A METHOD FOR DETERMINING TOTAL IRON IN AQUEOUS SOLUTIONS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit to U.S. Provisional Patent Application No. 62/000,780 titled "METHOD FOR DETERMINING TOTAL IRON IN AQUEOUS SOLUTION" to Dragna et al, filed May 20, 2014. The entire contents of the referenced patent application are incorporated into the present application by reference.

BACKGROUND 1. Field of the Invention

[0002] This invention relates to environmental chemistry and quantitative chemical analysis. Specifically, the invention concerns the detection of total iron in an aqueous medium using an a-diimine compounds as an indicator. 2. Description of Related Art

[0003] Bis(a-diimine)-iron complexes are important species because they are known to initiate catalytic atom-transfer radical polymerization, the dimerization of dienes, and the polymerization of olefins. Additionally, organic molecules containing a

[0004] -N=C-C=N- configuration have been known to react as bidentate ligands with ferrous ions (Fe ²⁺ ) to give colored complex species. The heterocyclic bases, 2,2'-bipyridine (bipy) and 1,10-phenanthroline (phen) form stable complexes with a variety of metal ions, including ferrous. The ferrous Fe(bipy) ₃ ²⁺ and Fe(phen) ₃ ²⁺ complexes display intense red colors and can be oxidized to the less intensely-colored ferric (Fe ³⁺ ) complexes.

[0005] The visible spectra associated with this type of iron complex is common to the iron-diimine organic nitrogen chelate. Iron is capable of complexing with a variety of diimine moieties to form chromophoric iron-diimine complexes. Electron derealization within the iron-diimine complex may be described in terms of a "back-donation" of electrons from filled iron d-orbitals into vacant 7T*-orbitals on the diimine moiety. For example, Stookey in J. Anal. Chemistry, 1970, Vol. 42, pp. 779-781 describes the use of diamine compounds for the detection of ferrous ions in water. This method suffers from a tedious multi-steps to obtain the iron concentration in the water sample.

SUMMARY

[0006] A discovery has been made that overcomes the multi-step titration methods for determining iron in a water sample. The discovery is premised in compositions and assay solutions that include an a-diimine compound, a reducing agent, and a buffer. Upon addition of the composition to a water solution, the ferrous ion, ferric ion, or total iron concentration are can be determined spectrometrically. The method can be done in the field and provides a rapid analysis of total iron.

[0007] In a particular aspect of the invention, a composition for the determination of iron concentration comprising an a-diimine compound, a reducing agent, and a buffer is described. The α-diimine compound is of the formula:

[0008] wherein Ri and R ₂ may be alkyl, cycloalkyl, aryl, heteroaryl or hydroxyl, and

R ₂ and R ₃ may be alkyl, cycloalkyl, aryl, or heteroaryl. Ri and R ₂ and/or R ₂ and R ₃ and/or R ₃ and R ₄ come together to form a fused cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocycle ring.

[0009] The reducing agent is a compound that reduces ferric ions to ferrous ions. The reducing agent may be ascorbic acid, hydroxylamine, zinc sulfide, or other reducing known to those of skill in the art that reduce ferric ions to ferrous ions. Any reducing agent that can reduce ferric ions to ferrous ions may be used.

[0010] The α-diimine compound complexes with ferrous ions to form an α-diimine - ferrous complex. The a-diimine-ferrous complex concentration may be measured spectrophotometrically. Spectrophotometric measurement of the a-diimine-ferrous complex concentration is used to determine iron concentration.

[0011] The α-diimine compound may comprise a single α-diimine moiety or multiple a-diimine moieties. In particular aspects of the invention, an α-diimine compound that comprises a single α-diimine moiety may be 3-(2-pyridyl)-5,6-diphenyl-l ,2,4-triazine-4',4"- disulfonic acid sodium salt, 1,10-phenanthroline, 4,7-diphenyl-l,10-phenanthroline, or phenyl 2-pyridyl ketoxime. In some embodiments, an a-diimine compound that comprises multiple a-diimine moieties may be 2,4,6-tris(2-pyridyl)l,3,5-triazine or 2,6-bis(2-pyridyl)- pyridine.

