WO2011046943A1 - Bromothymol blue composition for detection of free radicals - Google Patents

Abstract

Methods, compositions, and kits for detection and measurement of free radicals using sulfophthalein dyes.

Description

BROMOTHYMOL BLUE COMPOSITION FOR DETECTION OF FREE RADICALS

Cross Reference to Related Application

[0001] This application claims priority to U.S. Provisional Application No. 61/251,452 filed October 14, 2009, the entire contents of which are hereby incorporated by reference.

Field of the Invention

[0002] The present invention relates to methods, compositions, and kits for detecting and measuring free radicals in a sample using a sulfophthaleine dye. The invention also relates to methods of using sulfophthaleine dyes and test kits to monitor and optimize removal of organic contaminants in soils, groundwaters, wastewaters, and drinking water.

BACKGROUND

[0003] Free radicals are useful to destroy a wide array of organic chemicals associated with water, wastewater, waste, and surface and subsurface remediation processes. Free radicals are atoms or molecules with one or more unpaired electrons. Free radicals may be inorganic or organic. When electrons are added singly to molecular oxygen, reactive intermediates are formed. With the four electron reduction of oxygen to water, the superoxide anion, hydrogen peroxide, hydroxyl radical and water are sequentially formed.

[0004] Fenton (1894) first proposed Fe(II) salts could activate hydrogen peroxide to oxidize tartaric acid. Later, Haber and Weiss (1934) hypothesized that the oxidant generated in the Fenton reaction was the hydroxyl radical. The free radical chain reaction based on the decomposition of hydrogen peroxide was published by Barb (1949, 1951a and 1951b). The discovery of organic free radicals took place by Moses Gomberg in 1900. It is know well known that the hydroxyl radical can form carbon centered free radicals with organic compounds via hydrogen abstraction of carbon-, hydrogen- and oxygen-hydrogen bonds, adding to carbon double bonds or to aromatic rings.

[0005] The reactions of persulfate ions (S₂0₈ ^") with various organic and inorganic compounds have been extensively studied (House, 1962; Berlin, 1986). Highly reactive species such as sulfate free radicals (SO^4"-) and hydroxyl radicals (OH-) may be generated as a result of heat- or photo- or base- or metal- catalyzed decomposition of persulfate ions in aqueous phases (Berlin, 1986; Tanner and Osman, 1987). These species when placed in contact with organic compounds may initiate a series of radical chain reactions leading to oxidation of the organic substance.

[0006] Ozone has been used since 1893 in Netherlands to disinfect drinking water.

Hoigne and Bader (1976) reported the decomposition of ozone in aqueous systems and identified hydrogen peroxide, superoxide, the hydroxyl radical and the perhydroxyl radical in water.

[0007] Typically complex analytical instruments such as Electron Spin Resonance coupled to the spin trapping technique using the 5,5-dimethyl 1-pyrroline N-oxide (DMPO) as spin trap agent are used to measure free radical species. Selective radical quenching with radical traps such as: superoxide dismutase for the superoxide radical; deoxyguanosine, salicylic acid, 1-octanol, and ascorbic acid for the hydroxyl radical, followed by repeat reactions to examine changes in reaction rates, chemical or biochemical reaction mechanisms in the presence and absence of the radical scavengers are also used as ways to help determine the presence or absence of specific radicals.

SUMMARY OF THE INVENTION

[0008] This application presents embodiments of an invention which include methods of detecting a quantity of free radicals in a sample comprising preparing at least one aqueous solution containing a sulfophthalein dye and at least a portion of the sample, reacting the sulfophthalein dye with the sample, observing the solution(s) for a predetermined period of time and detecting the presence or absence of a change in absorbance of a wavelength in the solution(s) or a change in the intensity of the color of the solution(s) associated with the presence or absence of free radicals in the sample. In specific embodiments, the sulfophthalein dye is bromothymol blue.

[0009] This application also presents embodiments of an invention including methods for determing the amount of free radicals in a sample by preparing at least one aqueous solution containing a sulfophthalein dye and at least a portion of the sample and reacting the sulfophthalein dye with the sample and measuring the change in absorbance of a wavelength in the solution(s) or a change in the color intensity of the solution(s) between at least two time points to determine a rate of change associated with the amount of free radicals in the sample. In specific embodiments the sulfophthalein dye is bromothymol blue. [0010] This invention also includes compositions comprising a sulfophthalein dye, a water sample, and optionally an agent selected from the group consisting of an oxidant, an activator, a surfactant and a radical scavenger. In certain embodiments, the water sample may be a sample of wastewater or groundwater, or a soil sample, or a sample in which one or more organic contaminants are being removed by oxidative remediation.

[0011] Further embodiments of the invention include kits comprising a vessel, and a predetermined amount of dry weight of sulfophthalein dye sufficient to make a concentration of sulfophthalein dye between lOppm and lOOOppm when a volume of test sample is added or when added to a test sample, and a color intensity scale calibrated to different quantities of free radicals in the test sample. Other embodiments of the invention include kits comprising a vessel, and an amount of sulfophthalein dye in a concentrated solution sufficient to make a concentration of sulfophthalein dye between lOppm and lOOOppm when a volume of test sample is added or when added to a test sample and a color intensity scale calibrated to different quantities of free radicals in the test sample. In specific embodiments, the sulfophthalein dye is bromothymol blue.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Fig. 1 shows the structure of bromothymol blue (bottom left), the color of solutions of bromothymol blue at different pH (upper right), UV-Vis spectra for bromothymol blue at pH<6 for varying concentrations of bromothymol blue (bottom right), and a calibration curve comparing the concentration of bromothymol blue to the absorbance of 431nm (upper left).

[0013] Fig. 2 shows the UV-Vis absorbance spectrum for a solution of bromothymol blue and 2% hydrogen peroxide at different time intervals over a period of 60 minutes.

