DETERMINATION OF NITRATE IONS AT SUB-MIRCO LEVEL BY ATOMIC ABSORPTION SPECTROPHOTOMETRY

Abstract

An indirect, Atomic absorption spectrophotometric method has been developed for the determination of the nitrate ions. The method was based on the formation of the Cu(I)-neocuproine complex. First, the copper was reduced using the alkaline hydroxylamine sulfate, then nitrate sample was mixed with copper ions in aqueous phase. Mercuric sulfate was used to mask the chloride ions. After that whole mixture was allowed to react with the neocuproine in the organic phase. A quantitative proportional amount of Cu(I) ions was transferred to organic phase in form of [Cu(neocuptoinr)₂]NO₃^- complex. The copper in the organic phase was estimated by AAS at λ_max 240.8nm. A linear relationship was found between the amount of the nitrate ion added and the concentration of the copper estimated in organic phase. The detection limit was 0.1-0.01ppm. The effects of different anions like bicarbonate and hydroxyl ion were also determined.

Introduction

1.1.      General Description

          Nitrogen is the need of all living organism as it is used to build the essential components such as proteins, DNA, RNA, as well as enzymes and hormones. However, nitrogen cannot be used directly by biological systems to build the chemicals required for growth and reproduction. Before its incorporation into a living system, nitrogen  must first be combined with the other elements like hydrogen. This process of reduction of nitrogen commonly referred to as "nitrogen fixation" (N-fixation) may be accomplished chemically or biologically. As nitrogen is major component of the air so, it reacts with oxygen and ozone to produce nitrogen oxides and nitrate is one of them. Nitrogen oxidation also occurs in growing and decomposing biological systems. Nitrate is only one of several forms of nitrogen that occurs in surface waters. Nitrate tends to be the dominant form of nitrogen in waters with normal levels of dissolved oxygen. Nitrate is a natural constituent of plants and is found in vegetables at varying levels depending on the amount of fertilizer applied and on other growing conditions.

            The simple biological nitrogen cycle show that principle source of nitrate for terrestrial and aquatic ecosystem is from nitrification process. Nitrification is the biological oxidation of ammonia with oxygen into nitrite followed by the oxidation of thses nitrites into nitrates. whereas the assimilation and denitrification processes show the conversion of nitrate to organic nitrogen and then to nitrous oxide and moleculer nitrogen which is then released into atmosphere, soil and water.Nitrate can also get directly into surface waters through atmospheric deposition, from surface water runoff, or through the seepage of groundwater to streams and lakes. Nitrogen fixation is only an indirect source of nitrate in the biosphere. Various photochemical and thermal reactions are involved in production of nitrate compound from gaseous nitrogen oxides in the atmosphere.

1.2.         Brief history of nitrate

            Nitrate is an essential component of living things and is a major component of animal manure, human sewage waste and commercial fertilizers. Nitrates and nitrites have been used for centuries as fertilizers, as potassium nitrate (saltpeter) and ammonium nitrate are an important source of nitrogen in fertilizers and essential constituents of gunpowder.

            In the last third of the 18th Century, the nitrate, caliche (raw material of nitrate) had to cook clandestinely and a substance was extracted from it that was applied with such great success in the fabrication of gunpowder. Mariano Ollero, one of the Indians did great work in favor of nitrate. Nitrates were also extracted from certain sites⁽¹⁾.The use of nitrates(potassium nitrate) as fertilizer was first suggested by Galubar in 1656⁽²⁾.

1.3. Physiochemical properties

            The nitrate ion is a polyatomicmolecule and is negative ion.Some properties of   nitrate are;

·                     Chemical Name:        Nitrate

·                     Regulatory name:      Nitrate

·                     Molecular formula:   NO₃^-

·                     Molecular weight:     62.0049g/mol

An electrostatic potential map of nitrate ion.

1.4.         General chemistry

            Nitrate is the most fully oxidized compound of nitrogen and is stable to oxidation so, used as oxidizing agent. In a very dilute solution the nitrate remains chemically unreactive, and nearly all the transformations involving nitrate in natural water are mediated biochemically. The nitrate salts of all the common metals, in water have little tendency to form the coordination complexes with heavy metals. Organic chemists consider the nitrate as a functional group with general chemical formula RONO₂ where R stands for any organic residue.