[0012] The buffer prevents changes in pH. The operable pH range for the present composition is from about 1 to about 7. The preferred operating pH range is from about 1 to about 6. In some embodiments, a buffer with a pKa of less than 6 and greater than 1 may be used. Non-limiting examples of the buffer include citric acid, acetic acid, 2-(N- morpholino)ethanesulfonic acid, maleic acid, malic acid, formic acid, lactic acid, pivalic acid, pyridine, piperazine, picolinic acid, succinic acid, and histidine. In particular embodiments, the buffer is a phthalate buffer. In some embodiments, the buffer concentration ranges from about 100 nM to about 2 M. In other embodiments, the buffer concentration ranges from about 400 mM to about 1.2 M. In further embodiments, the buffer concentration is 600 mM.

[0013] Some embodiments of the invention relate to an assay solution for the determination of iron concentration comprising an α-diimine compound, a reducing agent, and a buffer in solution. The α-diimine compound is of the formula:

[0014] wherein Ri and R ₂ may be alkyl, cycloalkyl, aryl, heteroaryl or hydroxyl, and

R ₂ and R ₃ may be alkyl, cycloalkyl, aryl, or heteroaryl. Ri and R ₂ and/or R ₂ and R ₃ and/or R ₃ and R ₄ come together to form a fused cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocycle ring. The assay solution may include solution dispensed into 96-well plates. The assay solution may include a freeze-dried solution. A kit for the determination of dissolved iron concentration comprising an α-diimine compound, a reducing agent, and a buffer in a container containing multiple test locations, preferably a 96 well plate is described. The kit comprises the assay solution described. A method of determining the iron concentration in water, comprising employing the assay solution is described.

[0015] In some embodiments of the invention, a composition for the determination of ferrous concentration comprising an α-diimine compound and a buffer is described. The a- diimine compound is of the formula: — N U—

[0016] wherein Ri and R ₂ may be alkyl, cycloalkyl, aryl, heteroaryl or hydroxyl, and

R ₂ and R ₃ may be alkyl, cycloalkyl, aryl, or heteroaryl. Ri and R ₂ and/or R ₂ and R ₃ and/or R ₃ and R ₄ come together to form a fused cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocycle ring. In the absence of a reducing agent, ferric ions remain in the oxidized +3 state. The a-diimine compound does not complex an appreciable amount of ferric ions. Therefore, the resulting a-diimine-ferrous complex is a measure of only the ferrous ions in a solution comprising both ferrous and ferric ions.

[0017] In some embodiments of the invention, an assay solution for the determination of ferrous concentration comprising an a-diimine compound and a buffer in solution is described. The α-diimine compound is of the formula:

[0018] wherein Ri and R ₂ may be alkyl, cycloalkyl, aryl, heteroaryl or hydroxyl, and

R ₂ and R ₃ may be alkyl, cycloalkyl, aryl, or heteroaryl. Ri and R ₂ and/or R ₂ and R ₃ and/or R ₃ and R ₄ come together to form a fused cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocycle ring. In the absence of a reducing agent, ferric ions remain in the oxidized +3 state. The α-diimine compound does not complex an appreciable amount of ferric ions. Therefore, the resulting a-diimine-ferrous complex is a measure of only the ferrous ions in a solution comprising both ferrous and ferric ions.

[0019] In some instances, the compositions for iron determination can include an excipient. A non-limiting example of an excipient is a non-reducing disaccharide. In a preferred aspect, trehalose dihydrate is used as an excipient. Disaccharides are commercially available from many sources. A non-limiting example of a commercially available trehalose dihydrate is sold by Sigma Aldrich® (USA). The excipient can aid in the lyophilization process. An amount of excipient added to a titrant composition ranges from 0.1 to 10 wt.%, from 1.5 to 5 wt.%, or from 2 to 3 wt.% of the composition or assay solution for detection of iron.

