COMPOSITIONS, APPARATUS, AND METHODS FOR DETERMINING

CHLORIDE ION IN AN ANALYTE COMPOSITION

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 62/076,948, filed November 7, 2014, and U.S. Provisional Application No. 62/084,878, filed November 26, 2014, which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

A. Field of the Invention [0002] The invention generally concerns the determination of chloride ion concentration of a solution. In particular, the kits and methods of the present invention are used to determine the chloride ion concentrate in an analyte composition by adding the analyte composition to a lyophilized titrant composition in each microwell of the plurality of microwells to form a solution in each microwell having a detectable absorbance in response to the chloride ion in the analyte composition

B. Description of Related Art

[0003] Water produced concurrent with the extraction of crude oil or natural gas commonly contains chemical constituents with concentrations many times those found in fresh water or even sea water. When introduced to the surface environment through planned or unplanned releases produced water will adversely affect or destroy surface vegetation and can potentially contaminate fresh groundwater supplies. One of the contaminants in the produced water is chloride ion. High amounts of chloride can affect drinking water resources and vegetation. Many governmental agencies require that the water be treated prior to disposal, if the chloride concentration is above acceptable limits. Currently methods to test chloride concentration of water at remote sites include chloride test strips or titration methods using visual indicators. These methods use silver nitrate as the titrant with potassium chromate as the endpoint indicator. Other methods include electrochemical testing, which is expensive. While accurate, titration techniques suffer from many disadvantages and is time consuming. First, the visual technique requires the analyst to manually titrate a solution of acid, drop wise, into the analyte solution of interest. Secondly, the visual technique requires the subjective determination of a color change. These two disadvantages often result in errors resulting from the analyst overshooting the endpoint due to adding too much strong acid or misjudging the color change at the endpoint. Thus, the analyst has to in many cases redo the titration method.

SUMMARY OF THE INVENTION

[0004] A solution to the disadvantages of a visual titration method has been discovered. In particular, the solution resides in the use of lyophilized titrant samples in a microwell plate. Analyte samples are added to the lyophilized samples and the absorbance of the resulting samples is measured and the chloride concentration of the analyte composition is determined based on the measured absorbance value. Notably, the present invention eliminates the drawbacks of traditional manual titrations by eliminating the subjective naked- eye determination and provides a rapid analysis and accurate analysis of the analyte composition at the user site. The user simply has to add the analyte composition, or analyte solutions, to each microwell in the plate instead of manually titrating each analyte solution. Furthermore, the present invention removes the subjective naked-eye determination of an endpoint by using a spectrophotometer to determine the endpoint.

[0005] In one aspect of the invention, there is disclosed a chloride ion assay kit. The kit includes a) a microwell plate and b) a lyophilized titrant composition comprising an acid compound, an iron (III) compound, and mercury (II) thiocyanate. A plurality of microwells of the microwell plate contain the lyophilized titrant composition such that when an analyte composition is added to the lyophilized titrant composition in each microwell of the plurality of microwells a solution forms having an absorbance at a detectable wavelength in response to chloride ion comprised in the analyte composition. Without wishing to be bound by theory, it is believed that the chloride ion in the analyte composition causes the mercury (II) thiocyanate to dissociate. The free thiocyanate can then coordinate to iron (III) forming a colored complex. The amount of the colored complex formed is proportional to the amount of chloride present in the sample. By developing calibration curves based on these proportions, the chloride concentration can be determined quantitatively. The detectable wavelength can be between 440 nm and 460 nm, and preferably at 450 nm. The microwell plate can include 6, 24, 96, 384, or 1536 microwells. In some aspects of the invention, the microwell plate includes 6 microwells and each microwell contains the same amount of titrant composition or at least 2 microwells have the same amount of titrant and the rest of the microwells have a different amount of titrant composition. In other aspects of the invention, the microwell plate has at least 24 or 96 microwells and at least 10 microwells contain the same amount of titrant solutions or at least 10 microwells contain the same amount of titrant composition, and some of the microwells have a different amount of titrant composition as other microwells. The amounts of lyophilized titrant are used at a 1 :3 or a 1 :2 dilution with the analyte composition. The plurality of microwells can be sealed to prevent the titrant composition from exiting the plurality of microwells. In some instances, the plurality of microwells is sealed with a plastic film or a foil. The chloride ion assay kit can also include a spectrophotometer capable of measuring ultra violet and visible wavelengths.