            They are the esters of nitric acid and alcohols formed by nitroxylation⁽³⁾.Nitroglycerin is the trinitrate of glycerol, Guncotton is a nitrate of celluloss. Nitrite is also a negatively charged anion composed of one atom of nitrogen and two atoms of oxygen, it is a salt of nitrous acid. The nitrite, in contrast to nitrate, forms strong complexes with many transition metals. It is a weak reducing agent and can be oxidized to nitrate only by strong chemical oxidents and by nitrifying bacteria. Organic compounds containing the nitro functional group, which has same formula and structure as the nitrate ion but one of the O^-atoms is replace by R group, are known as nitro compounds.

            In a chemical analysis, a test for nitrate invovles the addition of a solution of ferrous sulfate to the substance to be tested, followed by the addition (without mixing) of few drops of concentrated sufuric acid. The presence of a nitrate is indicated by the formation of a brown rong, of Fe(NO₃)⁺² complex ion. The nitrate ion can be represented by resonance structures as;

Resonance hybride structures of nitrate ion.

1.5. Structure

          It is the conjugate base of nitric acid and has a chemical structure, in which nitrogen is central atom surrounded by three identical oxygen atoms in a symmetrical trigonal planar arrangement. The nitrate ion carries a formal charge of negative one while the nitrogen carries a positive charge. The N−O bonds are intermediate in length and strength between a single bond and a double bond.

The structure and bonding of nitrate ion.

1.6. Solubility of nitrate

         The salts formed by nitrate are generally water-soluble molecule, for this reason nitrate salts are often used when the water soluble salt of a metal is required. Nitrate salt forms when a positively charged ion attaches to one of the negatively charged oxygen atoms of the nitrate ion. The main cations in ground water are likely to be calcium, magnesium, potassium, sodium, iron and aluminium, and the salts they form with nitrate are all highly soluble. The extreme solubility of nitrate has two consequences, one is that all nitrates which are in environment are dissolved in water and second is that solubility does not limit the nitrate concentration in natural waters.

1.7. Mobility of nitrate ion

            Nitrate is highly mobile in the environment. It is often released in the form of a salt which quickly dissolve and liberate the free nitrate ion. Nitrate does not bind to soils or particles in the water and is therefore easily transported along with water. During rainy periods, nitrate not taken up by plants in surface soils will travel downwards through the soil and be carried away by groundwater, or will be carried overland to surface waters. If nitrate ends up in waters with very little dissolved oxygen, certain types of bacteria will convert it to nitrite, and ultimately to nitrogen gas, which can then escape to the atmosphere. In well-oxygenated waters, nitrate is readily taken up by aquatic plants and algae and used for growth ⁽⁴⁾.

            In the human body, the blood circulation transports nitrates to the salivary glands, where they are concentrated and oral absorption of nitrate is nearly 100%. When nitrate-rich food is eaten, saliva contains large amounts of nitrates which is converted partially to nitrite by bacteria of the oral cavity, stomach, and small intestine.When the nitrites are swallow they come into contact with acid gastric juice, and are then converted into the biologically active substance, nitric oxide⁽⁵⁾.Transport across the membrane is controlled by a nitrate/H⁺ co-transporter ⁽⁶⁾. Nitrateis readily found in the plasma, sweat, rarely occurs in the feces, and is primarily excreted by the mammalian kidney in the urine as nitrate or urea. Plasma and urine concentrations increase with dietary intake of nitrate ⁽⁷⁾_.

1.8. Nitrate level in Water

            The primary source of nitrate concentrations are surface runoff from agricultural or landscaped areas which have received excess nitrate fertilizer.Nitrates are also a by-product of septic systems. Specifically, they are naturally occurring chemical that is left after the break down or decomposition of animal or human waste and this affects the quality of ground water.

            The U.S. Environmental Protection Agency (EPA) has given maximum contaminant level (MCL) for nitrate in drinking water as 10 mg/L nitrate-nitrogen (nitrate-N) (equivalent to 45 mg/L as nitrate) and the World Health Organization (WHO) guideline (WHO 2004b) gives 50 mg/L of nitrate (equivalent to 11 mg/L as nitrate-N). According to the World Health Organization, most adults ingest 20-70 milligrams of nitrate- nitrogen per day with most of this coming from foods like lettuce, celery, beets, and spinach. When foods containing nitrate are eaten as part of a balanced diet, the nitrate exposure is not thought to be harmful⁽⁸⁾.

            The past few decades have seen a progressive increase in the general level of nitrate in the environment. This has been due to the formation of nitrogen oxides by fuel combustion and their deposition as a component of the acid rain, partly to increase in sewage recycling but mainly to the increased use of nitrate based fertilizers.