[0014] Fig. 3 shows the UV-Vis absorbance spectrum for a solution of bromothymol blue, 2% hydrogen peroxide, and 0.33mM GT-nZVI at different time intervals over a period of 8 minutes.

[0015] Fig. 4 shows a series of changes in bromothymol blue concentration in solutions having 2% hydrogen peroxide activated by different concentrations of GT-nZVI over a period of time.

[0016] Fig. 5 shows a table of experimental conditions for detecting free radical production in solutions having 0.1% hydrogen peroxide activated by different concentrations of Fe-TAML.

[0017] Fig. 6 shows the changes in bromothymol blue concentration over a period of time for the samples shown in Fig. 5.

[0018] Fig. 7 shows photographs of solutions of bromothymol blue added to groundwater samples obtained from different depths (feet below ground (fbg)) having low concentrations of sodium persulfate.

[0019] Fig. 8 shows UV-Vis absorbance spectra for the solutions shown in Fig. 7.

[0020] Fig. 9 shows photographs of solutions of bromothymol blue added to groundwater samples from different depths (fbg) having higher concentrations of sodium persulfate.

[0021] Fig. 10 shows UV-Vis absorbance spectra for the solutions shown in Fig. 9.

[0022] Fig. 11 shows photographs of groundwater samples obtained from different depths (fbg) having different concentrations of sodium persulfate.

[0023] Fig 12 shows UV-Vis absorbance spectra for the solutions shown in Fig 11.

DETAILED DESCRIPTION

[0024] Embodiments of the invention are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent parts can be employed and other methods developed without parting from the spirit and scope of the invention. All references cited herein are incorporated by reference as if each had been individually incorporated. For example, international application number PCT US2007/007517, filed March 27, 2007, and U.S. provisional application number 60/785,972, filed March 27, 2006, are hereby incorporated by reference.

[0025] Embodiments of the invention include methods for detecting a quantity of free radicals in at least one sample comprising preparing at least one aqueous solution containing a sulfophthalein dye and at least a portion of the sample and reacting the sulfophthalein dye with the sample for a predetermined period of time and detecting the presence or absence of a change in absorbance of a wavelength in the solution(s) or a change in the intensity in the color of the solution(s) associated with the with the presence or absence of free radicals in the sample. In this invention, the change in absorbance of a wavelength by the solution, or a change in the intensity in the color of the solution is not caused by a change in pH. Instead, the change in absorbance, or change in color intensity is caused by a chemical change in the dye, or degradation of the dye by free radials in the solution. Examples of free radicals that may be detected with this system include, but are not limited to, hydroxyl radicals, superoxide free radicals, hydroperoxide free radicals, sulfate free radicals, and organic free radicals.

[0026] Sulfophthalein dyes are useful probe molecules that are not degraded by direct oxidation and can be chemically degraded via free-radical pathways. Bisaminophenothiazine dyes, such as methylene blue, while not directly oxidized by H₂0₂ alone, do undergo direct oxidation by persulfate, rendering them unsuitable for use for detecting radicals produced by persulfate or produced in a system with a mixture of persulfate and other oxidants, for example, with hydrogen peroxide, with permangenate, with calcium peroxide or with ozone. In contrast, sulphophthalein dyes, such as bromothymol blue, are resistant to direct oxidation by both peroxide and persulfate, and make excellent probe compounds for the study of free radical oxidation using both H₂0₂ and sodium persulfate (Na₂S₂0g).

[0027] Sulfophthalein dyes, also called sulfonphthalein dyes, or sulfone phthalein dyes, are a class of commonly used pH indicator dyes having a common sulphophthalein structure (shown below). The dyes are commonly used for measuring pH of aqueous solutions, and for acid/base titrations. Aqueous solutions of sulfophthalein dyes change color with changes in pH, depending on the specific pk_a of the dye. The different substituents on the structure influence the pk_a of the dye, as well as the color of the dye in solution.

where each R group may be, independently H, halogen or Cj-C₄ alkyl. Examples of sulfophthalein dyes include bromothymol blue, phenol red, bromocresol green, bromocresol purple, bromophenol blue, and thymol blue.

[0028] As used here, the term "Ci-C₄ alkyl" include both straight and branched chains containing one to four carbon atoms, as well as cyclic structures such as cyclopropyl and cyclobutyl. Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (Pr) (including n- propyl (ⁿPr or n-Pr), isopropyl ('Pr or i-Pr) and cyclopropyl (^cPr or c-Pr)), butyl (Bu) (including n-butyl ("Bu or n-Bu), isobutyl ('Bu or i-Bu), tert-butyl (^lBu or t-Bu) and cyclobutyl (^cBu or c- Bu)). The term "halogen" means F, CI, Br, or I.

[0029] In some specific embodiments, the sulfophthalein dye is bromothymol blue.

[0030] One or more solutions may be prepared using at least a portion of the sample.

The sample may be a laboratory sample, or may be a sample of groundwater or wastewater or soil. Alternatively, the sample may be from a remediation site, from a wastewater treatment site, a drinking water treatment site, or groundwater remediation site.

[0031] A "remediation site" or "treatment site" is a location undergoing remediation or treatment. In other words, the area is currently being treated to reduce or remove the amount of one or more contaminant from the site. Contaminant remediation often uses radical generation to oxidize organic contaminants, thereby reducing their harmful effects. The method of the present invention is useful for monitoring the progress of contaminant remediation by measuring radical production in a remediation site. The site may be any location where the water is currently being treated, including in situ and ex situ treatment sites. Remediation or treatment may take place at the site of contamination, or may occur at a site remote from the contaminated area. As used herein, "site" also refers to the particular location where water treatment is taking place, for instance, for the treatment and purification of drinking water.

[0032] As used herein "remediation materials" or "treatment materials" refer to all of the materials used in the reduction of a contaminant or treatment of a site. The materials may be, for example, oxidants, oxidant activators, antioxidants, radical scavengers, surfactants, co-solvents, radical scavengers, oxidant stabilizers, or other materials (such as salts or structural materials), or combinations thereof.

