COMPLETE SPECIFICATION
1) Title of invention:
AN INNOVATIVE POTENTIOMETRIC METHOD OF DETERMINATION OF DISSOLVED OXYGEN OF WATER ADOPTING WINKLER'S BASIC PRINCIPLES.
2) Field of invention:
Analytical chemistry - A new unique and innovative potentiometric method of analysis of dissolved oxygen of water at a temperature.
3) Background of invention with regard to the drawback associated with
known art:
3.1 Introduction:
i) What is dissolved oxygen?
Dissolved Oxygen (DO) is the amount of oxygen dissolved in water. Adequate levels of DO are an important parameter for a water body's ability to support aquatic life. Just as humans need oxygen to breathe, aquatic animals need DO to "breathe" as it is absorbed through their gills. Oxygen enters the water by absorption directly from the atmosphere or by aquatic plant and algae photosynthesis. Oxygen is removed from the water through animal respiration and decomposition of organic matter. Low levels of DO can jeopardize the health of aquatic animals and cause other water quality impairments. ii) What affects the level of dissolved oxygen?
The amount of DO in water depends on several factors, including temperature, organic matter, the amount of organisms using oxygen for respiration, and nutrients. The colder the water, the more oxygen can be dissolved in the water. Therefore, DO concentrations are usually higher in the winter than in the summer.
Organic wastes that enter a body of water include leaves, grass clippings, dead plants or animals, animal droppings, and sewage are decomposed by bacteria; these bacteria remove dissolved oxygen from the water when they breathe. During photosynthesis, plants release oxygen into the water. During respiration, plants remove oxygen from the water. Bacteria and fungi use oxygen as they decompose dead organic matter in the stream. The type of organisms present (plant, bacteria, fungi) affect the DO concentration in a water body. If many plants are present, the water can be supersaturated with DO during the day, as photosynthesis occurs. Concentrations of oxygen can decrease significantly during the night, due to respiration. DO concentrations are usually highest in the late afternoon, because photosynthesis has been occurring all day.The temperature effects dissolved oxygen content of water in lakes, ponds and streams..
Dissolved Oxygen is one of the most important parameters to measure; it is essential for aquatic life . A low dissolved oxygen reading is a good indicator of pollution (Schaaf, 2003). Organisms are dependant on dissolved oxygen; without an adequate level of dissolved oxygen most organisms will die . The minimum dissolved Oxygen in water expected to be a minimum of5.0 mgO2/L at 25°C.
3.2 Analytical Background of DO:
Dissolved oxygen ( DO ) is central in studies on biological productivity and biochemical processes in the aquatic environments. Various methods are used for determining oxygen concentration. Because of

the highest accuracy and precision, the iodometric Winkler method (Winkler, 1888) and its modifications ( carpenter ,1965) have been widely used. Since the classical Winkler titration imposes a difficulty in visual determination of the endpoint of titration by means of the changes in the color of the starch-iodine complex, there have been attempts to apply instrumental detection of the endpoint to increase the precision of the method. Furthermore, automated titration techniques for the Winkler method have been developed in the last decade. Williams and Jenkinson (1982) proposed a system based on a photometric endpoint detector. Their system was applied successfully for measuring plankton primary productivity and community respiration in the oligotrophic ocean (Williams et al, 1983). Since the photometric endpoint detection can be affected by particles in the sample solution and bubbles generated during the measurement, Culberson and Huang (1987) proposed a system by using an amperometric endpoint detector. They reported that the precision of the method was 0.3M. Considering magnitudes of daily oxygen fluxes of plankton metabolism is in the order of 1M in the oligotrophic oceans, a better precision is required for productivity measurements. Compared to the photometric and amperometric endpoint detection, little attention has been paid to the potentiometric detection for use in automated titration probably because the electrochemical equilibrium at the platinum indicator electrode is considered to be established slowly (Grasshoff, 1981) and it is difficult to detect iodine at low concentration of lO-N (Potter, 1957). However, since potentiometry is simple, convenient and generally precise, it seems worth while exploiting potentiality of the potentiometric endpoint detection for analysis.
3.3 Accepted Methods for the determination of Dissolved Oxygen of Water: 3.3-1 Winkler Titration:
The Winkler Method was, at one time, commonly used to estimate the concentration of dissolved oxygen in aqueous solution. Dissolved oxygen meters have replaced this titration method for the routine analysis of D.O. in aquatic environments. The Winkler Method has been selected for this class because
• it is still used periodically to measure D.O.
• it is used to calibrate D.O. meters, and
• the method is a classic illustration of an oxidation reduction reaction used in volumetric analysis.
The Winkler titration procedure is the first recognized method for determination of oxygen concentrations in natural waters. The technique is a destructive chemical titration where aqueous samples are treated with manganous sulfate, potassium hydroxide, and potassium iodide to form manganous hydroxide, Mn(OH)2. Oxygen in the sample reacts with the Mn(II) species giving Mn(IIl). The Mn(III) is inherently unstable and will further react with another O2 molecule to form the Mn(IV) species. In order to fix the reaction, acidification is used to convert MnO(OH)2 to manganic sulfate which acts as an oxidizing agent to release free iodine, I2. This iodine is stoichiometrically equivalent to the dissolved oxygen in the sample and is titrated with sodium thiosulfate to its starch indicator endpoint. The Winkler method is subject to numerous interferences such as the presence of nitrite ion, ferrous and ferric iron, suspended solids, and organic matter. The method is prone to over reporting DO concentrations in anoxic and under reporting DO concentrations in hyperoxic environments . 3.3.2 Clark Cell Electrodes:
Membrane covered amperometric detectors are commonly used for the measurement of oxygen in natural waters, with most designs following principles described in a fundamental patent awarded to H. A. Clark. Clark was awarded US Patent 2,913,3866,"Electrochemical device for chemical analysis" in November 1959. "Clark cell" designs have a thin organic membrane covering a two-electrode cell, separating the cell and electrolyte solution from the test solution,

