Patent Description:
Ammonium nitrogen (NH<NUM>-N) is present in many above-ground water courses, in some ground waters, and in domestic and many commercial waste waters. Ammonia is present in the aquatic environment due to agricultural run-off and decomposition of biological waste. Ammonia thereby exists in water as either the ammonium ion (NH<NUM>+) or the un-ionized ammonia (NH<NUM>). Un-ionized ammonia is toxic to fish, even in low concentrations, while the ammonium ion is nontoxic except at extremely high concentrations. At neutral pH =<NUM> and ambient temperature, almost all of the ammonia exists as NH<NUM>+. As the pH and temperature increase, the amount of NH<NUM> increases and the amount of NH<NUM>+ decreases. Ammonium contents in water of <NUM> to <NUM>/l are therefore, depending on the pH of the water, classified as hazardous to fish. A water course is typically unsuitable for fishery purposes at ammonium contents of > <NUM>/l.

Ammonia is also toxic to all vertebrates causing convulsions, coma and death, probably because elevated NH<NUM>+ displaces K+ and depolarizes neurons, causing activation of NMDA type glutamate receptor, which leads to an influx of excessive Ca<NUM>+ and subsequent cell death in the central nervous system.

Ammonium compounds accordingly belong to the water-endangering substances, and limit values are set forth in many regulations. A regular monitoring of ammonium content in water and waste-water samples is therefore required.

Standardized methods for the determination of the ammonium content are frequently used. These include, for example, the Berthelot method where ammonia (NH<NUM>) reacts with a so-called "Berthelot's reagent" to form a blue product which is then used in a colorimetric method to determine ammonia. The method is carried out at an alkaline pH, at which ammonium is in the form of ammonia (NH<NUM>).

The reaction mechanism proposed for the Berthelot reaction consists of three steps. In a first step (<NUM>), ammonia reacts with hypochlorite to form monochloramine at basic pH:
The reaction mechanism proposed for the Berthelot reaction consists of three steps. In a first step (<NUM>), ammonia reacts with hypochlorite to form monochloramine at basic pH:.

In a second step (<NUM>), the monochloramine formed reacts with a phenol derivative in the presence of the catalyst nitroprusside to give a chloroquinone monoamine:
<CHM>.

The catalyst, nitroprusside, also called sodium nitroprusside, is a complex of the empirical formula Na<NUM>[Fe(CN)<NUM>NO]<NUM> • H<NUM>O. Various compounds are in principle suitable as the phenol derivative. Salicylates, thymol or <NUM>-chlorophenol can, for example, be used. In the final reaction step (<NUM>), the chloroquinone monoamine formed reacts with a further molecule of the phenol derivative to give a corresponding indophenol.

The formed indophenol has a blue color which is then measured photometrically, for example, at a wavelength in the region of its absorption maximum. In certain measurement ranges, indophenol correlates with the ammonium content of the sample.

The complete reaction mechanism has not been unambiguously clarified to date and proceeds via many intermediates.

The Berthelot method can in principle be used for various measurement ranges if the sample/reagent ratios are adapted correspondingly or the sample is pre-diluted.

Ammonium tests which use the Berthelot method are available from various manufacturers in the form of ready-to-use test sets. The Berthelot method is descripted in <CIT>, <CIT>, <CIT> and <CIT>.

The Berthelot method has the disadvantage that the blue color formed only correlates with the ammonium content in certain concentration ranges of the ammonium to be determined. The measurement signal (i.e., the extinction) of the photometric measurement generally increases with increasing ammonium content of the sample. However, the measurement signal (i.e., the extinction) then drops. <FIG> shows that the measurement signal of various concentrations of ammonium nitrogen (NH<NUM>-N) increasing and then decreasing. While no cause of this increase and decrease has to date been clarified with certainty, one theory is that the reaction mechanism no longer proceeds completely at unfavorable analyte/reagent ratios. A pH shift due to excessive monochloramine formed may be responsible. A reliable report of the ammonium content is not possible. A measurement undertaken at the wavelength in the region of absorption maximum expected for lower concentrations (i.e., at approximately <NUM>) would falsely report the concentration of ammonium nitrogen (NH<NUM>-N) which is <NUM>,<NUM> ppm in <FIG> to be between <NUM> and <NUM> ppm. This would result in a significant underestimation of the ammonium content, which might have serious repercussions for the environment.