[0033] As used herein, "wastewater" is any water that has been adversely affected in quality by anthropogenic influence. It comprises liquid waste discharged by domestic residences, commercial properties, industry, and/or agriculture and can encompass a wide range of potential contaminants and concentrations.

[0034] As used herein, "groundwater" is water located beneath the ground surface.

Groundwater also includes natural water removed from the ground, but without distillation or osmotic purification.

[0035] The sulfophthalein dye may be added to the sample, or the sample may be added to the sulfophthalein dye. The dye may be dry, i.e. powder, or may be in a concentrated solution. Generally, the solution is dissolved in water, i.e. an aqueous solution, but may also include co-solvents miscible with water, which do not hinder the detection of the sulfophthalein dye. Examples of suitable co-solvents include ethanol, methanol and acetonitrile.

[0036] When combined with a sulfophthalein dye, free radicals in the solution react with the dye, causing a chemical transformation. The chemical transformation may be detected as a change in the absorbance of a wavelength of the solution. The absorbance of the solution may be measured by any means known in the art. Most commonly, absorbance is measured by UV Vis spectrophotometry. The measurement may be made at a single wavelength or across a full spectrum. As the reaction between the free radical and the sulfophthalein dye occurs, the pH does not change significantly. Therefore, the change in absorbance of a wavelength is not caused by a change in pH. Sulfophthalein dyes have unique UV/Visible absorbance spectra, and the UV Visible spectra change depending on the pH of the solution. But if the pH does not change, the UV/Vis absorbance spectrum does not change, except when free radicals are present in the solution. One of ordinary skill may easily select a suitable monitoring wavelength for a particular sulfophthalein dye. Often, a useful wavelength is one having a significant absorbance for the particular sulfophthalein dye at the pH of the sample. For bromothymol blue, for example, when the pH is below 6, the wavelenth of 431 nm is useful for detecting the change in absorbance associated with the presence of free radicals in the solution. A useful wavelength may be a wavelength where the sulfophthalein dye has at least some absorbance, but where the sample (absent the sulfophthalein dye) does not absorb.

[0037] One may also detect a change in color intensity associated with the presence of free radicals in the sample. The change in color intensity may be detected visibly. The color intensity may be compared with a calibrated chart or color intensity scale. A calibrated chart may be easily prepared by preparing standardized solutions having a specific pH, using a particular sulfophthalein dye, and having a known quantity of free radicals. In this way the quantity of radicals in the sample may be estimated by comparing the color intensity to the calibrated chart. The ability to visually detect the change in color intensity allows the method to be used easily in the field, or at a remote remediation or treatment site where laboratory equipment may not be available. In certain specific embodiments, where the sulfophthalein dye is bromothymol blue, the color intensity scale includes a scale at a pH range of less than about 6.5, between about 6.5 and about 7.6, and greater than about 7.6 or combinations thereof. Since the color of the bromothymol blue solution changes based on pH, a calibrated color intensity scale may be provided for different pH ranges, so that the intensity of the measured sample may be easily compared.

[0038] A calibration chart may be prepared, for example, by adding a discreet quantity of a solution having a fixed concentration of sulfophthalein dye to a sample having a known quantity of free radicals. A series of samples with different known quantities of free radicals are used to prepare the calibration chart. The samples are allowed to react for a predetermined period of time, sufficient for the free radicals to produce a discernable change in color intensity. The color intensity for each sample after the set period of time is recorded on the calibration chart. One of ordinary skill may, based on this teaching, prepare calibration charts for many sulphophthalein dyes.

[0039] For example, to determine the amount of free radicals in a sample having an unknown amount of free radicals, the same quantity of a solution having the same concentration of sulfophthalein dye used to prepare the calibration chart is added to the sample. The sample is allowed to react for the same amount of time used for the preparation of the calibration chart, and then the color intensity is compared with the calibration chart. The color intensity at that time relates directly to the concentration of free radicals in the solution determined by the calibration chart.

[0040] The solutions are prepared using at least a portion of the sample of interest. The concentration of the sample in the prepared solution may vary. For instance, if the color change caused by the presence of free radicals occurs too quickly, the method may be easily repeated with using a smaller portion of the sample of interest. Often, the sample will be diluted to a preset volume for ease of detecting the change in color intensity. [0041] The concentration of sulfophthalein dye in the solution(s) may also vary. A number of factors may determine the concentration of sulfophthalein dye needed for the method. For instance, some dyes are more intensely colored in certain pH ranges. Therefore, if a sample has a pH where the dye is more intensly colored, then a smaller quantity of the dye may be required to detect free radicals in the sample. Likewise, if the color is less intense, a higher concentration of sulfophthalein dye may be required to detect a color change. In general, however, one of ordinary skill may easily determine useful concentrations of a particular sulfophthalein dye for use at a particular pH. Since the sulfophthalein dyes are also pH indicators, it is simple to determine whether a greater or lesser concentration of sulfophthalein dye is needed, since the relative pH can be easily determined by the initial color of the solution. If necessary, additional sulfophthalein dye may then be added to the sample to produce an effective concentration. For example, bromothymol blue may be used at a concentration of about 250ppm to about 500 ppm when the pH is less than 7.6, and may be used at a concentration of about 25ppm to about 50 ppm when the pH is greater than 7.6. If the solution, after the bromothymol blue is added is blue (indicating a pH greater than 7.6), it is apparent that a final concentration of about 25 ppm to about 50 ppm should be used. If the sample is yellow (indicating a pH below 7.6) a concentration of about 250ppm to about 500ppm should be used. Effective concentrations may be easily determined for different sulfophthalein dyes.