and keeping a thin layer of electrolyte in direct contact with the cathode. Oxygen diffuses through the membrane and is reduced on the cathode surface:
The reduction occurs because the cathode is held at a sufficiently negative voltage to reduce the oxygen, with careful consideration to keep the bias voltage sufficiently large to reduce the oxygen but not so high as to reduce other species. The dissolved oxygen in a given sample is calculated by measuring the cathodic current and sample temperature. A relative measure of dissolved oxygen compared to a fully saturated sample is determined using the cathodic current, temperature, barometric pressure, and salinity.
In a Clark cell electrode design, the greater the oxygen partial pressure, the greater the rate of oxygen diffusion through the membrane. Due to consumption of oxygen at the cathode and diffusion dependence of oxygen through the membrane, sufficient flow of fresh water is necessary to maintain accuracy and precision of DO analysis. Other interferences include organic growth or decay that can add or remove oxygen from the water prior to transfer of oxygen through the membrane. In addition, contamination from oils and other polymers can lead to a decrease in diffusion rates, changing the calibration function of the electrode. Some materials used in commercial Clark cell electrodes are susceptible to poisoning by contaminants, which leads to a decreased response. Over time, membranes in Clark cells deteriorate to the point of needing replacement, electrolytic solutions become less pure, and the electrodes are consumed to the point of limited response to oxygen exposure. All such issues result in the need for frequent servicing and refurbishing of the Clark cell style sensors, with associated material and labor costs. 3,3.3 Luminescence-based Optodes:

Figure 1 Optode design As shown in Figure 1, the luminescent dissolved oxygen sensor's active optical components consist of a pair of blue and red light-emitting diodes (LEDs) and a silicon photo-detector. The sensor cap has a coating of a platinum based luminophor that is excited by the light from the blue LED. The luminophor is coated on the outside with a carbon black polystyrene layer for optical insulation, providing excellent protection against photo-bleaching from external light sources when the sensor cap is attached to the sensor. The blue excitation LED is sinusoidally modulated at a frequency related to the luminophor's luminescence lifetime and the upper and lower lifetimes of analytical interest. The measured parameter of interest from the optode is the phase delay (essentially a time delay) between the exciting blue LED signal and the detected red emission from the luminophor, with the phase delay inversely related to the amount of dissolved oxygen near the luminophor, typically oxygen in the water of interest. This phase-modulation technique is used to measure the lifetime of the oxygen-dependent quenching of luminescence. The use of the phase-modulation technique means that intensity fluctuations of the blue LED or bleaching effects of the luminophor have no discernable impact

