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Nitrite-Indole Reaction: Spectrophotometric Study Thesis

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Updated: May 11th, 2021

Nitrates and nitrites are ions that consist of nitrogen covalently attached to three and two oxygen atoms respectively. These ions occur naturally and form part of the nitrogen cycle. Of the two ions, the nitrate ion (NO3) is more stable than the nitrite ion (NO2) but can be reduced by the actions of microorganisms to form the latter ion. The unstable nitrite ion is susceptible to redox reactions that can either oxidize it to form nitrates or reduce it into other chemical substances. The purpose of this chapter is to explain the Beer’s law, describe pertinent features of two spectrophotometers (UV-Visible Shimadzu and Lambda 365 UV), describe resources for NaNO2, the risk of NaNO2 in water and Zamzam water.

Beer’s Law

Chemical compounds can absorb or transmit ultraviolet or visible light. The amount of light absorbed or transmitted can be measured as transmittance or absorbance. Overall, absorbance is indirectly proportional to transmittance, meaning that less light is transmitted as more light is absorbed. Consequently, if light passes through a solution without any of it being absorbed, the net absorbance is 0 but the transmittance is 100%.

Conversely, the percent transmittance becomes 0 if all the light is absorbed. Beer Lambert’s law explains this relationship by relating absorbance to the concentration of a substance. Beer’s law states that A=ebc where A denotes the absorbance, e represents a constant known as molar absorptivity, which is measured in mol-1 cm-1 (Herzog, Schultheiss & Giesinger 2018). In the equation, the terms b and c denote the path length in centimeters and concentration of the substance in moles per liter.

Spectrophotometry is an analytical technique that uses Beer’s law to determine the concentration of solutions by measuring absorbance when the light at a specific wavelength is passed through a solution. The key instrument used in this procedure is a spectrophotometer. The following sections describe spectrophotometers from two popular manufacturers.

UV Visible Shimadzu

Shimadzu is a Japanese manufacturing company that is renowned for the production of analytical instruments and equipment for the last six decades. Among the most popular equipment are UV-Vis spectrophotometers that meet researchers’ needs for sturdiness, effortless use, and authentication. About six different models of UV-Vis spectrophotometers are available, each with its unique features.

They include the UV-1800, UV-1900, UV-2600, UV-2700, UV-1280, and BioSpec-Nano (Shimadzu 2019). UV-1900 has a double beam that improves usability, functioning, and conformity, whereas the starting research-grade UV-2600 boasts of an extended wavelength range to 1400 nm. Conversely, UV-2700 has a double monochromator that makes it suitable for analytical procedures with high absorbance values. These three systems have patented low-ray-light gratings, permit USB connections, and are available with a full-featured UVProbe software.

Other models include the multipurpose single-beam UV-1280 as well as a cost-effective BioSpec-nano that has low maintenance costs and micro-volume capability. The BioSpec-nano ideal for a life science laboratory and is capable of fast, simple quantification of nucleic acids in addition to protein analysis using very small analyte volumes in the range of 1µL to 2µL. The spectrophotometers provide high 1-nm resolution through a small double-beam appliance. They can be used as standalone units or connected to computers through the UVProbe software. Furthermore, they have inbuilt corroboration software to guarantee operational accuracy (Shimadzu 2019).

To understand the robustness of UV-Visible spectrophotometers from Shimadzu, details, and special features of their simplest UV-1800 are provided. The UV-1800 uses an accurate Czerny-Turner optical system (Davis Instruments 2019). Apart from the ability to be used as standalone instruments or PC-controlled devices through the UV Probe software, the UV-1800 also permits data printing through an elective screen-copy printer or printers that can use the Printer Command Language (PCL) control codes.

The spectrophotometer operates in seven different modes, the first being the photometric mode. In this mode, the instrument can measure an analyte’s transmittance or absorbance at single or multiple wavelengths simultaneously. Up to eight different wavelengths can be read. However, when obtaining multiple wavelengths, computations can only be done for data involving four wavelengths in addition to calculations of discrepancies between two wavelengths and their proportions.

The spectrum mode uses wavelength scanning data to generate spectra. Consequently, it is possible to detect and keep track of alterations in the sample by repetitive scans. Moreover, data processing procedures such as the expansion or reduction of spectra, determining the presence of peaks and their areas can be done. The quantitation mode uses standard samples to create a calibration curve that is used to determine the concentration of the analytes. Different combinations of wavelengths, including derivatives of one to three wavelengths, can be used. Subsequently, first-to-third regression standard curves can be developed.