[0042] The predetermined period of time for observation will also vary, and can be easily determined by one of ordinary skill in the art. For instance, standard solutions prepared using known concentrations of free radical may be used to determine the minimum amount of time required for a sufficient change in color intensity. Alternatively, a longer time interval may be used to allow maximum reaction to take place before detecting the color change. The predetermined time interval may be, for example, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 36 hours, 48 hours, 3days, 4 days, 5 days or longer. Color intensity for bromothymol blue, for example, is stable for periods of at least 5 days without degradation of color in the absence of free radicals. An extended interval, up to 5 days for example, may be used to allow maximum reaction of the sulfophthalein dye.

[0043] In other embodiments, the invention further comprises comparing the color intensity of the detected sample with a calibrated color scale. By comparing the color intensity with a calibrated color scale, the change in color intensity may be detected visually, without additional laboratory equipment. Applications

[0044] The method for detecting a quantity of free radicals may be used to monitor or optimize organic contaminant remediation processes. The method may be used, for instance to measure whether or not a particular oxidant and/or oxidant/activator combination is producing free radicals.

[0045] As used herein, the term "oxidant" includes all oxidizing compounds or compounds that decompose or react to form an oxidizing compound. For example, the term "oxidant" includes solid, liquid, or gaseous compounds that can decompose to liberate oxygen or an oxidizing species. For example, the term "oxidant" includes compounds such as persulfates, percarbonates, peroxides, hydrogen peroxide, and permanganates. For example, the term "oxidant" also includes oxidizing gases, such as oxygen, ozone, and air. For example, the term "oxidant" also includes dissolved gases, such as oxygen or ozone, dissolved in an aqueous or non-aqueous liquid.

[0046] An "activator" can be, for example, a chemical molecule or compound, or another external agent or condition, such as heat, temperature, or pH, that increases the rate of or hastens a chemical reaction. The activator may or may not be transformed during the chemical reaction that it hastens. Examples of activators which are chemical compounds include a metal, a transition metal, a chelated metal, a complexed metal, a metallorganic complex, and hydrogen peroxide. Examples of activators which are other external agents or conditions include heat, temperature, and high pH. Example activators include Fe(II), Fe(III), Fe(II)-EDTA, Fe(III)- EDTA, Fe(II)-EDDS, Fe(III)-EDDS, Fe(II)-citric acid, Fe(III)-citric acid, hydrogen peroxide, high pH, FeTAML, manganese oxides, include octahedral molecular sieves (OMS), and heat.

[0047] The method may be used, for example, in a laboratory setting. In a laboratory setting, the method to detect a quantity of free radicals in a sample may be used to determine whether a particular concentration of oxidant and/or oxidant/activator is producing free radicals. Various types of free radical generating oxidants and oxidant/activator combinations may be screened easily using the method of the invention, allowing for rapid determination of suitable materials for remediation of an organic contaminant.

[0048] The method may be used, for example, at a remediation or treatment site. At a remediation site, the method of detecting a quantity of free radicals in a sample may be used to determine if a composition administered to remediate an organic contaminant is functioning, i.e. whether free radicals are being produced in the remediation site or remediation zone at the site. Based on the results of the method, the administered composition may be altered. For instance, if no radicals are detected, additional oxidant and/or activator may be added to the remediation site until radical formation is detected. Likewise, if radicals are detected, then no further materials, such as oxidants and/or activators need to be added, thereby conserving remediation materials. The method can also be used to monitor the progress of a remediation or treatment process by testing periodically to test whether free radicals are still present in the remediation site. Based on the results, further materials may be added to the remediation site. In this way, the method may improve the efficiency of a remediation or treatment project by reducing the time or amount of materials required for the remediation or treatment project.

[0049] The method of the invention may also be used, for example, for rapid determination of the area where contaminant remediation is occurring (the zone of remediation), by testing the site at different locations, detecting the presence of free radicals in one or more locations. By accurately determining the zone of remediation, the remediation materials may be utilized efficiently and not wasted. The tested locations may be separated by distance along the surface, be at different depths, or both. Determining the zone of remediation allows the zone of remediation to be changed, if necessary. Changing the zone of remediation means altering the size or shape of the zone of remediation. When remediation materials are administered at a particular location, they may spread out from the site of administration to different degrees depending on the environment, establishing a zone of remediation. The zone may be made larger in width, length, depth or combination thereof. This may be accomplished by administering additional remediation materials to the same location, causing the remediation zone to grow. The remediation zone may also be expanded by administering remediation materials at additional locations. In other instances, remediation materials may be administered at different depths to expand the depth of the remediation zone. In this way, a larger contaminated area may be remediated effectively. If necessary, the zone of remediation may also be made smaller, in width, length, depth or combination thereof, e.g., if the zone of remediation encompasses an area larger than the contaminated area. This may be achieved, for example, by administering less remediation materials at a particular location, or administering remediation materials at fewer locations in the contaminated site.

[0050] Other embodiments of the invention include methods for determining the amount of free radicals in a sample comprising preparing at least one aqueous solution containing a sulfophthalein dye and at least a portion of the sample and reacting the sulfophthalein dye with the sample; measuring the change in absorbance of a wavelength in the solution(s) or a change in the color intensity of the solution(s) between at least two time points to determine a rate of change associated with the with the amount of free radicals in the sample. In specific embodiments, the sulfophthalein dye is bromothymol blue.

[0051] The solution(s) may be prepared as described previously. The measurement of the change in absorbance of a wavelength in the solution(s) or change in the color intensity of the solutions may be measured as described previously, e.g. by measurement of the UV- Visible absorbance spectrum or by visual inspection. The amounts of sample and amounts of sulfophthalein dye used in the method will vary, but should be sufficient to detect the change in absorbance of a wavelength or change in color intensity between at least two time points.

[0052] In this embodiment, at least two measurements are made to determine the amount of free radicals in a sample. The method may therefore be used to quantify the amount of free radicals in a sample. If desired, the method may be used to determine the absolute amount of free radicals by performing a calibration using standardized solutions having known concentrations of free radicals, and comparing the measured rate of change with the standardized solutions. Otherwise, relative amounts of free radicals may be easily determined by whether the rate of change is greater than or less than a desired value.