on the lifetime measurement throughout the life of the O2 + 2H20 + 4e —> 40H" . In addition, because of the inverse relationship between oxygen concentration and phase delay of the emitted red light, the signal-to-noise ratio is particularly advantageous for measuring very low dissolved oxygen concentrations. Finally, the blue and red LEDs are alternatively switched between measurement cycles, allowing the red LED to provide an internal reference for the optical and electronic signal paths8. This internal reference provides measurement stability by correcting for temperature or time induced changes in the phase measurement electronics. 3.3.4. Dissolved Oxygen Electrode and Amperometry: 3.3.4.1. Principle of Amperometry (Polarography)
Polarogram - When an electrode of noble metal such as platinum or gold is made 0.6 to 0.8 V negative with respect to a suitable reference electrode such as Ag/AgCl or an calomel electrode in a neutral KG solution (see Fig. 2.1), the oxygen dissolved in the liquid is reduce at the surface of the noble metal. This phenomenon can be observed from a current-voltage diagram -called a polarogram - of the electrode. A negative voltage applied to the noble metal electrode (called the cathode) is increased, the current increases initially but soon it becomes saturated. In the plateau region of the polarogram, the reaction of oxygen at the cathode is so fast that the rate of reaction is limited by the diffusion of oxygen to the cathode surface. When the negative bias voltage is further increased, the current output of the electrode increases rapidly due to other reactions, mainly, the reduction of water to hydrogen.
If a fixed voltage in the plateau region (for example, - 0.6V) is applied to the cathode, the current output of the electrode can be linearly calibrated to the dissolved oxygen. It has to be noted that the current is proportional not to the actual concentration but to the activity or equivalent partial pressure of dissolved oxygen, which is often referred to as oxygen tension. A fixed voltage between -0.6 and -0.8 V is usually selected as the polarization voltage when using Ag/AgCl as the reference electrode.
DO Sensor - When the cathode, the reference electrode, and the electrolyte are separated from the measurement medium by a polymer membrane, which is permeable to the dissolved gas but not to most of the ions and other species, and when most of the mass transfer resistance is confined in the membrane, the electrode system can measure oxygen tension in various liquids. This is the basic operating principle of the membrane covered polarographic DO probe (Fig 2.2).


Fig. 2.2. Membrane covered polarographic oxygen sensor.
3.3.5 References:
• Winkler, L. W. (1888): Die Bestimmung des im Wasser gelosten Sauerstoffen. Ber. Dtsche. Chem. Ges., 21, 2843- 2855.
• Carpenter, J. H. (1965a): The accuracy of the Winkler method for dissolved oxygen analysis. Limnol. Oceanogr.,10, 135-143.
• Carpenter, J. H. (1965b): The Chesapeake Bay Institute technique for the Winkler dissolved oxygen method. Limnol.Oceanogr., 10, 141-143.
• Culberson, C. H. (1991): Dissolved Oxygen. WHP Operations and Methods, Unpublished manuscript, 15 pp.
• Culberson, C. H. and S, Huang (1987): Automated amperometric oxygen titration. Deep-Sea Res., 34, 875-880.
• Culberson, C. H., G. Knapp, M. C. Stalcup, R. T. Williams and F. Zemlyak (1991): A comparison of methods for the determination of dissolved oxygen in seawater. WOCE Report 73/91, 77 pp.
• Grasshoff, K. (1981): The electrochemical determination of oxygen, p. 329-420. In Marine Electrochemistry, ed.by M. Whitfield and D. Jagner, John Wiley and Sons, New York.
• Knowles, G. and G. F. Lowden (1953): Methods for detecting the end-point in the titration of iodine with thiosulfate. Analyst, 78, 159-164.
• Potter, E. C. (1957): Microdetermination of dissolved oxygen in water. I. Nature of the problem. J. Appl. Chem., 7, 285-297.
• Williams, P. J. LeB, and N. W. Jenkinson (1982): A transportable microprocessor-controlled precise Winkler titration suitable for field station and shipboard use. Limnol. Oceanogr., 27, 576-584.
• Bradbury, J. H. and A. N. Hambly (1952): An investigation of errors in the amperometric and starch indicator methods for the titration of millinormal solutions of iodine and thiosulfate. Austral. J. Sci. Res., Ser. A, 5, 541-554.
• Strickland, J.D.H., and Parsons, T.R. (1968). Determination of dissolved oxygen, in A Practical Handbook of Seawater Analysis. Fisheries Research Board of Canada, Bulletin, 167,71-75.