1.8. a.         Removal of Nitrate from Drinking Water

            Nitrate is a tasteless, colorless and odorless compound that cannot detect unless water is chemically analyzed. Heating or boiling water containing nitrate will not remove the nitrate, but may actually concentrate it. Options to consider if the water supply is contaminated with nitrate above the 10 mg/l level include using bottled water for drinking, and for food and beverage preparation, or installing a home water treatment unit. Charcoal filters, water softeners and chemical disinfection, such as chlorination, do not remove nitrate from water. Nitrate may successfully be removed from water using treatment processes such as ion exchange, distillation, and reverse osmosis. These treatment techniques require careful maintenance and sampling to achieve and confirm effective operation⁽⁹⁾.

1.9. Nitrate in Pakistan

            Excessive nitrogen fertilization and high nitrate content water for crop irrigation are common problems in Pakistanas the use of nitrogen fertilizers for improved crop yields has been increasing rapidly in Pakistanand this results in increased nitrate levels in drinking water. Other sources of nitrate contamination of water include intensive livestock operations that produce large amounts of animal waste, sub-standard septic systems and municipal waste streams. Shallow and poorly constructed wells in rural areas of Pakistanare at great risk of nitrate contamination. Drinking water being the main source, other sources of inorganic nitrate/nitrite exposure to children and adults are considered to be vegetables (spinach, cabbage, and carrots), meat preservatives, burn creams, industrial salts and anticorrosive. Sources of organic nitrate/nitrite include inhalants, pharmaceuticals, laundry ink, industrial solvents and antibiotics⁽¹⁰⁾.In Pakistan the vegetables are major contributors of nitrate and nitrite while the cereal and pulses contribute marginally in the human diet.

1.9. a. Fertilizer use and its impact in Pakistan

            In Pakistan, the fertilizer use level is low and there is low rainfall and high evaporation. Moreover, the organic matter in soils is low and the topography is almost flat in the major agricultural areas of Sindh and Punjab, where bulk of the fertilizers is consumed. Given these conditions, nitrate movement downward to cause ground water contamination and subsequent accumulation in steams is unlikely to be significant. Long term experiments, using chemical fertilizers, conducted at Ayub Agricultural Research Institute, Faisalabad, did not show any significant accumulation and movement of nitrates in soil.

            In Pakistan bulk of the fertilizers is nitrogenous and urea is the single most important nitrogenous fertilizers, amounting to 80% of nitrogen used in the country. Urea is applied to soil at the time of planting when soil moisture level is low and urea is also used as top dressing when its application is followed by irrigation. When urea is applied to soil at the time of plantation, it is mixed well into the soil. The urea is hydrolyzed into ammonium carbonate by enzymatic reactions. The ammonium carbonate undergoes ionization and ammonium ions are adsorbed on the soil particles. The ammonium ions are then oxidized to nitrates by microbes. The nitrate ions thus formed are either taken up by the plants and micro organisms, or move downward. But due to low moisture and arid environment there is little downward water movement and hence no significant downward movement of nitrate takes place⁽¹¹⁾.

1.10. Uses of nitrate

            Nitrate is used extensively in the production of fertilizers because it is readily taken up by plants for growth. When mixed with hydrocarbons or carbohydrates, nitrates can form a flammable or even explosive mixture as; potassium nitrate is the oxidizing ingredient in black gunpowder. Nitrates and nitrites are used in pickling and for preserving meats, certain machine oils and antirust tablets. In medicine, organic nitrates are widely used as drugs and therapeutic agents. Nitrate is also used in a variety of industrial applications, including as oxidizing agents in explosives, matches and pyrotechnics. Other uses are in photography, glass making, engraving, textile dyes and as a raw material for manufacturing nitric acid.

1.11. The Biological Activity of Nitrite and Nitrate

            The biological reduction of nitrate to nitrite has been observed in plants, animals and micro-organisms. The physiological meaning of the reduction is self-evident at least in plants, which can use the nitrate as source of nitrogen. Stephenson has shown that E.Coli and some other anaerobes which contain a specific enzyme reducing the nitrate are able to grow in the lactate medium in anaerobiosis only in the presence of the nitrate. So, in this case the nitrate acts not as a nitrogen source but as an ultimate hydrogen acceptor in place of molecular oxygen. In some biological oxidations only nitrate but neither quinine nor methylene blue can replace molecular oxygen^﴿12﴾.Nitrite and nitrate in blood is widely used as an index of endothelial NO synthase activity as routine indirect measures of NO levels⁽¹³⁾.Recently nitrite was thought to be an inert oxidative breakdown product of endogenous NO synthesis. Nitrite is now considered a central homeostatic molecule in NO biology and may serve as an important signaling molecule⁽¹⁴⁾. Dietary nitrate has been recently shown to reduce diastolic blood pressure in healthy persons⁽¹⁵⁾.