[0053] The method may further include the step of comparing the rate of change in absorbance of a wavelength or color intensity with a desired predetermined value. The predetermined value will vary based on the application. The predetermined value may be measured in a laboratory, e.g. as the amount of free radicals required to oxidize a particular concentration of a contaminant in a particular amount of time. The predetermined value may be, for example, 1) a minimum threshold for contaminant remediation or effective water treatment, 2) a maximum threshold to prevent undesired oxidation of compounds which should not be oxidized, such as biomolecules or living matter, 3) an optimized level for efficient use of remediation materials to prevent waste of materials or manpower.

[0054] Based on the method, an iterative process may be used to minimize the difference between the measured amount of free radicals in the sample and the predetermined value by modifying the amount of free radicals in the sample. In this context, modifying the amount of free radicals in the sample may mean changing the concentration of oxidants, activators, antioxidants, radical scavengers, surfactants, co-solvents, other materials, or combinations thereof (also known as remediation or treatment materials) in the sample being measured. In other instances, modifying the amount of free radicals in the sample may mean changing the concentration of the remediation materials in the remediation or treatment site from which the sample is obtained. In either case, modifying the amount of free radicals in the sample may mean increasing or decreasing the amount, depending on the difference between the measured value and the desired predetermined value. After the amount of free radicals are modified in the sample (or site from which the sample is obtained) the steps of preparing, reacting, measuring, comparing and modifying are repeated until the measured amount agrees with the predetermined value. The measured value may "agree" with the predetermined value when the two are equal, or when the difference between the two is within a desired margin for error, or when the difference between the measured value and the predetermined value is minimized.

[0055] In some embodiments, the predetermined value is the amount of free radicals needed to effectively reduce the amount of a contaminant in a remediation site. The effective amount of free radicals may be determined in a laboratory setting based on controlled experiments. Conditions may therefore be optimized for a particular contaminant prior to introducing remediation materials into a contaminated site. In these embodiments, the method may be used to measure the optimized amount, thereby determining the predetermined value. The method may also be used to optimize the reduction of a contaminant by determining whether conditions at the remediation site are in agreement with the optimized conditions determined in the laboratory. If necessary, the conditions at the remediation site may be modified using the methods described to reduce the difference between the measured amounts and the predetermined value. For instance, the amounts of remediation materials may be changed to increase or decrease the amount of free radicals produced at the remediation site.

[0056] In further embodiments, the sample may include an oxidant and an oxidant stabilizing chemical. The method may then be used to determine the effectiveness of the oxidant stabilizing chemical by comparing the amount of free radicals in the sample with the amount of free radicals in an identical sample lacking the oxidant stabilizing chemical. In this case, an effective stabilizing chemical is one where a lower amount of free radicals are present in the sample containing the stabilizing chemical compared with a sample lacking the stabilizing chemical. [0057] The method may be extended to determine the effective amount of an oxidant stabilizing chemical by preparing and measuring multiple samples having different quantities of oxidant stabilizing chemical and comparing the amount of free radicals in those samples with a predetermined value. The effective amount of oxidant stabilizing chemical is selected as the quantity of oxidant stabilizing chemical producing a sample with an amount of free radicals in agreement with the predetermined value. The predetermined value may be any particular value, based on the application. Again, the predetermined value may be measured in a laboratory under controlled conditions. For instance, the predetermined value may be the amount necessary for effective remediation of an organic contaminant from a contaminated site. The predetermined value is not always the minimum, since an oxidant stabilizing chemical which completely prevents formation of free radicals would not be useful in radical mediated remediation processes.

[0058] The method may also be extended to screen oxidant stabilizing chemicals by preparing and measuring multiple solutions from samples having different oxidant stabilizing chemicals, comparing the amounts of free radicals in each solution with a predetermined value, and selecting the oxidant stabilizing chemicals having an amount of free radicals lower than the predetermined value. The method of the invention enables the rapid screening of different chemicals to determine which compounds stabilize a particular oxidant. The predetermined value may be any desired value, and may be determined experimentally or selected for a particular application. For instance, if a particular application requires free radicals to be produces at a rate lower than the normal rate, for instance, because the rate of solubilization of the contaminant is slower than the rate of oxidation, then the predetermined value may be measured as the amount of free radicals that give the slower rate of oxidation f the contaminant. The predetermined value will vary based on the specific contaminant, as well as the remediation or treatment processes used.

[0059] Oxidant stabilizing compounds may include plant-based surfactants or combinations of biodegradable surfactants and co-solvents such as those described in concurrently filed US provisional patent application entitled "Stabilized Surfactant-Oxidant Composition and Methods for Remediation" (Attorney Docket No. 65637-281591, filed October 14, 2009), which is incorporated by reference in its entirety. [0060] The surfactant and/or cosolvent may be, for example, ALFOTERRA 123-8S,

ALFOTERRA 145-8S, ALFOTERRA L167-7S, ETHOX HCO-5, ETHOX HCO-25, ETHOX CO-40, ETHOX ML-5, ETHAL LA-4, AG-6202, AG-6206, ETHOX CO-36, ETHOX CO-81, ETHOX CO-25, ETHOX TO- 16, ETHSORBOX L-20, ETHOX MO- 14, S-MAZ 80K, T-MAZ 60 K 60, TERGITOL L-64, DOWFAX 8390, ALFOTERRA L167-4S, ALFOTERRA L123-4S, ALFOTERRA L145-4S, VeruSOL-1 surfactant, VeruSOL-2 surfactant, VeruSOL-3 surfactant, VeruSOL-4 surfactant, VeruSOL-5 surfactant, VeruSOL-6 surfactant, Citrus Burst 1 , Citrus Burst 2, Citrus Burst 3, and E-Z Mulse., Citrus Burst 1, Citrus Burst 2, Citrus Burst 3, E-Z Mulse, or combinations thereof. VeruSOL surfactants are available from VeruTEK, Inc. ALFOTERRA surfactants are available from Sasol North America. Citrus Burst surfactants are available from Florida Chemical. Ethox, Ethal, and Ethsorbox surfactants are available from Ethox Chemicals. S- Maz and T-Maz surfactants are available from BASF. Tergitol and DOWFAX are available from Dow Chemicals.