• Adams, R. C, Barnett, R. E. & Keller, D. E. (1943). Field and laboratory determination of dissolved oxygen. Amer. Soc. Test. Mat. Proc. 464/1 Meet. 43, 1240-57.
• Briggs, R., Knowles, G. & Scragg, L. J. (1954). A continuous recorder for dissolved oxygen in water. Analyst, 79, 744-52.
• Fox, H. M. & Wingfield, C. A. (1938). Determination of oxygen dissolved in a small quantity of water. J. Exp. Biol. 15, 437-45.
• Ohlf, W. (1953). Die chemische und die elektrochemische Bestimmung des molekular gelfoten Saueratoffs der Binnengew&sser. Mitt. int. Ver. Lirnnol., no. 3, 1-44.
• Ovhnston, T. C. J. & Watson, J. H. E. (1954). The spectrophotometric determination of small amounts of oxygen in water*. Analyst, 79, 383—7.
4) Object of invention:
Objective of the investigation
The goal of this experiment is:
1. to understand that the best indicators of the health of a water ecosystem is the dissolved
oxygen parameter,
2. to appreciate that dissolved oxygen levels change and vary according to the time of day, the weather and the temperature
3. to appreciate that a decrease in the dissolved oxygen levels is usually an indication of an
influx of some type of organic pollutant.
4. for advice: to monitor the amount of dissolved oxygen in pond, river, and lake waters.
5) Statement of invention:
5.1 Introduction:
The concentration of dissolved oxygen can be readily, and accurately, measured by the method originally developed by Winkler in 1888 (Ber. Deutsch Chem. Cos., 21, 2843). Dissolved oxygen can also be determined with precision using oxygen sensitive electrodes; such electrodes require frequent standardization with waters containing known concentrations of oxygen. They are particularly useful in polluted waters where oxygen concentrations may be quite high. In addition, their sensitivity can be exploited in environments with rapidly-changing oxygen concentrations. However, electrodes are less reliable when oxygen concentrations are very low. For these reasons, the Winkler titration is often employed for accurate determination of oxygen concentrations in aqueous samples.
It is evident from the background of analytical approach of DO determination in water by different methods has its own merits and de-merits in each case. This has prompted us exploit the basics of Winkler's principle(1888) and to show how the potential variations due to the redox reactions at the indicator Pt -electrode could quantitatively relate to the amount of DO present in water. We have designed a cell in such away both potentials and titrimetric methods can be executed in a single process of DO determination in the given volume of water. One of the most useful titrations involving iodine is that originally developed by Winkler to determine the amount of oxygen in samples of water. The dissolved oxygen content is not only important with respect to the species of aquatic life which can survive in the water, but is also a measure of its ability to oxidise organic impurities in the water.
In the Winkler Method, a strong alkali (NaOH or KOH) is added to a divalent manganese solution containing the dissolved oxygen water sample. In such conditions, any dissolved

oxygen in the sample rapidly oxidizes an equivalent amount of divalent manganese (Mn+2) to a higher valence state (Mn+4). The Mn02(s) exists as a precipitate in the solution. When the solution is acidified in the presence of iodide (KI), free iodine (I2) is produced at a concentration equivalent to the original concentration of dissolved oxygen in the sample. The series of reactions can be written stoichiometricallv as:
5.2 Theory and the basic principle of the determination of DO in water in the present
potentiometric method:
Compared to the photometric and amperometric end point detection, little attention has been paid to the potentiometric detection for use in titration probably because the electrochemical equilibrium at the platinum indicator electrode is considered to be established slowly (Grasshoff, 1981) and it is difficult to detect iodine at low concentration of 10-6 N (Potter, 1957). However, since potentiometry is simple, convenient and generally precise, it seems worthwhile exploiting potentiality of the potentiometric analysis.
5.3 Electrochemical cell set up:

In the present investigation of DO analysis of water by potentiometric method, a electrochemical cell is constructed with a redox potential electrode, as given
Hg/Hg2Cl2/KCl (Saturated) // Redox chemical system /Pt
( reference electrode) (Indicator electrode)
The emf of the cell , Ecell = [ E1Indi - Eref] = EIndi - 0.2422.

Where, Eref is a saturated calomel electrode of constant potential,!).2422 Volts. 5.4 Theory involved in potentiometric measurement of DO in water based on Winkler's basic principle*
In the present potentiometric method of investigation, redox chemistry of reactions of only two stages are given prime importance to determine the DO of water. This can be illustrated in the following reactions on the basis of Winkler method.
Manganous sulfate reacts with the potassium hydroxide at pH 12.0 to produce a white flocculent precipitate of manganous hydroxide:
MnS04 + 2 KOH -> Mn(OH)2 + K2S04 (white) If there is any DO in the water ,a second reaction between the Mn(OH)2 and DO occurs immediately to form a brownish manganic oxide precipitate.
2Mn(OH)2 + 02 -> 2MnO(OH)2 (brown) When the samples are ready to investigate , a requisite amount of H2S04 (1:1 sulphuric acid) is added at p = 1.0-2.0 to dissolve completely the brownish precipitate with magnetic stirrer swirling. Manganic sulphate ,Mn(S04)2, is yielded as the product of this reaction.
2MnO(OH)2 +2 H2S04 --> 2Mn(S04)2 + 6H20 At this stage, it is realized that amount of Mn equivalent to the amount of DO in water is quantitatively oxidized to Mn4+ state.. This results in a steady potential at the platinum indicator electrode of the cell which can be observed using a digital potentiometer or interfaced to a computer system. The standard oxidation potential of the indicator electrode , Pt/ Mn2+;Mn4+, for 1M MnS04