The standards and test set manufacturers therefore prescribe that further analyses with a diluted sample (various pollution steps) should be carried out in addition to analysis of the sample in order to check the plausibility of the measurement result. This is, however, inconvenient and time-consuming for the user.

<CIT> attempted to address the above problem.

<CIT> describes a method to determine the plausibility of a measurement of the ammonium content using the Berthelot method where, besides the measurement of the extinction in the absorption region of the blue indole dye formed, a measurement of the extinction in the absorption region of the nitroprusside employed as catalyst is additionally performed on the sample. If the measurement in the absorption region of the nitroprusside employed as catalyst indicates that sufficient nitroprusside is present, the result of the ammonium determination is determined to be plausible. If the measurement in the absorption region of nitroprusside indicates that little or no catalyst is present, the plausibility of the result of the ammonium determination must be doubted. The plausibility of the actual measurement result of the ammonium determination can be checked via an additional measurement of the extinction in the absorption region of nitroprusside carried out directly on the same sample. Further measurements of dilutions of the sample are thereby normally unnecessary.

A disadvantage of this method is that a measurement of two different chemical compounds is required, i.e., a first measurement of the blue indole dye formed, and a second measurement of the absorption region of the nitroprusside employed as catalyst.

An object of the present invention is to provide an alternative method for the determination of the ammonium content of aqueous samples which directly provides information on the plausibility of the measurement result without the need to measure further samples, for example, as part of a dilution series. A further object of the present invention is to provide a method for the determination of the ammonium content of aqueous samples which directly provides information on the plausibility of the measurement result while only measuring the absorption regions of one chemical compound, i.e., the formed blue indole dye.

The invention provides a method for the determination of a plausibility of a measurement result of a determination of an ammonium content of an aqueous sample. The method includes providing a blue indole dye mixture of the aqueous sample with an alkaline pH by providing the aqueous sample; adding a base, a chlorinating agent, a phenol derivative, and nitroprusside, so as to obtain the blue indole dye mixture, whereas the method further comprises:· determining a first extinction value of the blue indole dye mixture in a first absorption region from <NUM> to <NUM>, the first extinction value being the measurement result,· determining a second extinction value of the blue indole dye mixture in a second absorption region from <NUM> to <NUM>, wherein the second absorption region is distant from the first absorption region,· dividing the first extinction value by the second extinction value so as to obtain a quotient; and· determining the measurement result to not be plausible if the quotient > <NUM>.

The present invention is described in greater detail below on the basis of embodiments and of the drawing in which:
<FIG> shows the measured extinction in the case of the determination of the ammonium content of a sample by the Berthelot method for various ammonium concentrations.

The present invention provides a method for the determination of a plausibility of a measurement result of a determination of an ammonium content of an aqueous sample. The method includes providing a blue indole dye mixture of the aqueous sample with an alkaline pH by providing the aqueous sample; adding a base, a chlorinating agent, a phenol derivative, and nitroprusside, so as to obtain the blue indole dye mixture, whereas the method further comprises:· determining a first extinction value of the blue indole dye mixture in a first absorption region from <NUM> to <NUM>, the first extinction value being the measurement result,· determining a second extinction value of the blue indole dye mixture in a second absorption region from <NUM> to <NUM>, wherein the second absorption region is distant from the first absorption region,· dividing the first extinction value by the second extinction value so as to obtain a quotient; and· determining the measurement result to not be plausible if the quotient > <NUM>.

An "aqueous sample" as used in the present invention is a sample which contains one or typically more components dissolved in water. The aqueous sample generally contains no further solvents apart from water. It may, however, contain up to <NUM> % of one or more water-miscible solvents, such as, for example, ethanol. An aqueous sample can be a water sample, a food or drink, or a body fluid. The aqueous sample is preferably a water sample, such as, for example, a sample taken from a water course, a waste-water sample, a ground water sample, a tap water sample, a sample of water fed to an industrial process or discharged from an industrial process as waste water, etc..

In order to determine the ammonium content quantitatively, the extinction of the sample after addition of the requisite reagents and after formation of the blue to blue-green indophenol (i. e, the blue indole dye mixture) is determined at a certain wavelength in the absorption region of the indophenol. The measurement is typically carried out at the absorption maximum of the blue indole dye mixture. The quantitative determination can then be carried out with the aid of a calibration curve. This procedure is known to the person skilled in the art. Indophenols generally have an absorption maximum in the wavelength range between <NUM> and <NUM>.