[0061] For example, blends of biodegradable citrus-based solvents (for example, d- limonene) and degradable surfactants derived from natural oils and products can be used. Other examples include compositions of surfactant and cosolvent containing at least one citrus terpene and at least one surfactant. A citrus terpene may be, for example, CAS No. 94266-47-4, citrus peels extract (citrus spp.), citrus extract, Curacao peel extract (Citrus aurantium L.), EINECS No. 304-454-3, FEMA No. 2318, or FEMA No. 2344. A surfactant may be a nonionic surfactant. For example, a surfactant may be an an ethoxylated soybean oil, an ethoxylated castor oil, an ethoxylated coconut fatty acid, or an amidified, ethoxylated coconut fatty acid, an alkyl polyglucoside or an alkyl polyglucoside-based surfactant, a decylpolyglucoside, or an alkyl decylpolyglucoside-based surfactant or combinations thereof. An ethoxylated castor oil can include, for example, a polyoxyethylene (20) castor oil, CAS No. 61791-12-6, PEG (polyethylene glycol)-10 castor oil, PEG-20 castor oil, PEG-3 castor oil, PEG-40 castor oil, PEG-50 castor oil, PEG-60 castor oil, POE (polyoxyethylene) (10) castor oil, POE(20) castor oil; POE (20) castor oil (ether, ester); POE(3) castor oil, POE(40) castor oil, POE(50) castor oil, POE(60) castor oil, or polyoxyethylene (20) castor oil (ether, ester). An ethoxylated coconut fatty acid can include, for example, CAS No. 39287-84-8, CAS No. 61791-29-5, CAS No. 68921-12-0, CAS No. 8051-46-5, CAS No. 8051-92-1, ethoxylated coconut fatty acid, polyethylene glycol ester of coconut fatty acid, ethoxylated coconut oil acid, polyethylene glycol monoester of coconut oil fatty acid, ethoxylated coco fatty acid, PEG- 15 cocoate, PEG-5 cocoate, PEG-8 cocoate, polyethylene glycol (15) monococoate, polyethylene glycol (5) monococoate, polyethylene glycol 400 monococoate, polyethylene glycol monococonut ester, monococonate polyethylene glycol, monococonut oil fatty acid ester of polyethylene glycol, polyoxyethylene (15) monococoate, polyoxyethylene (5) monococoate, or polyoxyethylene (8) monococoate. An amidified, ethoxylated coconut fatty acid can include, for example, CAS No. 61791-08-0, ethoxylated reaction products of coco fatty acids with ethanolamine, PEG- 11 cocamide, PEG-20 cocamide, PEG-3 cocamide, PEG-5 cocamide, PEG-6 cocamide, PEG-7 cocamide, polyethylene glycol (11) coconut amide, polyethylene glycol (3) coconut amide, polyethylene glycol (5) coconut amide, polyethylene glycol (7) coconut amide, polyethylene glycol 1000 coconut amide, polyethylene glycol 300 coconut amide, polyoxyethylene (11) coconut amide, polyoxyethylene (20) coconut amide, polyoxyethylene (3) coconut amide, polyoxyethylene (5) coconut amide, polyoxyethylene (6) coconut amide, or polyoxyethylene (7) coconut amide.

[0062] Other embodiments of the invention include methods for optimizing the formation of free radicals. In this case, the methods used to measure the amount of free radicals present in a sample may be used to optimize the production of free radicals. The optimization process may be performed in a laboratory or field setting, based on the particular application. Optimizing the formation of free radicals may involve maximizing the rate of free radical production, for instance by optimizing the ratio between oxidant and activator. Optimization may also involve decreasing the rate of free radical production to match the rate of solubilization of a contaminant, or optimizing free radical production to use the least amount of materials, such as oxidant, activator, surfactant, antioxidant, etc. In certain instances, optimal conditions for remediating a particular contaminant may be determined in a laboratory. The amount of free radicals produced by the optimized conditions may be measured using the methods for determining the amount of free radical in a sample. The remediation process may then be moved to a remediation site. After remediation begins, the amount of free radicals produced at the remediation site may be measuring using the method of the invention, and compared with the optimized amounts from the laboratory. Field conditions may then be adjusted as needed to match the optimal conditions.

[0063] Other embodiments of the invention include methods for optimizing a free radical treatment process. Treatment or remediation processes includes reducing the amount of a contaminant in contaminated groundwater or wastewater or soil, or treating drinking water using free radicals. Again, the methods of the invention may be used to optimize the processes. In this case, optimization my include increasing the rate of remediation, or reducing the amount of remediation materials required for the remediation or treatment process. The type of optimization will depend on the particular application.

[0064] Features of this method include the ability to qualitatively and quantitatively screen for the presence of free radicals in water and waste samples in an easy manner that does not require complex and expensive instrumentation and can easily be conducted in the field. This is an improvement in the application of advanced oxidation processes that are dependent on the successful generation of free radicals to effect desired chemical reactions intended for the destruction of organic compounds or oxidation of inorganic compounds

[0065] Other embodiments of the invention include compositions created by practicing the methods of the invention. Embodiments of the invention include compositions comprising a sulfophthalein dye and a water sample. The water sample may be groundwater, contaminated groundwater, wastewater. The compositions may optionally include oxidants, activators (also called oxidant activators), surfactants and/or co-solvents, such as the oxidant stabilizing compounds described previously, radical scavengers or combinations thereof.