addition of KI solution. The Mn(S04)2 immediately reacts with the potassium iodide (KI) added, liberating the number of moles of iodine exactly equivalent to the number of moles of dissolved oxygen present in the sample. The release of iodine (I2) imparts a brown coloration to the water typical of iodine.
2Mn(S04)2 + 4KI --> 2MnS04 + 2K2S04 + 2I2 The above redox reaction takes place spontaneously at the Platinum indicator electrode of the cell and a resultant potential is developed at the indicator electrode for the two competitive



5.5 Experimental procedure:
i) Preparation of standard MnSO4 solution for calibration plot:
From the stoichiometric relationship of Winkler method at t° C, prepare the following standard solutions of MnSO4.6H20 M.F =169),as given in the table.-1.

Note: To obtain a linear calibration plot, all the solutions are to be prepared in the given water sample containing the DO in water and should be kept in a closed bottles.
ii) Set up a electrochemical cell as represented

Hg/Hg2CI2/KCl (Saturated) // Redox chemical system /Pt
( reference electrode) (Indicator electrode)
iii) A closed container with a calomel and an indicator Pt -electrode is taken. A thermometer is
also inserted to record the temperature of the DO water sample . An additional pH
electrode is also dipped to controller the desired pH of the solution. Pipette out 25ml of the
above prepared stock solution into this container containing the electrodes set up carefully
and quickly, iv). Connect a digital potentiometer / or to a computer system to interface, v). Make use of a magnetic stirrer to obtain homogenous mixing of reagents vi). Add 0.5ml of 4N NaOH (Note : pH =12.0).
Keep the closed solution for 10-15 minutes, vii). Add 6.0 ml of 40% H2SO4 into this closed container and keep it for 15-20 minutes with
magnetic stirrer rotating. Note the temperature, t°C and pH =1.0 -2.0. viii) Record the equilibrium potential developed( E 1 mv ) using a potentiometer / or
interface it with a computer. ix). Add 0.40ml of 10% KI solution into the container having the electrodes. Keep it for
another 10-15 minutes to record a steady potential ( E2 mv) using potentiometer / or
interface it with computer, x). Find the difference between E1 and E2 for a particular standard MnSO4 solution of the
prepared series. It can be stated that the below mentioned stochiometry holds good for the determination of DO of water sample by potentiometric analysis (table -2).
1 mole of 02 = 2mo!es of I2 = 4 moles of Na2S203 = [ E1 - E2 ] Volts.

Figure -3 A calibration plot for DO measurement
xii). 25 ml of unknown DO sample + add 0.0265g of MnS04 H20 [ Molecular Weight
=169] + 0.5 ml of NaOH- keep for few minutes + Add 6.0 ml of H2S04 + dissolve the ppt -keep for 15 minutes and record its E1 value. Add 4.0 ml of KI and take E2 value after 10 minutes. Find the difference, [ E\ - E2 ] for the sample .
xiii) Report the DO value of the water sample from the calibration graph.

5.6 Titrimetry by Winkler's method in the same cell set up:
xvi) The above DO values of water samples are further corroborated by Winklers titration of
the same prepared solutions in the same cell set up:
The solutions are then titrated in the above set up as follows.
6) A summary of invention:
The dissolved oxygen (DO) values obtained as above from both Potentiometric( the present investigation) and titrimetric( Winkler's volumetric) methods in the same cell set up are nearly identical; and corroborates that the Winkler's principle also can be conveniently adopted to determine DO of water potentiometrically as discussed elsewhere. The results of the above method are reproducible and hence , a new unique and innovative method for DO of water determination can be adopted with accuracy as compared to different methods described elsewhere in section (3).

7) A brief description of the accompanying drawing:
Figure as given
8) Detailed description of the invention with reference to drawing/examples,
Figure description as given

9) Claim(s):
The contents of the entire section (5.0 to 5.6) including
• the theory and cell set up for the above determination of DO of water by potentiometry.,
• Experimental working procedure contents,
• apparatus set up design for electrometric and titrimetric, and
• the calibration graph for DO determination, are to be patented.