The kinetics mode can measure how the absorbance of an analyte changes with time to compute enzyme activity. The two possible options under this function are the rate measurement or kinetics measurement. The time-scan mode makes it possible to find how attributes such as absorbance, energy, or transmittance change as time progresses. Another unique style is the multi-component quantitation mode that can measure and differentiate up to eight different compounds mixed in one sample. Pure versions of the various analytes can work as standards.

UV-1800 has a biomethod mode that can analyze proteins and DNA through various quantitation techniques. For example, the absorbance ratio at 260 and 230 nm is useful for protein analysis, whereas the ratio at 260 and 280 nm is used for DNA analysis. Other protein quantitation methods that can be used in the biomethod mode include the Biuret, Lowry, bicinchoninic acid assay (BCA), Coomassie Brilliant Blue (CBB), and direct measurement approaches (Davis Instruments 2019).

Shimadzu UV-Visible spectrophotometers also guarantee data security through pre-programmed security and authentication functionality. Functions can be limited based on three user levels, namely developer, administrator, and operator (or user). Automated validation functions encompass the exactness, repeatability, and resolution of wavelengths, photometric precision, stray light, and noise levels. An important maintenance attribute that can be documented in the operating time of the lamp to help the user to expect a replacement and prevent unnecessary interruptions.

Lambda 365 UV Spectrophotometers (PerkinElmer)

The Lambda 365 is a UV-Vis spectrophotometer that provides high-tech performance that meets the expectations of drug companies, geneticists, and analytic chemists as well as quality assurance and quality control requirements of manufacturing companies. The Lambda system boasts of 21 CFR part 11 compliant software to support a wide range of analytical procedures ranging from conventional techniques to applications that need regulatory observance (PerkinElmer 2019).

It offers an array of spectral bandwidth between 0.5 nm and 20 nm to suit different application requirements. Additionally, the system has the capacity for several accessories to enhance its usability. Examples of such accessories include multicell changers, optical fiber probes to facilitate distant measurement, an integrating sphere to measure color, and different cuvette holders to match diverse sampling needs.

Furthermore, the sample partition is large enough to hold at least ten sampling fixture combinations that are aligned automatically by the software to improve the sample positioning. All attachments are easy to install thus minimizing the time spent on setup. These provisions make the Lambda 365 double-beam system the most appropriate in instances that require high stability and minimal stray light.

Resources of NaNO2

In agricultural practices, inorganic fertilizers contain nitrogen as nitrates. Industrially, nitrates are used as oxidizing agents in various processes (Canter 2019). They are also used as raw materials in the manufacture of glass (as pure potassium nitrate) and explosives. In the food sector, sodium nitrite serves as a food preservative, particularly in the curing of meats. Plants also contain nitrates, which is a key nutrient for healthy growth. Mammals produce nitrate and nitrites endogenously. Furthermore, oral microflora is capable of converting nitrates secreted into saliva to nitrites.

Air contains approximately 78% of nitrogen (Canter 2019). These levels can be raised through man-made processes that generate nitrogen such as agriculture, industrial activities, and motor vehicles. Consequently, nitrogenous compounds are created in the atmosphere when lightning catalyzes the chemical conversion of this nitrogen to oxides of nitrogen. Nitrate can be found in the air in the form of nitric acid, organic acids, nitrate radicals, inorganic aerosols, as well as organic gases.

Risk of NaNO2 in Water

Agricultural activities such as excessive use of nitrogen-containing fertilizers and manure lead to the accumulation of nitrates in the soil, which leach and gain access to surface and groundwater. Furthermore, wastewater treatment in septic tanks and other treatment plants leads to the oxidation of nitrogenous substances from human and animal excreta. During this process, treated wastewater containing nitrites is distributed in galvanized pipes under anaerobic conditions.

Nitrosomonas bacteria take advantage of these conditions to convert the nitrates to nitrites. Additionally, poorly controlled chloramination processes in the residual disinfection process of wastewater can also lead to the formation of nitrites.

In soil, nitrogen-containing fertilizers have inorganic nitrogen while waste products from animals contain organic nitrogen that is first converted to ammonia through a decomposition step. The resultant ammonia is then changed to nitrates and nitrites by oxidation. Plants can absorb the excess nitrates and nitrites and use them as precursors for the biosynthesis of organic nitrogenous compounds. However, in aquifers without plants to absorb the nitrates and nitrites, aerobic conditions lead to the percolation of the compounds in large amounts. The rate of filtration is usually higher if the movement of soil water to the aquifer is downhill.

Under such conditions, denitrification or breakdown of the nitrates occurs at very low levels in the soil and rocks that make up the aquifer. When oxygen is absent, nitrate can undergo total denitrification to form free nitrogen. Other factors that determine the fate of nitrate in soil and whether or not it reaches water bodies include the quantity of rainwater, the height of water tables, availability of organic matter, and physicochemical attributes (Rebolledo et al. 2016).