1.12. Toxic effects of nitrate

            The major known ecological effects of nitrate are those related to nutrient enrichment of the ecosystem or acidification of precipitation, direct toxic effects of nitrate, nitrite, and nitrosoamine on plants and animals. Nitrogen discharges rainwater has also been associated with algal blooms, fish kills and anoxic conditions in many freshwater systems.

            Nitrate can reach at a such high levels that can potentially cause the death of fish, in freshwater that are close to land, While nitrate is much less toxic than ammonia or nitrite, levels over 30 ppm of nitrate can inhibit growth, impair the immune system and cause stress in some aquatic species⁽¹⁶⁾.Some organic nitrate aerosols are known to have direct toxic effects on animals, plants and microorganism.

Nitrates and their role in human health has been the subject of study since the 12th century. In the human body, the nitrate is stable, inert compound that cannot be metabolized by human enzymes. The nitrate reducing activity of bacteria may convert nitrate into nitrite and other bioactive nitrogen compounds that affect physiological processes and human health.

            Contamination of drinking water by nitrates is an evolving public health concern since nitrate can undergo reduction to nitrite and nitrosation of nitrites can form N-nitroso-compounds, which are carcinogens (cancer causing). The carcinogenic feature of N-nitroso compounds has been well established. Methemoglobinemia(blue baby syndrome) is a well-recognized hazard of ingestion of nitrates and nitrites⁽¹⁷⁾. The first reported case of fatal acquired methemoglobinemia in an infant due to ingestion of nitrate‑contaminated well water was first reported in 1945 by Comly⁽¹⁸⁾. Hemoglobin molecules contain iron within a porphyrin heme structure. The iron in hemoglobin is normally found in the Fe²⁺state. The iron of hemoglobin can be oxidized to the Fe³⁺ state to form methemoglobin. Actually, Nitrates do not cause methemoglobinemia but nitrite formed from nitrate convert ferrous iron in haemoglobin to ferric iron resulting in methemoglobin, abnormal dark brown haemoglobin incapable of carrying oxygen. Once it is formed, the molecule loses its ability to carry molecular oxygen. Because red blood cells are bathed in oxygen, a certain amount of physiologic methemoglobin formation occurs continuously. Several endogenous reduction systems exist to maintain methemoglobin in the reduced state. In normal individuals only about 1% of total hemoglobin is methemoglobin at any given time ⁽¹⁹⁾.Methemoglobin can be reduced back to hemoglobin by both spontaneous (NADH-dependent and to a lesser degree by NADPH-dependent) methemoglobin reductase enzymes. Ezeonu et al.⁽²⁰⁾ suggested that nitrate in drinking water probably plays an important role in gastric carcinogenesis. According to Gupta et al.⁽²¹⁾ a review of the literature indicated an association among high nitrate ingestion, methaemogloinemia and pathologic changes in bronchi and lung parenchyma.

            Hypotension is the main cardiovascular effect seen with nitrate and nitrite medications. It is not ingestion of nitrates and nitrites in food and water⁽²²⁾. Some study results have raised concern about the cancer-causing potential of nitrates and nitrites used as preservatives and color-enhancing agents in meats ⁽²³⁾. Nitrates can react with amino acids to form nitrosamines, which have been reported to cause cancer in animals ⁽²⁴⁾.Elevated risk of cancers of the esophagus, nasopharynx, and bladder have been reported. Animal studies suggest that nitrate at high doses can competitively inhibit iodine uptake and induce hypertrophic changes in the thyroid⁽²⁵⁾.

1.13. Nitrate analysis in forensic samples

            Quantification of nitric oxide in vivo is difficult because of its short half-life. Instead the oxidized products, nitrite and nitrate, are commonly used as indices of nitric oxide generation. A simple method for the measurement of nitrite and nitrate in plasma using high performance capillary electrophoresis was described. Plasma standard curves gave regression coefficients of 0.98 for nitrite and 0.99 for nitrate with an intra-assay coefficient of variance of 4.6% for 50 μM nitrite (n=10) and 1.2% for 50 μM nitrate (n=10). The assay allowed measurement of nitrite and nitrate in a single analysis requiring minimal sample preparation, and with the sensitivity to detect both basal and physiological changes in plasma nitrite and nitrate⁽²⁶⁾.