[0006] The titrant can be a composition that includes an acid compound, an iron (III) compound, and mercury (II) thiocyanate, where the composition has an absorbance at a detectable wavelength in response to chloride ion comprised in the solution. The acid compound can be camphor sulfonic acid, p-toluenesulfonic acid 1,4- piperazinediethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, 3-(N- morpholino)propanesulfonic acid, or any combination thereof, with 2-(N- morpholino)ethanesulfonic acid and camphor sulfonic acid being preferred. The iron (III) compound can be iron (III) nitrate, iron (III) sulfate, iron (III) chloride, iron (III) triflate, or any combination thereof, with iron (III) nitrate being preferred. In a preferred embodiment, the titrant composition consists essentially of a 2-(N-morpholino)ethanesulfonic acid, iron nitrate, and mercury (II) thiocyanate. The composition can be a powder. The powder can be made by providing an aqueous solution of the titrant composition to one or more containers and subjecting at least one of the containers to lyophilizing conditions sufficient to remove the water from the aqueous solution to form the powder. In some instances, the one or more containers are microwells of a microwell plate. The powder can be packaged (for example, a bag, vial, or encapsulated). The powder can be sold separately from the kit.

[0007] The chloride ion assay kit of the present invention can be used to determine the chloride concentration of an analyte composition or a plurality of analyte compositions. The method can include a) obtaining any one of the chloride ion assay kits described throughout this Specification; b) obtaining an analyte composition; c) adding substantially the same volume of the analyte composition to each of the plurality of microwells of the microwell plate to form solutions from the analyte composition and the lyophilized titrate compositions in each of the plurality of microwells; and d) measuring the absorbance value for each solution in each of the plurality of microwells at a wavelength and determining the chloride ion concentration of the analyte composition based on the measured absorbance values in response to chloride ion comprised in the analyte composition. The pH of the formed solution can be from 6 to 9. The measured absorbance ranges from 0.1 to 1.1 and the concentration of chloride ions ranges from greater than 0.1 up to 250 mg/L at a wavelength of 450 nm. In some instances, the measured absorbance can be 0.1932 and the concentration of chloride ions is 1 mg/L and/or the measured absorbance can be 0.9903 and the concentration of chloride ions is 200 mg/L at a wavelength of 450 nm. The analyte can be obtained from a variety of sources such as a subsurface well, a hydrocarbon subsurface, a water well in a subsurface hydrocarbon formation, or a wastewater reservoir or tank. In some instances, the analyte composition is obtained from a hydrocarbon drilling or fracking process. In some instances, a plurality of solutions having the same analyte is obtained, and each analyte composition is obtained from a different well of a plurality of subsurface wells or a plurality of different wastewater units.

[0008] The chloride ion assay kits described throughout the Specification can be made by a) obtaining a microwell plate; b) obtaining a lyophilized titrant composition comprising an acid compound, an iron (III) compound and mercury (II) thiocyanate; wherein a plurality of microwells of the microwell plate contain the lyophilized titrant composition such that when an analyte composition is added to the lyophilized titrant composition in each microwell of the plurality of microwells a solution forms having an absorbance at a known wavelength in response to chloride ion comprised in the analyte composition. In some instances, the lyophilized titrant composition can be obtained by providing an aqueous solution of the titrant composition to one or more microwells of the microwell plate and subjecting the microwell plate to lyophilizing conditions sufficient to remove the water from the aqueous solution and form a powder.