[0020] The compositions for iron determination 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.

[0021] Other aspects of the invention relate to a method for determining ferric ion concentration in a solution comprising both ferrous and ferric ions. The method comprises the steps of measuring the total iron concentration, measuring the ferrous ion concentration, and subtracting the measured ferrous ion concentration from the total iron concentration to give the ferric ion concentration. The method may employ the inventive compositions or assay solutions.

[0022] In one aspect of the invention, an iron detection assay kit is described. The iron detection assay kit can include a microwell plate, a lyophilized composition comprising an a- diimine compound, a buffer, an excipient, and, optionally, a reducing agent, wherein a plurality of the microwells contain the lyophilized composition such that when an analyte composition is added to the lyophilized composition in each well of the plurality of wells, as solution forms and the total iron, ferrous ion, ferric ion, or a combination thereof, is determined based on the response of the a-diimine compound to the solution.

[0023] The term "alkyl" when used without the "substituted" modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, and no atoms other than carbon and hydrogen. Thus, as used herein cycloalkyl is a subset of alkyl. The groups -CH ₃ (Me), -CH ₂ CH ₃ (Et), -CH ₂ CH ₂ CH ₃ (rc-Pr), -CH(CH ₃ ) ₂ (wo-Pr), -CH(CH ₂ ) ₂ (cyclopropyl), -CH ₂ CH ₂ CH ₂ CH ₃ (n- Bu), -CH(CH ₃ )CH ₂ CH ₃ (sec-butyl), -CH ₂ CH(CH ₃ ) ₂ (wo-butyl), -C(CH ₃ ) ₃ (ierf-butyl), -CH ₂ C(CH ₃ ) ₃ (neo-pentyl), cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl are non-limiting examples of alkyl groups. The term "alkanediyl" when used without the "substituted" modifier refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched, cyclo, cyclic or acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups, -CH ₂ - (methylene), -CH ₂ CH ₂ - -CH ₂ C(CH ₃ ) ₂ CH ₂ - CH2CH2CH2-, and , are non-limiting examples of alkanediyl groups. The term "alkylidene" when used without the "substituted" modifier refers to the divalent group =CRR' in which R and R' are independently hydrogen, alkyl, or R and R' are taken together to represent an alkanediyl having at least two carbon atoms. Non-limiting examples of alkylidene groups include: =CH ₂ , =CH(CH ₂ CH ₃ ), and =C(CH ₃ ) ₂ . When any of these terms is used with the "substituted" modifier one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, - H ₂ , -N0 ₂ , "C0 ₂ H, -C0 ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -OCH ₂ CH ₂ OH, -C(0)CH ₃ , -NC(0)CH ₃ , -N(CH ₃ ) ₂ , -C(0)NH ₂ , -OC(0)CH ₃ , - S(0) ₂ NH ₂ , or imidazolidinone. The following groups are non-limiting examples of substituted alkyl groups: -CH ₂ OH, -CH ₂ C1, -CF ₃ , -CH ₂ CN, -CH ₂ C(0)OH, -CH ₂ C(0)OCH ₃ , -CH ₂ C(0)NH ₂ , -CH ₂ C(0)CH ₃ , -CH ₂ OCH ₃ , -CH ₂ OC(0)CH ₃ , -CH ₂ NH ₂ , -CH ₂ N(CH ₃ ) ₂ , and -CH ₂ CH ₂ C1. The term "haloalkyl" is a subset of substituted alkyl, in which one or more hydrogen atoms has been substituted with a halo group and no other atoms aside from carbon, hydrogen and halogen are present. The group, -CH ₂ C1 is a non- limiting examples of a haloalkyl. An "alkane" refers to the compound H-R, wherein R is alkyl. The term "fluoroalkyl" is a subset of substituted alkyl, in which one or more hydrogen has been substituted with a fluoro group and no other atoms aside from carbon, hydrogen and fluorine are present. The groups, -CH ₂ F, -CF ₃ , and -CH ₂ CF ₃ are non-limiting examples of fluoroalkyl groups. An "alkane" refers to the compound H-R, wherein R is alkyl.