[0066] Other embodiments of the invention include test kits. The test kit may comprise a vessel, and a predetermined amount of dry weight of sulfophthalein dye sufficient to make a concentration of sulfophthalein dye between lOppm and lOOOppm when a volume of test sample is added or when added to a test sample, and a color intensity scale calibrated to different quantities of free radicals in the test sample. The color intensity scale is calibrated based on solutions having known concentrations of free radicals measured after a predetermined period of time, sufficient for the detection of the free radicals according to the method of the invention. The color intensity scale may be a printed chart, graph or table. Instructions may also be included to inform the user of the specific concentration of sulfophthalein dye, volume of sample, and time interval for the measurement. The tested sample may then be compared with the calibrated color intensity scale to determine whether free radicals are present, or to determine the amount of free radicals present in the sample, using the methods of the present invention. In specific embodiments, the sulfophthalein dye is bromothymol blue.

[0067] In other embodiments, the kit comprises a vessel, and an amount of sulfophthalein dye in a concentrated solution sufficient to make a concentration of sulfophthalein dye between lOppm and lOOOppm when a volume of test sample is added or when added to a test sample and a color intensity scale calibrated to different quantities of free radicals in the test sample. The calibrated color intensity scale is discussed previously. In this embodiment, the sulfophthalein dye is present in a concentrated solution sufficient to make an effective concentration (i.e. between lOppm and lOOOppm) when combined with a test sample. Again, the kit may include instructions as to the amount of sulfophthalein dye solution, sample, and time interval. The tested sample may be compared with the color intensity scale to determine whether free radicals are present, or to determine the amount of free radicals present in the sample. In some embodiments, the sulfophthalein dye is bromothymol blue.

[0068] In some embodiments, the concentration of bromothymol blue in the vessel after addition of or addition to the test sample is between 25 ppm and 50 ppm when the pH is greater than 7.6. In other embodiments, the concentration of bromothymol blue in the vessel after addition of or addition to the test sample is between 250 ppm and 500 ppm when the pH is less than 7.6.

[0069] In other embodiments, the color intensity scale includes a color intensity scale for bromothymol blue at a pH range of less than about 6.5, between about 6.5 and about 7.6, and greater than about 7.6 or combinations thereof. Since the color of the bromothymol blue solution changes based on pH, a calibrated color intensity scale may be provided for different pH ranges, so that the intensity of the measured sample may be easily compared.

[0070] The new feature of this method is the ability to qualitatively and quantitatively screen for the presence of free radicals in water and waste samples in an easy manner that does not require complex and expensive instrumentation and can easily be conductive in the field. This is an improvement in the application of advanced oxidation processes that are dependent on the successful generation of free radicals to effect desired chemical reactions intended for the destruction of organic or oxidation of inorganic compounds.

EXAMPLES

[0071] Bromothymol Blue can be quantatively determined using UV/VIS spectroscopy, as shown in Fig. 1. The lack of degradation of bromothymol blue by direct oxidation in a 2% solution of hydrogen peroxide with a 500 mg/L bromothymol blue is shown in Fig 2. In contrast when hydrogen peroxide is catalyzed to form free radicals, the bromothymol blue will degrade rapidly, as shown in Fig. 3. Green Tea synthesized nano-scale zero-valent iron (GT-nZVI) are prepared according to the methods described in U.S. Provisional Application 61/071,785 and PCT Application PCT/US2009/044402 which are incorporated by reference in its entirety. The relationship between the rate of bromothymol blue concentration can be directly related to the rate of free radical generation as shown in Fig 4.

[0072] Experimental conditions of free radical degradation of bromothymol blue catalyzed by various Fe-TAML concentrations are shown in Fig. 5. The free radical degradation of bromothymol blue catalyzed by various Fe-TAML concentrations over time are shown in Fig. 6. Fe-TAML catalysts are described, for example, in U.S. Patents 5,847,120, 5,876,625, 6,011,152, 6,051,704, 6,054,580, 6,099,586, 6,100,394, 6,136,223, 6,241,779, and 7,060,818 which are incorporated by reference in their entirety.

EXAMPLE 1

[0073] The ability of bromothymol blue to measure the presence of free radicals is demonstrated by using groundwater samples obtained from a site in which the Surfactant Enhanced In Situ Chemical Oxidation (S-ISCO®, VeruTEK, Inc.) process using sodium persulfate, activated with Fe-EDTA and VeruSOL-3 acting as a surfactant was in the process of being implemented at a Field Site. S-ISCO® is described in U.S. Pre-Grant publication 2008/0207981, which is incorporated by reference in its entirely. At this site, various groundwater sampling locations located across the site were used to collect depth discrete groundwater samples. Groundwater samples obtained from the field were sampled for sodium persulfate concentrations and had bromothymol blue added to samples. Photographs of samples were taken immediately after the bromothymol blue was added to the samples and after a 5 day period. Additionally, sodium persulfate concentrations were measured after the 5 day period. An example of depth discrete groundwater samples from a zone without significant detections of persulfate is shown in Fig 7. There was no significant decrease in the color of the various samples and there was no evidence of bromothymol blue concentration decreases over the five day period from review of the UV VIS scans shown in Fig. 8.

[0074] In contrast depth discrete groundwater samples were obtained from a sampling location at the same site when free radical generation was expected based on detections of significant sodium persulfate concentrations in the 10 foot to 28 foot depth intervals. It is evident that free radicals were present in samples with greater than 1 g/L of sodium persulfate based on the Day 0 and Day 5 sample photographs and UV/VIS scans in Fig. 9 and Fig. 10, respectively. In all samples with sodium persulfate concentrations greater than 1 g/L, the color due to the presence of bromothymol blue in the sample was completely absent after the 5 day period. Additionally, all samples with sodium persulfate concentrations greater than 1 g/L had no remaining UV VIS absorption after the five day period. It is also evident that the one sample with less than 1 g/L of sodium persulfate did not experience any color change and did not have a change in the UV/VIS absorption, both indicating no free radicals were present.