[0009] In the context of the present invention, 41 embodiments are described. Embodiment 1 describes a composition for determining the chloride content of a solution. The composition can include an acid compound, an iron (III) compound, and mercury (II) thiocyanate and the composition can have an absorbance at a detectable wavelength in response to chloride ion contained in the solution. Embodiment 2 is the composition of embodiment 1, where the acid compound can include camphor sulfonic acid, p- toluenesulfonic acid 1 ,4-piperazinediethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, or any combination thereof, with 2-(N- morpholino)ethanesulfonic acid, or any combination thereof. Embodiment 3 is the compositions of any one of embodiments 2 or 3 where the acid compound is camphor sulfonic acid, 2-(N-morpholino)ethanesulfonic acid, or both. Embodiment 4 is the compositions of any one of embodiments 1 to 3 where the iron (III) compound can include iron (III) nitrate, iron (III) sulfate, iron (III) chloride, iron (III) triflate, or any combination thereof. Embodiment 5 is the composition of embodiment 4, wherein the iron (III) compound is iron (III) nitrate. Embodiment 6 is the composition of embodiment 1, wherein the composition consists essentially of 2-(N-morpholino)ethanesulfonic acid, iron (III) nitrate, and mercury (II) thiocyanate. Embodiment 7 is the compositions of embodiments any one of 1 to 6, wherein the composition is a powder. Embodiment 8 is the composition of embodiment 7, wherein the powder is made by providing an aqueous solution of the titrant composition to one or more containers and subjecting at least one of the containers to lyophilizing conditions sufficient to remove the water from the aqueous solution to form the powder. Embodiment 9 is the composition of embodiment 8, wherein the one or more containers are microwells of a microwell plate. Embodiment 10 is the compositions of any one of embodiments 8 or 9, wherein the container is a bag or a vial.

[0010] Embodiment 11 describes a chloride ion assay kit. The chloride ion assay kit can include (a) a microwell plate; and (b) a lyophilized titrant composition that includes an acid compound, an iron (III) compound, and mercury (II) thiocyanate. A plurality of microwells of the microwell plate contain the lyophilized titrant composition such that when an analyte composition is added to the lyophilized titrant composition in each microwell of the plurality of microwells a solution forms having an absorbance at a detectable wavelength in response to chloride ion comprised in the analyte composition. Embodiment 12 is the chloride ion assay kit of embodiment 11, wherein the microwell plate comprises 6, 24, 96, 384, or 1536 microwells. Embodiment 13 is the chloride ion assay kit of any one of embodiments 11 or 12, wherein the microwell plate comprises 6 microwells and each microwell contains the same amount of titrant composition. Embodiment 14 is the chloride ion assay kit of any one of embodiments 11 to 12, wherein the microwell plate comprises 24 microwells or 96 microwells, and at least 10 microwells contain the same amount of titrant composition. Embodiment 15 is the chloride ion assay kit of any one of embodiments 11 to 12, wherein the microwell plate comprises 6 microwells and at least 2 microwells have the same amount of titrant and the rest have of the microwells have a different amount of titrant composition. Embodiment 16 is the chloride ion assay kit of any one of embodiments 11 to 15, wherein the amounts of lyophilized titrant are used at a 1 :3 or a 1 :2 dilution with the analyte composition. Embodiment 17 is the chloride ion assay kit of any one of embodiments 11 to 16, wherein the micro well plate comprises 24 microwells or 96 microwells, and at least 10 microwells contain the same amount of titrant composition, and some of the remaining microwells have a different amount of titrant composition. Embodiment 18 is the chloride ion assay kit of embodiment 17, wherein the acid compound camphor sulfonic acid, p-toluenesulfonic acid 1 ,4-piperazinediethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, 3-(N- morpholino)propanesulfonic acid, or any combination thereof. Embodiment 19 is the chloride ion assay kit of embodiment 18, wherein acid compound is 1,4- piperazinediethanesulfonic acid. Embodiment 20 is the chloride ion assay kit of any one of embodiments 11 to 19, wherein the iron (III) compound is iron (III), nitrate, iron (III) sulfate, iron (III) chloride, iron (III) triflate, or any combination thereof. Embodiment 21 is the chloride ion assay kit of embodiment 20, wherein the iron (III) compound is iron nitrate. Embodiment 22 is the chloride ion assay kit of any one of embodiments 11 to 22, wherein the detectable wavelength is between 440 nm and 460 nm, and preferably at 450 nm. Embodiment 23 is the chloride ion assay kit of any one of embodiments 11 to 22, wherein the plurality of microwells are sealed to prevent the titrant composition from exiting the plurality of microwells. Embodiment 24 is the chloride ion assay kit of embodiment 23, wherein the plurality of microwells are sealed with a plastic film or a foil. Embodiment 25 is the chloride ion assay kit of any one of embodiments 11 to 24, further comprising a spectrophotometer capable of measuring ultra violet and visible wavelengths.