[0024] The term "cycloalkenyl" refers to a cyclic structure comprising carbon-carbon single bonds and at least one carbon-carbon double bond.

[0025] The term "heterocycle" when used without the "substituted" modifier refers to a cyclic, aromatic group wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heterocycle group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the ring or ring system. If more than one ring is present, the rings may be fused or unfused. Non-limiting examples of heterocycle groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, and pyranyl. When the term "heterocycle" used with the "substituted" modifier one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br,

-I, -NH ₂ , -N0 ₂ , -C0 ₂ H, -C0 ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -C(0)CH ₃ , -N(CH ₃ ) ₂ , -C(0)NH ₂ , -OC(0)CH ₃ , or -S(0) ₂ NH ₂ . [0026] The term "aryl" when used without the "substituted" modifier refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of a one or more six-membered aromatic ring structure, wherein the ring atoms are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl group (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C ₆ H ₄ CH ₂ CH ₃ (ethylphenyl), naphthyl, and the monovalent group derived from biphenyl. The term "arenediyl" when used without the "substituted" modifier refers to a divalent aromatic group, with two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one or more six-membered aromatic ring structure(s) wherein the ring atoms are all carbon, and wherein the monovalent group consists of no atoms other than carbon and hydrogen. As used herein, the term does not preclude the presence of one or more alkyl group (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Non- limiting examples of arenediyl groups include:

[0027] When these terms are used with the "substituted" modifier one or more hydrogen atom has been independently replaced by -OH, -F, -CI, -Br, -I, -NH ₂ , -N0 ₂ , -C0 ₂ H, -C0 ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -C(0)CH ₃ , -N(CH ₃ ) ₂ , -C(0)NH ₂ , -OC(0)CH ₃ , or -S(0) ₂ NH ₂ . An "arene" refers to the compound H-R, wherein R is aryl.

[0028] The term "heteroaryl" when used without the "substituted" modifier refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more aromatic ring structures wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. As used herein, the term does not preclude the presence of one or more alkyl, aryl, and/or aralkyl groups (carbon number limitation permitting) attached to the aromatic ring or aromatic ring system. If more than one ring is present, the rings may be fused or unfused. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl, pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term "heteroarenediyl" when used without the "substituted" modifier refers to an divalent aromatic group, with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the two points of attachment, said atoms forming part of one or more aromatic ring structure(s) wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. As used herein, the term does not preclude the presence of one or more alkyl, aryl, and/or aralkyl groups (carbon number limitation permitting) attached to the aromatic ring or aromatic ring system. If more than one ring is present, the rings may be fused or unfused. Non-limiting examples of heteroarenediyl groups include:

[0029] The terms "a" and "an" are defined as one or more unless this disclosure explicitly requires otherwise. The term "substantially" is defined as being largely but not necessarily wholly what is specified (and include wholly what is specified) as understood by one of ordinary skill in the art. In any disclosed embodiment, the term "substantially" may be substituted with "within [a percentage] of what is specified, where the percentage includes .1, 1, 5, and 10 percent.

[0030] The terms "comprise" (and any form of comprise, such as "comprises" and

"comprising"), "have" (and any form of have, such as "has" and "having"), "include" (and any form of include, such as "includes" and "including") and "contain" (and any form of contain, such as "contains" and "containing") are open-ended linking verbs.

[0031] The assay solution 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 phrase "consisting essentially of in one non-limiting aspect, a basic and novel characteristic of the assays of the present invention are their abilities to detect iron in an aqueous solution.