The processes of nitrification and denitrification can also happen in surface water if the temperatures and pH are conducive. Nonetheless, the most effective way of reducing nitrate concentrations in the surface water is by establishing plant cover. This way, the levels of nitrate that move with groundwater can also be reduced.

Given the probability of finding nitrites and nitrate in potable water, the Environmental Protection Agency (EPA) has established the Maximum Contaminant Level (MCL) of nitrate at 10 mg/L or 10 parts per million in drinking water (Agarwal 2015).

Rainwater has been shown to have up to 5 mg/L of nitrate, whereas surface water contains between 0 and 18 mg/L of the ion. However, surface runoff from agricultural activities and animal waste can elevate nitrate levels further. Seasons also play a role in the concentrations of nitrates in the water. For example, during the rainy season, nitrate-rich aquifers can empty into water bodies such as rivers thus increasing the levels of nitrates. The concentration often fluctuates with the season and may increase when the river is fed by nitrate-rich aquifers.

Zamzam Water

Water is an essential solvent for the sustenance of life due to its involvement in physiological functions. It is possible for a human being to go without food for at least 30 days However, the same cannot be said for water. The maximum period that one can survive without water is only seven days (Abu-Taweel 2017). Freshwater, which is the usable form of water only constitutes approximately 2.8% of the water on the entire earth is freshwater.

Therefore, water is a crucial but scarce resource. Even though the world’s water sources are rationed in the light of rapidly depleting resources, there exists a source of water called Zamzam (Khalid et al. 2014). Zamzam water is situated in Mecca, which is a sacred city for Muslims in the western region of Saudi Arabia, approximately 70 kilometers south of Jeddah. The coordinates of the city are a latitude of 21° 26′ 48″ N and longitude of 39° 53′ 46′′ E (Khalid et al. 2014). The altitude of the area is approximately 1399 feet above sea level.

Zamzam water is colorless and odorless. However, it has a characteristic taste and a pH of between 7.9 and 8.0, which shows that it is basic. Studies to determine the mineral content of Zamzam water indicate that sodium, calcium, potassium, magnesium, chloride, bicarbonate, sulfate, fluoride, and nitrate at concentrations of 133, 96, 43.3, 38.88, 163.3, 195.4, 124, 0.72, and 124.8 milligrams per liter in that order (Badar, Bamosa, Salahuddin & Al Meheithif 2019).

Short narratives that talk about Zamzam are recounted in the holy books of different religions such as the Quran, the Bible, and the Torah (Old Testament). Zamzam is described as a valuable gift from God. Among the Muslims, it is believed to be a branch from a holy spring in the inhospitable desert adjoining Mecca. It is used to aid recovery from various diseases as advised by Prophet Muhammad, who asserted that Zamzam water was good for whatever reason it had been drunk (Abu-Taweel 2017). In a separate Hadith, the Prophet held that Zamzam water could heal every disease and that its healing properties arise from the fact that angel Jibril dug it and made Ismail be the first partaker.

Apart from the religious beliefs regarding the healing properties of Zamzam water, several scientific inquiries have been made to ascertain or refute the claims made about the water. It has been shown that Zamzam water has strong anti-inflammatory properties. For instance, it displays inhibitory effects against tumor necrosis factor-alpha (TNFα) and interleukin 1 (IL1) (Abu-Taweel 2017).

Its analytic action has been demonstrated through an indirect effect on the immunology of the endocrine system and growth patterns of the human body. In a separate investigation to determine the effect of Zamzam water on dental health in children, it was observed that the average decayed, missing, or filled teeth score was lower in children with permanent teeth using Zamzam water than in the mixed dentition group using regular water (Vani et al. 2016). However, the differences were not statistically significant.

The effect of Zamzam water on the endocrine system has been verified by its application in the remedy of implantation malfunction, stimulation of prolactin from the endometrium, production of luteinizing hormone, angiopoietin receptors, and endometrial vascular endothelial growth factor (Abu-Taweel 2017).

Additionally, Zamzam water leads to the upregulation of intercellular communications in the gap junction as well as connexin 43 antibodies within the endometrium. Stem cell differentiation in the endometrium is also prompted by Zamzam water. This effect is attributed to its high concentrations of calcium and magnesium and support for biochemical reactions and coenzyme activity in the formation of antibodies (Al-Barakah, Al-jassas & Aly 2017).