[0011] Embodiment 26 is a method of determining the chloride ion concentration of an analyte composition. The method can include (a) obtaining any one of the compositions as of any one of embodiments 1 to 10 or an one of the chloride assay kits of embodiments 11 to 25; (b) obtaining an analyte composition; (c) adding substantially the same volume of the analyte composition to each of the plurality of microwells of the microwell plate to form solutions from the analyte composition and the lyophilized titrate compositions in each of the plurality of microwells; and (d) measuring the absorbance value for each solution in each of the plurality of microwells at a wavelength and determining the chloride ion concentration of the analyte composition based on the measured absorbance values in response to chloride ion contained in the analyte composition. Embodiment 27 is the method of embodiment 26, wherein a ratio of the amount of titrant to sample is from 1 :2 to 1 :3. Embodiment 28 is the method of any one of embodiments 26 to 27, wherein the wavelength is 450 nm and the measured absorbance ranges from 0.1 to 1.1 and the concentration of chloride ions ranges from greater than 0.1 up to 250 mg/L. Embodiment 29 is the method of embodiment 28, wherein the wavelength is 450 nm and the measured absorbance is 0.1932 and the concentration of chloride ions is 1 mg/L. Embodiment 30 is the method of embodiment 28, wherein wavelength is 450 nm and the measured absorbance is 0.9903 and the concentration of chloride ions is 200 mg/L. Embodiment 31 is the method of any one of embodiments 26 to 30, wherein the acid compound is 1 ,4-piperazinediethanesulfonic acid. Embodiment 32 is he method of embodiment 26, wherein the titrant composition consists essentially of a 1,4- piperazinediethanesulfonic acid, iron nitrate, and mercury (II) thiocyanate. Embodiment 33 is the method of any one of embodiments 26 to 32, wherein the analyte in is an aqueous composition obtained from a subsurface well. Embodiment 34 is the method of any one of embodiments 26 to 33, wherein the analyte composition comprises a plurality of solutions having the same analyte, and each analyte composition is obtained from a different well of a plurality of subsurface wells. Embodiment 35 is the method of embodiments 33 or 34, wherein the well is a hydrocarbon well or a water well in a subsurface hydrocarbon formation. Embodiment 36 is the method of any one of embodiments 26 to 35, wherein the analyte composition is obtained from a drilling process or fracking process. Embodiment 37 is the method of any one of embodiments 26 to 36, wherein the analyte composition is obtained from a wastewater tank or reservoir.

[0012] Embodiment 38 is a method of making any one of the chloride assay kits of embodiments 12 to 25. The method can include (a) obtaining a microwell plate; and (b) obtaining a lyophilized titrant composition that includes an acid compound, an iron (III) compound and mercury (II) thiocyanate, wherein a plurality of microwells of the microwell plate contain the lyophilized titrant composition such that when an analyte composition is added to the lyophilized titrant composition in each microwell of the plurality of microwells a solution forms having an absorbance at a known wavelength in response to chloride ion contained in the analyte composition. Embodiment 39 is the method of embodiment 38, wherein obtaining a lyophilized titrant composition can include providing an aqueous solution of the titrant composition to one or more microwells of the microwell plate and subjecting the microwell plate to lyophilizing conditions sufficient to remove the water from the aqueous solution and form a powder. Embodiment 40 is the method of any one of embodiments 38 or 39, wherein the plurality of microwells are sealed to prevent the titrant composition from exiting the plurality of microwells. Embodiment 41 is the method of embodiment 40, wherein the plurality of microwells are sealed with a plastic film or a foil.