[0032] Furthermore, a composition that is configured in a certain way is configured in at least that way, but may also be configured in ways that are not listed. Metric units may be derived from the English units provided by applying a conversion and rounding to the nearest milliliter. The feature or features of one embodiment may be applied to other embodiments, even though not described or illustrated, unless expressly prohibited by this disclosure or the nature of the embodiments.

[0033] Details associated with the embodiments described above and others are presented below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The following drawings illustrate by way of example and not limitation. For the sake of brevity and clarity, every feature of a given structure may not be labeled in every figure in which that structure appears.

[0035] FIG. 1 illustrates an embodiment for determining total iron in aqueous solution, wherein the a-diimine compound is 3-(2-pyridyl)-5,6-diphenyl-l,2,4-triazine-4',4"- disulfonic acid sodium salt. Ferric ions, in a mixture of ferric and ferrous ions, are reduced to ferrous ions. The a-diimine complexes with the ferrous ions and is used to determine the total iron concentration.

[0036] FIG. 2A is raw calibration data for the iron-sensing solution. Standard iron concentrations are given in mg/L and absorbance (A565) is measured at 565 nm.

[0037] FIG. 2B is the processed calibration data for the iron sensing solution.

Average absorbances, standard deviations (SD), and relative standard deviations (RSD) are listed for each iron concentration.

[0038] FIG. 3 is a calibration curve for the iron sensing solution using linear regression. The line of best fit for the iron sensing solution. R ² =0.99997.

DETAILED DESCRIPTION [0039] Iron is present in many water sources and can be detrimental to equipment and piping used in many industrial applications, for example, oil-field applications. The presence of iron (III) can indicate pipe corrosion. Iron can also catalyze decomposing processes for fracturing fluids used in oil production. In acidizing, the presence of iron (III) may indicate excessive corrosion from improper acid buffering. Iron (II) can also contribute to scaling, when it combines with sulfide, and also oxidizes to iron (III) during ozone treatment, which reduces the effectiveness of the treatment. Excessive scaling can permanently damage a valuable hydrocarbon formation, in addition to decreasing the flow within the tubular pipes used for transporting hydrocarbons or treatment fluids. Thus, the rapid determination of iron (III), iron (II), and total iron is vital data that can drive a number of decision points in production of gases and liquids from hydrocarbon formations. The present inventor provides such a rapid determination of iron. The compositions and assay solutions of the present invention can provide the total concentration of iron (III), iron (II), and total iron.

[0040] In the following description, numerous specific details are provided to provide a thorough understanding of the disclosed embodiments. One of ordinary skill in the relevant art will recognize, however, that the invention may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention. The phrases "coordinates to," "coordinates with," "complexes with," "forms a complex with," and "chelates to" are used interchangeably herein. The terms "coordinate," "complex," and "chelate" are used interchangeably herein. The terms "assay solution" and "iron sensing solution" are used interchangeably herein.

[0041] A composition and an assay solution for the determination of dissolved iron concentration comprising an a-diimine compound, a buffer, and a reducing agent are described. The a-diimine compound includes a pair of vicinal imine functional groups. This arrangement of imines allows for strong binding of ferrous (Fe ²⁺ ), and results in a chromophoric ferrous-diimine complex. The chromophoric iron-diimine complex absorbs certain wavelengths of visible light and transmits other wavelengths of light. Analysis of the colored complex can be used to build calibration curves and determine the concentration of ferrous (Fe ²⁺ ) in solutions with unknown concentration. FIG. 1 is a schematic depicting the mechanism of the determination of total iron in a solution. In FIG. 1 a solution containing Fe ²⁺ and Fe ³⁺ is treated with a reducing agent (for example ascorbic acid) in the presence of an α-diimine compound of the present invention. The a-diimine compound, 3-(2-pyridyl)- 5,6-diphenyl-l,2,4-triazine-4',4"-disulfonic acid sodium salt is shown in FIG 1, however any α-diimine compound described throughout the specification can be used. The ferric ions Fe ³⁺ in the mixture of ferric are reduced to ferrous ions Fe ²⁺ , and the Fe ²⁺ complexes with the a- diimine compound to form an α-diimine -iron compound, which has an known absorbance, for example, between 560 and 570 nm, preferably 565 nm.

