi juice on insect bites banishes itching and irritation. It bleaches freckles and helps to clear up acne. The most popular medicinal use of calamansi is when taken orally as a cough remedy. The fruit juice is slightly diluted and drunk warm. Aside from the food and medicinal uses of calamansi, the fruit juice is used to bleach ink stains from fabrics and serve as a body deodorant. According to Jesse M. Pine, PSTD, DOST-Oriental Mindoro, The Calamansi Processing Facility in Barangay San Miguel, Roxas was successfully launched last September 8, 2012 through the collective efforts of the Oriental Mindoro Calamansi Producers Association (OMCPA), the Department of Science and Technology – MIMAROPA (DOST-MIMAROPA), the Department of Labor and Employment Region IV-B (DOLE IV-B), and the Office of Oriental Mindoro 2nd District Rep. Reynaldo V. Umali.

The project aims to develop new and competitive products from calamansi as well as address the problems long besetting the calamansi producers: low prices during peak seasons through value-adding which would guarantee steady income for the farmers and offer employment opportunities for the women in the area. DOST-MIMAROPA provided technological assistance amounting to PhP 417,500.00 for the acquisition of a hydraulic press, double-jacketed kettle, and dispenser; DOLE IV-B was tapped to provide production accessories and additional capital worth PhP 300,000.00 ; and the Office of Rep.

Reynaldo V. Umali provided the building, including electricity and water supply estimated at around PhP 1.4M. According to the same source, Dr. Ma. Josefina P. Abilay, Regional Director of DOST-MIMAROPA emphasized that the technical assistance given is a way to add value to calamansi, thus making it sold for competitive prices in the market and would benefit the members of the OMCPA. Said Dr. Abilay, “Hindi lamang ready-to-drink juice o concentrate ang maaaring gawin dito. Ang balat at iba pang bahagi ng calamansi ay marami pa ring by-products na magagawa. Maaari pa rin itong i-alok sa mga health-conscious na mamimili kapag nilagyan ng pulot o coco sugar at maipagbibili pa sa mas mataas na halaga.”(The processing facility will not just produce ready-to-drink calamansi juice or concentrate. The peel and other parts of the calamansi fruit have other uses as well. And we can sell our products at higher prices when sweetened with honey or coco sugar as added ingredients.) Citric Acid

According to Science DAILY EDITION 2012, Citric acid is a weak organic acid found in citrus fruits. It is a natural preservative and is also used to add an acidic (sour) taste to foods and soft drinks. In biochemistry, it is important as an intermediate in the citric acid cycle and therefore occurs in the metabolism of almost all living things. It also serves as an environmentally benign cleaning agent and acts as an antioxidant. Citric acid exists in a variety of fruits and vegetables, but it is most concentrated in lemons and limes, where it can comprise as much as 8 percent of the dry weight of the fruit. Clayton Kim’s “HOW TO EXTRACT CITRIC ACID FROM FRUIT” became the chief basis of the procedure of the researchers in making the product by extracting the citric acid that is found naturally in citrus fruits. In the work done by Clayton Kim, he extracted the acid form the Lemon fruit which is substituted by Calamondin in this study because of availability and abundance matters. Additional variations in point in time were also done by the researchers all for the success of the study. (Columbia University; Material Safety Data Sheet Citric Acid; March 2002)

According to Chemistry Professor Bassam Z. Shakhashiri of University of Wisconsin-Madison, a chelate is a chemical compound composed of a metal ion and a chelating agent. A chelating agent is a substance whose molecules can form several bonds to a single metal ion. In other words, a chelating agent is a multidentate ligand. An example of a simple chelating agent is ethylenediamine. EDTA is a versatile chelating agent. It can form four or six bonds with a metal ion, and it forms chelates with both transition-metal ions and main-group ions. EDTA is frequently used in soaps and detergents, because it forms a complexes with calcium and magnesium ions. These ions are in hard water and interfere with the cleaning action of soaps and detergents. The EDTA binds to them, sequestering them and preventing their interference. In the calcium complex, [Ca(EDTA)]2–, EDTA is a tetradentate ligand, and chelation involves the two nitrogen atoms and two oxygen atoms in separate carboxyl (-COO–) groups. EDTA is also used extensively as a stabilizing agent in the food industry. Food spoilage is often promoted by naturally-occurring enzymes that contain transition-metal ions.