[0013] The term "acidic solution" or "acid compound" refers to a solution that has a concentration of hydrogen ions greater than the concentration of hydroxide ion ([H+] > [OH ]).

[0014] The terms "basic solution" or "alkaline solution" refers to a solution that has a concentration of hydrogen ions less than the concentration of hydroxide ion ([H+] < [OH ]).

[0015] The term "pH" refers to the measurement of the concentration of hydrogen ions in water or other media. pH is generally expressed as a log scale based on 10 where pH = -log[H+].

[0016] The term "about" or "approximately" are defined as being close to as understood by one of ordinary skill in the art, and in one non-limiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.

[0017] The term "substantially" and its variations are defined as being largely but not necessarily wholly what is specified as understood by one of ordinary skill in the art, and in one non-limiting embodiment substantially refers to ranges within 10%, within 5%, within 1%, or within 0.5%. [0018] The terms "inhibiting" or "reducing" or "preventing" or "avoiding" or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.

[0019] The term "effective," as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. [0020] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." [0021] The words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0022] The chloride ion assay kits and the methods of using and making the chloride ion assay kits of the present invention can "comprise," "consist essentially of," or "consist of particular ingredients, components, compositions, etc. disclosed throughout the specification. With respect to the transitional phase "consisting essentially of," in one non-limiting aspect, a basic and novel characteristic of the kits of the present invention is the ability to determine the chloride concentration of an aqueous solution using spectrometric analysis.

[0023] Other objects, features and advantages of the present invention will become apparent from the following figures, detailed description, and examples. It should be understood, however, that the figures, detailed description, and examples, while indicating specific embodiments of the invention, are given by way of illustration only and are not meant to be limiting. Additionally, it is contemplated that changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS [0024] FIGS. 1A-1C are schematics of chloride ion assay kits of the present invention.

[0025] FIG. 2 is a flow chart depicting a method of determining chloride concentration of a water body.

[0026] FIG. 3 is a graph of chloride concentration (mg/L) versus absorbance.

DETAILED DESCRIPTION OF THE INVENTION [0027] Conventional technologies used to determine the chloride concentration of a solution involve visual titration methods that are time-consuming and often inaccurate. Many time, manual visual titrations result in error resulting from the analyst overshooting the endpoint due to adding too much strong acid or misjudging the color change at the endpoint. A discovery has been made that avoids overshooting the endpoint and eliminating the need for a visual titration of adding a silver nitrate solution drop wise into a water solution that includes the analyte. The discovery lies in the use of a lyophilized titrant sample in a microwell plate. The titrant sample can include an acid compound, an iron (III) compound and mercury (II) thiocyanate. Each microwell of the microwell of the microwell plates has at least two microwells having the same amount of titrant composition. The analyte composition is added to the titrant to form a solution and the chloride concentration of the solution is determined by measuring the absorbance value for each solution in each of the plurality of wells at determining the chloride concentration of the analyte composition based on the measured absorbance values on a calibration curve. [0028] These and other non- limiting aspects of the present invention are discussed in further detail in the following sections.

A. Titrant Composition

[0029] The titrant composition can be made by preparing an aqueous solution of titrant solution and then subjecting the solution to lyophilizing conditions to remove the water and produce a powder. A saturated solution of mercuric thiocyanate saturated in water can be prepared by adding mercuric thiocyanate. The saturated solution can be filtered. To the filtered solution the acid composition and iron (III) compound and diluted to a known volume to produce the titrant solution. The amounts of acid, iron compound and mercuric thiocyanate can be determined using known stoichiometric calculations. The titrant solution can have a pH from 3.3 to 3.7, preferable 3.5 and a concentration of 250 to 350 mM of acid, 25 to 35 mM iron (III) and saturated Hg(SCN) ₂ . The titrant solution can be lyophilized and then specific amounts of the resulting powder can be added to each microwell of a microwell plate. In some embodiments, the titrant solution is not lyophilized. In a preferred instance, a known volume of titrant solution is added to the microwells of the microwell plate and the microwell plate subjected to lyophilizing conditions. Lyophilizing conditions include -60 °C and 100 mtorr. For example, f a 1 :3 dilution with the analyte composition was to be used, the microwells of a 96-microwell plate can be filled with 100 microliters of aqueous titrant composition. If a 1 :2 dilution the analyte composition was to be used, the microwells of a 96-microwell plate can be filled with 150 microliters of aqueous titrant composition. B. Chloride Ion Assay Kit