[0042] The iron concentration of a sample can be determined by treating the sample with the present composition, measuring the absorbance, and comparing the absorbance to the absorbance of assay standards of known concentration. The iron concentration of a sample can be determined by placing a volume of the sample in an assay solution of the present invention and measuring the absorbance of the resulting solution. The a-diimine compound acts as a colorimetric iron sensor.

[0043] The buffer prevents changes in pH. The operable pH range for the present composition is from about 2 to about 7. In particular embodiments, the buffer is a phthalate buffer. In some embodiments, the phthalate concentration ranges from about 100 nM to about 2 M. In other embodiments, the phthalate concentration ranges from about 400 mM to about 1.2 M. In further embodiments, the phthalate concentration is 600 mM.

[0044] Total dissolved iron is the sum of ferrous (Fe ²⁺ ) and ferric (Fe ³⁺ ) ions in solution. The reducing agent reduces ferrous ions to ferric ions. The absorbance of the resulting ferrous-diimine solution is measured, and is a function of the analyte's ferrous concentration. Measured ferrous concentration is therefore indicative of the total iron concentration in a sample.

[0045] In other embodiments, a composition and an assay solution for the determination of dissolved iron concentration comprising an a-diimine compound and a buffer are described. In the absence of a reducing agent, the α-diimine compound acts as a colorimetric iron sensor of the ferrous ions in the sample.

[0046] An iron assay kit described throughout the Specification can be made by a) obtaining a microwell plate; b) obtaining a lyophilized titrant that includes the iron indicating composition α-diimine compound, optionally a reducing agent, a buffer, and, optionally, an excipient; c) adding sequentially increasing amounts of the lyophilized titrant composition to a plurality of microwells of the microwell plate such that when an analyte composition is added to the lyophilized titrant composition in each microwell of the plurality of microwells a solution forms. The plurality of microwells can be sealed with foil or a plastic film to inhibit or prevent the titrant composition from exiting the plurality of microwells. In some embodiments, the titrant composition consists essentially of 3-(2-pyridyl)-5,6-diphenyl-l,2,4- triazine-4',4"-disulfonic acid sodium salt, ascorbic acid, citric acid and trehalose dihydrate. In some embodiments, the composition can include polyethylene glycol compounds as another excipient. For example, the poly ethylene glycol compound can be p-(l, 1,3,3- tetramethylbutyl)-phenyl ether having from 9 to 10 ethylene oxide units, commonly sold under the trade name Triton X-100 (Dow Chemical, U.S.A.).

[0047] The iron assay kit can be used to determine the total iron, ferrous ion, ferric ion content. The microwell plate containing the lyophilized titrant compositions in microwells can be obtained. In some embodiments, one series of microwells (for example 8 microwells in one strip of a 96 microwell plate) can include the composition to determine total iron content and another series of microwells can be include the composition to determine ferrous ion content (e.g., the composition without a reducing agent). A known amount of analyte solution (for example 300 microliters) can be added to the lyophilized titrant composition in the microwells using a delivery apparatus (for example, multichannel pipette). After the solids in the plate have fully dissolved, the microwell plate can be placed in a detector (for example, a plate reader) and the absorbances of each microwell at the wavelengths of the iron complex are measured. In embodiments, when the iron indicator composition is 3-(2- pyridyl)-5,6-diphenyl-l,2,4-triazine-4',4"-disulfonic acid sodium salt, the absorbance at 565 nm.

[0048] In other embodiments, a method for the determination of ferric ions in a sample are described. The method comprises the steps of measuring the total iron concentration, measuring the ferrous ion concentration, and subtracting the measured ferrous ion concentration from the total iron concentration.