These enzymes catalyze the chemical reactions that occur during spoilage. EDTA deactivates these enzymes by removing the metal ions from them and forming stable chelates with them. It promotes color retention in dried bananas, beans, chick peas, canned clams, pecan pie filling, frozen potatoes, and canned shrimp. It improves flavor retention in canned carbonated beverages, salad dressings, mayonnaise, margarine, and sauces. It inhibits rancidity in salad dressings, mayonnaise, sauces, and sandwich spreads. EDTA salts are used in foods at levels ranging from 33 to 800 ppm. In other applications, EDTA dissolves the CaCO3 scale deposited from hard water without the use of corrosive acid. EDTA is used in the separation of the rare earth elements from each other. The rare earth elements have very similar chemical properties, but the stability of their EDTA complexes varies slightly.

This slight variation allows EDTA to effectively separate rare-earth ions. EDTA is used as an anticoagulant for stored blood in blood banks; it prevents coagulation by sequestering the calcium ions required for clotting. As an antidote for lead poisoning, calcium disodium EDTA exchanges its chelated calcium for lead, and the resulting lead chelate is rapidly excreted in the urine. The calcium salt of EDTA, administered intravenously, is also used in the treatment of acute cadmium and iron poisoning. Dimercaprol (2,3-dimercapto-1-propanol) is an effective chelating agent for heavy metals such as arsenic, mercury, antimony, and gold. These heavy metals form particularly strong bonds to the sulfur atoms in dimercaprol. Related Studies

Citric Acid
The study entitled “CITRIC ACID PRODUCTION BY ASPERGILLUS NIGER USING MOLASSES AND PUMPKIN AS SUBSTRATES” is conducted by Ibrahim Khalil of Department of Biochemistry and Molecular Biology of Jahangirnagar University in benefit of his country Bangladesh. The study aims to help in the economic development of the country in ways such as having it own production using its own products to prevent having to import in other countries that causes much amount of money. Bangladesh, like Philippines, is in great need of Citric Acid as it is a very essential chemical and extensively used in food and pharmaceutical industries. The study entitled “PRODUCTION OF CITRIC ACID BY FUNGI” is the study by Mustafa Yigitoglu that helped the researchers in terms of knowing some information about the early production of Citric Acid which is more related than prior study. The researchers have collected information regarding the very first production of Citric Acid which is isolated and crystallized from lemon juice by Scheele. In addition, there is also information presented in the study that a small amount of citric acid, approximately less than 1% of total world production, is still produced from citrus fruits in Mexico and South America where citrus fruits are available economically.

More information on Citric Acid production through extracting from citrus fruits was provided by the study “FERMENTATION OF PINEAPPLE TASTE JUICE FOR THE PRODUCTION OF CITRIC ACID USING CANDIDA LIPOLYTICA ATCC” by Joyce Koshy. Citric acid extracted from fruits is commercially known as natural citric acid in contrast to the citric acid produced by microbial fermentation. Until the early days of this century, citric acid was produced from lemon juice although Wehmer had described this organic acid as a metabolic product of moulds of the genera Penicillium and Mucor. Today, most of the citric acid used in food and other industries come from fungal fermentations. Although chemical synthesis of this organic acid is possible, no competitive synthetic process that is superior to fungal fermentation has been developed (Kapoor, et al, 2002). In the study “PRODUCTION OF CITRIC ACID FROM CITRUS FRUIT WASTES” by Local Isolate and MTCC, they used citrus fruit wastes as the substrates, which are easily available raw materials. Four substrates – sweet lime pulp, orange pulp, sweet lime peel and orange peels were selected.