[0030] FIGS. 1A-1C depict schematics of embodiments of chloride ion assay system 100. The chloride ion assay system or kit includes microwell plate 102 having a plurality of microwells 104. The plurality of microwells 104 can be assembled in the removable holders 106. Holders 106 may include members 108 that position on top of the side wall 110. Holders 106 may rest on, or be suspended above, bottom wall 112 of the microwell plate 102. As shown, holder 106 includes eight (8) microwells 104, however, it should be understood that the number of microwells can be adjusted to the size of the microwell plate 102. For example, the number of the microwells 104 can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. As shown in FIG. 1A, the microwell plate 102 does not include any titrant composition. FIG. IB depicts all of the microwells having titrant composition 114 and FIG. 1C depicts some of the microwells having titrant composition 114. The microwells 104 can hold a volume of 20, 50, 300, 500 microliters, preferably 300 microliters or 400 microliters. The microwell plate 102, microwells 104, holders 106, can be made of any chemical resistant material. Non-limiting examples of materials include polymers, copolymers of polymers, polystyrene, polypropylene, cyclo-olefms and the like. The holders 106 may be polymeric or plastic tape with the microwells 104 embossed on the tape. Microwell plates are commercially available from Thermo Fisher Scientific (Waltham, MA, USA). [0031] As shown in FIG. IB, the microwells 104 can be filled with the same amount of lyophilized titrant composition. In other embodiments, the microwells 104 in each holder 106 can have the same amount of titrant composition, but a total amount of titrant composition in the holders 106 can be different. For example, microwells 104-1 to 104-8 can have a different amount of titrant composition than microwells 104-9 to 104-16. It should be understood, that configuration of the amount titrant in the microwells can be any chosen configuration that correlates to a calibration curve. In some instances, the microwells 104 are filled with a known amount of an aqueous solution of titrant composition and then microwell plate is positioned in a lyophilizing unit and lyophilized under conditions sufficient to remove the water from the solution. The microwells 104, microwell holders 106, and/or the microwell plate can be sealed with a known sealing agent (for example, plastic film or foil) to allow the microwell plate 102 or the microwell holders 106 to stored or transported. In some embodiments, the chloride assay system includes a spectrophotometer that is capable of measuring the absorbance of the chosen colorimetric dye and/or a calibration curve. The calibration curve depicts the amount of chloride ion versus absorbance value. In some instances, a calibration curve is provided for each holder 106.

C. Method of Determining Chloride Concentration [0032] The chloride ion assay system and kit described throughout the specification can be used to determine the chloride concentration of a solution. The solution can be a sample from a water body such as a subsurface water well in a hydrocarbon formation, a wastewater storage unit, a wastewater reservoir, a lake, a river, a canal or the like. Referring to FIG. 2, a flow chart for determining chloride concentration is depicted. In method 200, the microwell plate 102 containing the lyophilized titrant composition 114 is obtained in step 202. In step 204, a known amount of analyte composition (for example 300 microliters) is added to the lyophilized titrant composition 114 reagents in the microwells 104 using a delivery apparatus (for example, multichannel pipette). In step 206, after solids in the plate have fully dissolved, the microwell plate 102 is placed in a spectrophotometer (for example, a plate reader) and the absorbance at the known wavelength (for example 450 nm) for each microwell is measured. The chloride concentration is determined by referring to a calibration curve and selecting the chloride concentration that correlates to the absorbance value.