[0049] In some embodiments, a controller system can be coupled to the detector. The controller system can include a software algorithm that is used to determine the total iron content, ferrous ion content or ferric ion content. The data collected at the wavelength of the iron-complex is collected and plotted against the calibration curves. If the microwell plate includes a composition containing a reducing agent, the controller returns the total amount of iron in the sample. If ferric ion content is desired, the total iron content is determined using the composition that includes a reducing agent and the ferrous ion content is determined using the composition that does not include the reducing agent. The controller can compute the ferric ion concentration by subtracting the ferrous ion concentration from the total iron concentration. The two results can be displayed on a screen. Using the method and iron assay kit of the present invention, the total iron concentration of the analyte taken from a single well can be determined using one sample rather than using multiple samples and many indicators and/or titrant systems. The controller system 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. The measured absorbance from microwell plate 204 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 an alkalinity 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.

[0050] The claims are not to be interpreted as including means-plus- or step-plus- function limitations, unless such a limitation is explicitly recited in a given claim using the phrase(s) "means for" or "step for," respectively.

EXAMPLES

[0051] 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.

[0052] Example 1: The iron sensor solution was prepared by combining 25 g 3-(2- pyridyl)-5,6-diphenyl-l,2,4-triazine-4',4"-disulfonic acid sodium salt (PDT disulfonate) and 10 g of sodium ascorbate, 120 g 2-(N-morpholino)ethanesulfonic acid (MES) hydrate in 700 mL distilled water. The pH was adjusted to 5.5 with IN sodium hydroxide. The solution was transferred to a 1000 mL volumetric flask and diluted to the mark with distilled water, resulting in the final sensor solution: 50 mM PDT disulfonate, 60 mM ascorbate, 600 mM MES, pH 5.5.

[0053] Calibration Curves: The iron sensing solution of Example 1 can be used to develop a calibration curve for the determination of total iron in a solution. FIG. 2A is a table of raw calibration data for the iron-sensing solution. Standard iron concentrations are given in mg/L and absorbance (A565) is measured at 565 nm. FIG. 2B is a table of the processed calibration data for the iron sensing solution. Average absorbances, standard deviations (SD), and relative standard deviations (RSD) are listed for each iron concentration. From the data, a calibration curve can be developed. FIG. 3 is a calibration curve for the iron sensing solution using linear regression. The line of best fit for the iron sensing solution was R ² =0.99997.

[0054] Example 2. Total Iron Assay Kit. Micro wells (300 micro liter) of a 96- microwell plate were filled with 33% (100 microliters) of 10 mM of the a-diimine compound (3-(2-Pyridyl)-5,6-diphenyl-l,2,4-triazine-4',4"-disulfonic acid monosodium salt hydrate, Sigma Aldrich®), 30 mM ascorbic acid (free acid), 150 mM citric acid (free acid), 3% w/w trehalose dihydrate, 0.01% w/v Triton X-100, pH 3.2. The reagents listed in Table 1 are diluted with water to approximately 75% of the desired batch size with agitation until dissolved. The pH of the solution was adjusted to 3.2 using concentrated (3-30%>) sodium hydroxide prepared from ACS reagent grade sodium hydroxide and distilled water. The mixture was dilute up to approximately 95% of the batch size with distilled water, the pH was monitored, and then diluted with water to the final volume. The solution was filtered using a bottle top or vacuum capsule filter of 0.22 microns. The resulting aqueous titrant solution was lyophilized at -60 °C and 100 mtorr.

Table 1

Reagent for 1 L for 2 L for 5 L for 10 L a-diimine compound 4.93 g 9.86 g 24.65 g 49.3 g

Ascorbic Acid (free) 5.29 g 10.58 g 26.45 g 52.9 g

Citric Acid (free) 28.85 g 57.7 g 144.25 g 288.5 g

Trehalose dihydrate 30 g 60 g 150 g 300 g