The citric acid production was carried out by submerged fermentation. This study has been proven that pulp was found to be more effective than peels. The researchers found this data useful in choosing to hand-squeezed the Calamondin fruit pulp to get its juice. In the study “EFFECT OF DIFFERENT EGTA CONCENTRATIONS ON DENTIN MICROHARDNESS” by Braz Dent J, the effect of 1%, 3% and 5% EGTA (ethylene glycol-bis-(beta-amino-ethyl ether) N,N,N’,N’-tetra-acetic acid) on the microhardness of root dentin of the cervical third of human teeth was studied. Five newly extracted maxillary incisors were sectioned transversely at the cementoenamel junction, and the crowns were discarded. The roots were embedded in blocks of high-speed polymerized acrylic resin and cut transversely into 1-mm sections. The second section of the cervical third of the root of each tooth was sectioned and divided into four parts. Each part was placed on an acrylic disc that was used as a base for microhardness measurement. Fifty microliters of 1% EGTA, 3% EGTA, or 5% EGTA were applied to the dentin surface. Deionized and distilled water was used as control.

Dentin microhardness was then measured with a load of 50 g for 15 s. Statistical analysis showed that the three concentrations of the chelating solution EGTA significantly reduced dentin microhardness when compared with water, and that there was a statistically significant difference among the three solutions. In the study “EVALUATION OF THE EFFECT OF EDTA, EDTAC AND CITRIC ACID ON THE MICROHARDNESS OF ROOT DENTINE” by Mauricio MH of Department of Endodontics, Rio de Janeiro State University, Rio de Janeiro, Brazil, microhardness decreased with increasing time of application of chelating solutions. There were no significant (P > 0.05) differences between initial microhardness for the three groups as well as after 1 min of application of the substances. After 3 min, EDTA produced a significantly greater reduction in microhardness. However, there was no significant difference between EDTA and EDTAC after 5 min. Citric acid caused significantly less reduction in microhardness. Overall, citric acid was least effective in reducing dentine hardness whilst EDTA had the strongest effect.

To evaluate the effect of citric acid, ethylenediaminetetraacetic acid (EDTA) and ethylenediaminetetraacetic acid plus Cetavlon (EDTAC) solutions on the microhardness of human root canal dentine. The Methodology was sixteen maxillary human canines were sectioned transversely at the cemento-enamel junction and the crowns were discarded. Subsequently, each root was embedded in an epoxy resin cylinder and their middle third sectioned horizontally into 4 mm thick slices. The samples were randomly divided into three groups according to the chelating agent employed, as follows (n = 6): group 1: EDTA 17%, group 2: EDTAC 17% and group 3: citric acid 10%. Dentine microhardness was then measured with a load of 50 g for 15 s. At the beginning of the experiment, reference microhardness values were obtained for samples without any etching (t = 0 min). The same samples were then exposed to 50 microL of the chelator solution for 1, 3 and 5 min. The Student’s t-test (P < 0.05) was used to compare results for different times for each chelator and different chelators for each time.

In the study “EFFECT OF EDTA AND CITRIC ACID SOLUTIONS ON THE MICROHARDNESS AND THE ROUGHNESS OF HUMAN ROOT CANAL DENTIN” by Belli S. of Department of Endodontics, Faculty of Dentistry, University of Selcuk, Turkey, The purpose of this study was to evaluate the effect of citric acid and EDTA solutions on the microhardness and the roughness of human root canal dentin. Forty-five human teeth sectioned longitudinally were used. Specimens were randomly divided into three groups of 30 teeth each and were treated as follows: (a) one molar (19%) citric acid (C6H8O7) for 150 s followed by 5.25% NaOCl; (b) 17% EDTA for 150 s and rinsed with 5.25% NaOCl; (c) rinsed with distilled water and served as control. Three groups were then divided into two subgroups of 15 specimens each. The specimens, in first subgroup were subjected to Vicker’s testing whereas the second subgroup underwent surface roughness testing. The results were analyzed using one-way ANOVA and Tukey tests. Significant differences were observed in microhardness among the test groups, citric acid group being the least hard (p 0.05). Also, citric acid significantly increased surface roughness.