[0033] The system 100 can be automated to acquire data. The acquired data can be transmitted to one or more computer systems. The computer systems can include components such as CPUs or applications with an associated machine readable medium or article which may store an instruction or a set of instructions that, if executed by a machine, may cause the machine to perform a method and/or operations in accordance with the methods of the present invention. For example, the microwell plate 102 can be put in a plate reader and the spectrophotometer can automatically measure the absorbance of each sample. The measured absorbance can be stored in a computer system in the spectrophotometer and/or transmitted to another computer system. Either computer may be capable of processing the absorbance and displaying or printing a chloride ion value for a series of analytes. Such a machine may include, for example, any suitable processing platform, computing platform, computing device, processing device, computing system, processing system, computer, processor, or the like, and may be implemented using any suitable combination of hardware and/or software. The machine-readable medium or article may include, for example, any suitable type of memory unit, memory device, memory article, memory medium, storage device, storage article, storage medium and/or storage unit, for example, memory, removable or non-removable media, erasable or non-erasable media, writeable or re-writeable media, digital or analog media, hard disk, floppy disk, Compact Disk Read Only Memory (CD-ROM), Compact Disk Recordable (CD-R), Compact Disk Rewriteable (CD-RW), optical disk, magnetic media, magneto-optical media, removable memory cards or disks, various types of Digital Versatile Disk (DVD), a tape, a cassette, or the like. The instructions may include any suitable type of code, such as source code, compiled code, interpreted code, executable code, static code, dynamic code, and the like. The instructions may be implemented using any suitable high-level, low-level, object- oriented, visual, compiled and/or interpreted programming language, such as C, C++, Java, BASIC, Perl, Matlab, Pascal, Visual BASIC, assembly language, machine code, and so forth. The computer system may further include a display device such as monitor, an alphanumeric input device such as keyboard, and a directional input device such as mouse.

EXAMPLES [0034] The present invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. Example 1

(Chloride Assay Kit)

[0035] Titrant Composition. A saturated solution of mercuric thiocyanate saturated in water was prepared by adding mercuric thiocyanate (2 grams, ACS grade, Alfa) to distilled water (1L) and let stir overnight. The remaining slurry was filtered through a 0.22 micron filter and used to prepare all further reagents. 2-(N-morpholino)ethanesulfonic acid (14.8 g, (MES), ACS grade, Alfa) and iron (III) nitrate nonahydrate (3.1 g, ACS, grade, Alfa) were dissolved in the aqueous saturated Hg(SCN) ₂ solution and diluted up to 250 mL volumetrically. The resultant solution was filtered through a 0.22 micron filter and constitutes the aqueous titrant composition: 300 mM MES, 30 mM iron (III), sat. Hg(SCN) ₂ , pH 3.5.

[0036] If a 1 :3 dilution with the analyte composition was to be used, the microwells of a 96-microwell plate were filled with 100 microliters of aqueous titrant composition. If a 1 :2 dilution the analyte composition was to be used, the microwells of a 96-microwell plate were filled with 150 microliters of aqueous titrant composition. The aqueous titrant composition was lyophilizing to remove water to obtain the lyophilized sample in the microwell plate at - 60 °C and lOO mtorr. [0037] Calibration Curve. A calibration curve was produced by diluting a chloride standard (1000 mg/L, Hach) to the concentrations in Table 1, then filling a freeze-dried plate of a 100 microliter fill of the titrant composition with 300 microliters of sample. The data was then fit with a four-parameter Marquadt non- linear function as shown in FIG. 3.

Table 1

The lyophilized titrant sample, the microwell plate, and, optionally, the calibration curve, constitute the chlorine assay kit.

Example 2

(Determination of Chloride Concentration of a Water Body)

[0038] Chloride Assay. Analyte compositions (300 microliters) containing an unknown amount of chloride ion was added to 7 microwells of the 96-microwell plate prepared in Example 1. After dissolution of the lyophilized titrant sample, the microwell plate was positioned in a plate reader and the absorbance value of the plate was determined. The absorbance value was plotted against the calibration curve. The chloride concentration were compared to chloride concentrations determined using ion chromatography as shown in Table 2. Comparison of the results show that the values determined by the method of the invention are acceptable as compared to known methods. Table 2