Zamzam water is also beneficial in agricultural processes such as planting. For example, in a study conducted by Mutwally et al. (2015), the authors observed that using Zamzam water alone or mixing it with tap water led to substantial increases in the rates of germination, shoot dimensions, as well as the dry and wet matter of shoots. The rate of flowering in broad bean plants was also higher in plants watered using Zamzam water compared to other water treatments.

Studies also show that Zamzam water contains nitrates and nitrites that pose adverse health risks. For example, Badar et al. (2019) reported that mice given Zamzam water exclusively had higher levels of methemoglobin in their blood compared to mice that received ordinary and standardized bottled water. Hemoglobin is the standard oxygen transport protein that is found in the red blood cells of mammals. It comprises four heme molecules each containing a ferrous iron ion (Fe2+) at its center.

Methemoglobin is usually formed when the ferrous iron is oxidized to the ferric state (Fe3+). This aberrant form of the oxygen transport protein is incapable of transporting oxygen because it lacks an electron that is crucial for oxygen binding. Methemoglobinemia, which is the presence of methemoglobin in the blood, usually occurs following exposure to drugs or chemical substances with oxidizing potential. These substances can either be nitrites or amines. Methemoglobinemia causes the deprivation of vital organs of oxygen. This condition is attributed to the oxidation of the Fe2+ into Fe3+ by nitrites and becomes lethal when the methemoglobin levels reach 70% (Ferrari & Giannuzzi 2015).

Conclusion

Spectrophotometry applies Beer’s law to determine the concentration of substances, particularly colored ones by measuring their absorbance and comparing it to the concentration of known substances. The concentration of NaNO2, which is a harmful substance when ingested in large quantities, can be determined this way by reacting it with indole to produce a colored product. Zamzam is a popular source due to its reported healing properties. However, studies suggest that it may contain significant quantities of nitrite ions, which make it a possible health hazard. Therefore, there is a need to ascertain these claims by testing Zamzam water for nitrite ions.

Reference List

Abu-Taweel, GM 2017, ‘Effects of perinatal exposure to Zamzam water on the teratological studies of the mice offspring,’ Saudi Journal of Biological Sciences, vol. 24, no. 4, pp. 892-900.

Agarwal, R 2015, ‘Nitrate contamination in ground water of Jaipur District, Rajasthan, India: its impact on human health: a review’, Ground Water, vol. 9, pp. 0-68.

Al-Barakah, FN, Al-jassas, AM & Aly, AA 2017, ‘Water quality assessment and hydrochemical characterization of Zamzam groundwater, Saudi Arabia’, Applied Water Science, vol. 7, no. 7, pp. 3985-3996.

Badar, A, Bamosa, AO, Salahuddin, M & Al Meheithif, A 2019, ‘Effect of Zamzam water on blood methemoglobin level in young rats,’ Journal of Family & Community Medicine, vol. 26, no. 1, pp. 30-35.

Canter, LW 2019, Nitrates in groundwater, Routledge, Abingdon, UK.

Davis Instruments 2019, Web.

Ferrari, LA & Giannuzzi, L 2015, ‘Assessment of carboxyhemoglobin, hydrogen cyanide and methemoglobin in fire victims: a novel approach’, Forensic Science International, vol. 256, pp. 46-52.

Herzog, B, Schultheiss, A & Giesinger, J 2018, ‘On the validity of Beer–Lambert law and its significance for sunscreens’, Photochemistry and Photobiology, vol. 94, no. 2, pp. 384-389.

Khalid, N, Ahmad, A, Khalid, S, Ahmed, A & Irfan, M 2014, ‘Mineral composition and health functionality of Zamzam water: a review’, International Journal of Food Properties, vol. 17, no. 3, pp. 661-677.

Mutwally, HMA, Omar, SAM & Bedaiwy, M 2015, ‘Effect of water types on some growth parameters of wheat and broad bean plants under Al-Baha, KSA environmental condition’, Journal of Kuwait Chemical Society, vol. 33, pp. 25-27.

PerkinElmer 2019, . Web.

Rebolledo, B, Gil, A, Flotats, X & Sánchez, JÁ 2016, ‘Assessment of groundwater vulnerability to nitrates from agricultural sources using a GIS-compatible logic multicriteria model’, Journal of Environmental Management, vol. 171, pp. 70-80.

Shimadzu 2019, . Web.

Vani, NV, Idris, AM, Abuhaya, AH, Jafer, M & Almutari, DA 2016, ‘Assessment of calcium, magnesium, and fluoride in bottled and natural drinking water from Jazan Province of Saudi Arabia and a brief review on their role in tooth remineralization’, Journal of International Oral Health, vol. 8, no. 11, pp. 1012-1015.

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