Chapter III
Methodology
This chapter discusses the methods used in this study, as well as the materials and equipment gathered by the researchers. A. Method of Researched Used

The study used an experimental method of research. This is the method used by the researchers since the study aims to produce Citric Acid extracted from Calamansi fruit and use it as chelating agent. The Experimental Method explains and gives thorough analysis on the effectiveness of a certain variable with a definite period of time. Considering this, the experimental research method is appropriate to the study. B. Materials and Equipment

Eight kilograms of Calamansi fruit was collected. 120 milliliters of Calcium Hydroxide and 150 milliliters of Sulfuric Acid were also purchased. Measuring cups and tablespoons were also prepared. Two glass containers, one 10×10 white cloth and one metal spoon were also collected. One thermometer was also purchased. C. General Procedure

Eight kilos of Calamansi fruit was hand-squeezed to get its juice. Two liters of the extracted fluid was combined with eight tablespoon of calcium hydroxide in a glass container. The liquid solution was then mixed in a circular motion and was heated at medium-high heat in the microwave for one minute. The process was done to turn citric acid into calcium citrate. After heating, the container was placed in the freezer for 15 minutes to let the solution cool down. After the cooling process, the solution was filtered using a white cloth. The remains of calcium citrate were scraped from the cloth using a spoon and were separated in another container. The whole process was repeated until 300 ml of calcium citrate was collected. After accumulating the desired amount of calcium citrate, it was combined with 150 ml of sulphuric acid and was transferred into a cooking pot. The new solution was cooked in 150° F for 10 minutes. The cooking process was done to let the carbon evaporate. After cooking, the 25 grams solution was set aside to cool for 30 minutes. The resulting product is the solid form of Citric acid. Chunky pieces were crushed with the back of metal spoon to achieve the powder form. D. Application of Treatments

For Treatment 1, four kilos of Calamansi fruit was hand-squeezed to get its juice. One liter of the extracted fluid was combined with four tablespoon of calcium hydroxide in a glass container. The liquid solution was then mixed in a circular motion and was heated. After heating, the container was placed in the freezer. After the cooling process, the solution was filtered using a white cloth. The whole process was repeated until 150 ml of calcium citrate was collected. After accumulating the desired amount of calcium citrate, it was combined with 75 ml of sulphuric acid and was transferred into a cooking pot. The new solution was cooked in 150° F for 10 minutes. After cooking, the 25 grams solution was set aside to cool for 30 minutes. The resulting product called chelating agent is to be poured into a liter of tap water. For Treatment 2, four kilos of Calamansi fruit was hand-squeezed to get its juice.

One liter of the extracted fluid was combined with four tablespoon of calcium hydroxide in a glass container. The liquid solution was then mixed in a circular motion and was heated. After heating, the container was placed in the freezer. After the cooling process, the solution was filtered using a white cloth. The whole process was repeated until 150 ml of calcium citrate was collected. After accumulating the desired amount of calcium citrate, it was combined with 75 ml of sulphuric acid and was transferred into a cooking pot. The new solution was cooked in 150° F for 10 minutes. After cooking, the 25 grams solution was set aside to cool for 30 minutes. The resulting product called chelating agent is to be poured into a liter of groundwater. For Treatment 3, which could be considered the controlled in terms of the utilization of calamansi as chelating agent, is a liter of tap water without the chelating agent. Treatment 4 is a liter of groundwater without the chelating agent.