[0075] In an additional set of depth discrete groundwater samples taken from another location, the same trend in change of color to clear and reduction of UV/VIS absorption is evident in samples with greater than 1 g/L of sodium persulfate initially present in the sample, as shown in Fig. 11 and Fig. 12, respectively.

[0076] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Nothing in this specification should be considered as limiting the scope of the present invention. All examples presented are representative and non-limiting. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

Claims

Claim 1. A method for detecting a quantity of free radicals in at least one sample comprising

Combining at least a portion of the sample and a sulfophthalein dye in at least one aqueous solution and reacting the sulfophthalein dye with the sample for a predetermined period of time;

detecting the presence or absence of a change in absorbance of a wavelength in the solution(s) or a change in the intensity in the color of the solution(s) associated with the presence or absence of free radicals in the sample.

Claim 2. The method of claim 1, wherein the sulfophthalein dye is bromothymol blue.

Claim 3. The method of any one of claims 1-2 wherein the at least one sample is from a remediation site.

Claim 4. The method of any one of claims 1 -2, wherein the at least one sample is from a wastewater treatment process.

Claim 5. The method of any one of claims 1-2, wherein the at least one sample is from a drinking water treatment process.

Claim 6. The method of any one of claims 1-2 wherein the at least one sample is a groundwater sample.

Claim 7. The method of any one of claims 1-2 wherein the at least one sample is from a groundwater treatment process.

Claim 8. The method of claim 3 further comprising preparing more than one sample from different locations of the remediation site and measuring the zone of effective remediation as encompassing locations where free radicals are detected and not encompassing locations where free radicals are not detected.

Claim 9. The method of Claim 8, further comprising changing the zone of remediation.

Claim 10. The method according to claim 1, further comprising measuring the change in absorbance of a wavelength in the solution(s) or a change in the color intensity of the solution(s) between at least two time points to determine a rate of change associated with the amount of free radicals in the sample.

Claim 11. The method of claim 10, wherein the sulfophthalein dye is bromothymol blue.

Claim 12. The method of any one of claims 10-11 further comprising comparing the rate of change in color intensity or change in absorbance of a wavelength with a desired predetermined value.

Claim 13. The method of claim 12, further comprising minimizing the difference with the predetermined value by modifying the amount of free radicals in the sample and repeating the preparing, reacting, measuring, comparing and modifying steps until the measured amount agrees with the predetermined value.

Claim 14. The method of any one of claims 10-11, wherein the predetermined value corresponds to the amount of free radicals needed to effectively reduce the amount of a contaminant in a remediation site.

Claim 15. The method of any one of claims 10-14, wherein the sample further includes an oxidant and an oxidant stabilizing chemical.

Claim 16. The method of claim 15, further comprising determining the effectiveness of the oxidant stabilizing chemical by comparing the amount of free radicals in the sample with the amount of free radicals in an identical sample lacking the oxidant stabilizing chemical, and associating a lower amount of free radicals in the sample with an effective oxidant stabilizing chemical.

Claim 17. The method of Claim 15, further comprising

determining an effective amount of oxidant stabilizing chemical by

preparing and measuring multiple solutions from samples having different

quantities of oxidant stabilizing chemical, and

comparing the amounts of free radicals in each solution with a predetermined value; and

selecting the amount of oxidant stabilizing chemical having an amount of free radicals in agreement with the predetermined value.

Claim 18. The method of claim 14 further comprising

screening oxidant stabilizing chemicals by

preparing and measuring multiple solutions from samples having different

oxidant stabilizing chemicals,

comparing the amounts of free radicals in each solution with a predetermined value, and

selecting the oxidant stabilizing chemicals having an amount of free radicals in agreement with the predetermined value.

Claim 19. A method for optimizing the formation of free radicals comprising performing the method of claim 13

Claim 20. A method for optimizing a free-radical treatment process comprising performing the method of claim 14.

Claim 21. A composition comprising a sulfophthalein dye, a water sample, and one or more agents selected from the group consisting of an oxidant, an activator, a surfactant, and a radical scavenger.

Claim 22. A kit comprising a vessel, and an amount of sulfophthalein dye sufficient to make a concentration of sulfophthalein dye between lOppm and lOOOppm when a volume of test sample is added or when added to a test sample, and a color intensity scale calibrated to different quantities of free radicals in the test sample.

Claim 23. The kit according to claim 22 wherein the sulfophthalein dye is dry.

Claim 24. The kit according to claim 22 wherein the sulfophthalein dye is in a concentrated solution.

Claim 25. The kit of any one of claims claim 22-24, wherein the sulfophthalein dye is bromothymol blue.

Claim 26. The kit of claim 25, wherein the concentration of bromothymol blue in the vessel after addition of or addition to the test sample is between about 25 ppm and 50 ppm when the pH is greater than 7.6.

Claim 27. The kit of claim 25, wherein the concentration of bromothymol blue in the vessel after addition of or addition to the test sample is between about 250 ppm and about 500 ppm when the pH is less than 7.6.

Claim 28. The kit of any one of claims 23-27 wherein the color intensity scale is calibrated at pH ranges of less than about 6.5, between about 6.5 to about 7.6, greater than about 7.6, or combinations thereof.

Claim 29. The method of any one of claim 1-9, wherein the absorbance of a wavelength or color intensity is compared to a calibrated chart.

Claim 30. The method of claim 2, wherein the color intensity after a period of reaction is compared to a calibrated chart.

Claim 31. The method of claim 30, wherein the calibrated chart is calibrated at pH ranges of less than about 6.5, between about 6.5 to about 7.6, and greater than about 7.6.

Claim 32. The method of claim 2, wherein the color intensity of the solution after a period of reaction is compared to solutions of predetermined concentrations of bromothymol blue.

Claim 33. The method of claim 32, wherein the color intensities in the sample are compared to solutions of predetermined concentrations of bromothymol blue at pH ranges of less than about 6.5, about 6.5 to about 7.6, and greater than about 7.6.