            Tsikas.D,et.al,⁽²⁷⁾developed a gas chromatographic—mass spectrometric
(GC—MS) method in which [15N]nitrite and [15N]nitrate were used as internal standards. Endogenous nitrite and [15N]nitrite added to acetone-treated plasma and urine samples were converted into their pentafluorobenzyl (PFB) derivatives using PFB bromide as the alkylating agent. For the analysis of endogenous nitrate and [15N]nitrate they were reduced to nitrite and [15N]nitrite, respectively, by cadmium in acidified plasma and urine samples prior to PFB alkylation. Reaction products were extracted with toluene and 1-μL aliquots were analyzed by selected-ion monitoring at m/z 46 for endogenous nitrite (nitrate) and m/z 47 for [15N]nitrite ([15N]nitrate). The intra- and inter-assay relative standard deviations for the determination of nitrite and nitrate in urine and plasma were below 3.8%. The detection limit of the method was 22 fmol of nitrite. Healthy subjects (n = 12) excreted into urine 0.49 ± 0.25 of nitrite and 109.5 ± 61.7 of nitrate (mean ± S.D., μmol/mmol creatinine) with a mean 24-h output of 5.7 μmol for nitrite and 1226 μmol for nitrate. The concentrations of nitrite and nitrate in the plasma of these volunteers were determined to be (mean ± S.D., μmol/l) 3.6 ± 0.8 and 68 ± 17, respectively.

            A simple and sensitive method for analysis of nitrate and nitrite in blood was developed using gas chromatography with a nitrogen-phosphorus detector (GC-NPD). Nitrate and nitrite were analyzed after the mesitylene derivative(2-nitromesitylene).
2-Nitromesitylene was derived by condensation of mesitylene with NO₃^-. For the analysis of the GC-NPD, calibration curves in the range 0.3-5.0.MU.g/mL were found to be linear for nitrate and nitrite. The limit of quantitation of the assay was 300ng/mL for nitrates and nitrite. Moreover, this method using GC-NPD is useful in forensic toxicology as evidenced in actual case⁽²⁸⁾.

            A forensic investigation was carried out⁽²⁹⁾ and cyanide was first detected in an evidence sample of a canned coffee drink. The detectivity of IBN and related compounds by headspace gas chromatography and capillary electrophoresis was examined.
IBN decomposed to isobutyl alcohol (ⁱBuOH) and nitrite in aqueous solution and under higher temperature and more acidic conditions.  IBN was also produced by the esterification of ⁱBuOH with nitrite below pH 5. An IBN-spiked canned coffee drink solution was stored at 4°C and periodically analyzed for IBN, ⁱBuOH, nitrite, nitrate, and cyanide. The nitrate level reached a plateau of 60% molar recovery. A highly sensitive and virtually specific method has been developed for the trace and ultra trace 5 ng ml^-1-1 μg ml fluorimetric analysis of nitrate and nitrite. The method was based on the quenching action of nitrite on the native fluorescence of murexide (ammonium purpurate) [λ_ex= 349.0 nm, λ_em = 444.5 nm] in the acid range of 0.045-0.315 (M) H₂SO₄. The method was very precised and accurate (S.D. = ±0.4877 and R.S.D. = 0.4878% for the determination of 0.1 μg ml^-1 of nitrite in 11 replicates). Relatively large excesses of over 35 cations and anions did not interfere. The proposed technique had been successfully applied for the determination of nitrite and nitrate in ground water, surface water and sea water, nitrate in forensic samples⁽³⁰⁾. Copyright 2007 American Academy of Forensic Sciences

            Urea nitrate (uronium nitrate, UN) is a powerful improvised explosive. It is difficult to identify UN, because in the presence of water it readily decomposes to its original components, urea and nitric acid. A method for the recovery and detection of urea nitrate in traces was described. The residues were extracted with hot acetone, and the extracted chromatographed on chromosorb G-HP. The eluent was analyzed by liquid chromatography mass spectrometry using atmospheric pressure chemical ionization (APCI).It was found that UN can be also formed during the analytical procedure, by certain combinations of urea, nitrate ions, and a source of protons and, hence, the presence of the characteristic adduct ion did not necessarily indicate an "authentic" urea nitrate⁽³¹⁾. In the biological or forensic samples the nitrate ion is present in very little quantity and the estimation of the nitrates in forensic samples is of great importance, as very small surge in nitrate level may lead to undesirable change in living tissue. The aim of this study is to develop a method for estimation of nitrate, with better detection limit without sacrifying the selectivity of the method and also to avoid the too much effects of the interfering factors.

To accomplish the aim, the objectives of the research are;

1. Review of literature for estimation of nitrate directly or indirectly.
2. Determine the most suitable method and modification of method to minimize the interference.
3. Experimental work for estimation of nitrate.

Reporting of results and their discussion.

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Introduction

Literature Review

Experimental Work

Results and Discussion

References