The quotient of the present invention will necessarily depend on the wavelengths chosen for the first absorption region and the second absorption region. The quotient can, for example, be > <NUM>, preferably > <NUM>, very preferably > <NUM>. The quotient can, for example, be greater than or equal to the quotients listed in Table <NUM>.

The first absorption region is chosen at a wavelength in a region which is expected for the absorption maximum of the blue indole dye mixture. This can for example, be anywhere from <NUM>-<NUM>, preferably from <NUM>-<NUM>, very preferably at approximately <NUM> or exactly at <NUM>. Possible first absorption regions are listed in Table <NUM>.

The second absorption region is chosen to be distant from the first absorption region. While the present invention is in no way limited thereto, it has been observed that the extinction of the blue indole dye mixture is more gradual when lower concentrations of ammonium nitrogen (NH<NUM>-N) are measured. Reference to <FIG> shows, for example, that the low concentrations of ammonium nitrogen (NH<NUM>-N) (i.e., the uppermost curve = <NUM> ppm, third curve from the bottom = <NUM> ppm, the curve second from the bottom = <NUM> ppm, and the bottommost curve = <NUM> ppm) have a more gradual extinction than does the high concentration of ammonium nitrogen (NH<NUM>-N) (i.e., the curve second from the top = <NUM>,<NUM> ppm). The curve showing the highest concentration of ammonium nitrogen (NH<NUM>-N) additionally has an earlier absorption maximum (i.e., about <NUM>) than does the other lower concentrations (i.e., about <NUM>). The curve showing the highest concentration of ammonium nitrogen (NH<NUM>-N) also shows an extinction having a steeper decline. A core aspect of the present invention is that a measurement in the second absorption region for a high concentration of ammonium nitrogen (NH<NUM>-N) will therefore result in a quotient which is significantly different from the lower concentrations of ammonium nitrogen (NH<NUM>-N). A high concentration of ammonium nitrogen (NH<NUM>-N) as used herein is understood to be a concentration > <NUM> ppm NH<NUM>-N, for example, > <NUM> ppm NH<NUM>-N, for example, > <NUM> ppm NH<NUM>-N, for example, > <NUM>,<NUM> ppm NH<NUM>-N.

The second absorption region can, for example be from <NUM>-<NUM>, preferably from <NUM>-<NUM>, preferably from <NUM>-<NUM>, preferably from <NUM>-<NUM>, preferably from <NUM>-<NUM>, preferably from <NUM> to <NUM>, very preferably at approximately <NUM> or exactly at <NUM>. Possible second absorption regions are listed in Table <NUM>.

The selection of the correct quotient will therefore depend on the selection of the first absorption region and on the second absorption region. A person skilled in the art will therefore know which quotient to choose in order to determine whether the measurement result is plausible. For example, if the first absorption region is selected at the expected absorption maximum of the blue indole dye mixture, i.e., at approximately <NUM> or, preferably, at exactly <NUM>, then:.

In the determination of the ammonium content by the Berthelot method, a chlorinating agent, a phenol derivative and nitroprusside as catalyst are added to the sample in the alkaline region, and a certain time, which is generally between <NUM> and <NUM> minutes, preferably between <NUM> and <NUM> minutes, is typically awaited in order that the detection reaction can take place. A blue-colored indophenol thereby forms over several reaction steps. The extinction determined in the absorption region of the indophenol correlates with the ammonium content of the sample in certain measurement ranges known to the person skilled in the art. The measurement of the ammonium content can be carried out in accordance with the present invention using any variant of the Berthelot method in which nitroprusside is used as a catalyst.

The ammonium content of a sample is a sample's content of ammonium compounds present which can be converted into ammonia when the sample is rendered alkaline or are in the form of ammonia in an alkaline sample. The alkaline pH can thereby be between pH <NUM> and <NUM>, preferably between pH <NUM> and <NUM>, very preferably about pH <NUM>. The alkaline pH of the aqueous sample with an alkaline pH can, for example, be provided by adding a base or hydroxide solutions. Use can, for example, be made of aqueous LiOH solutions, NaOH solutions and KOH solutions, preferably a sodium hydroxide solution. Examples of ammonium compounds are therefore ammonium salts, ammonium hydroxide and ammonia.

The chlorinating agent can, for example, be dichloroisocyanurate (DIC), dichloroisocyanuric acid or sodium hypochlorite. The phenol derivative can, for example, be phenol or <NUM>-substituted phenols, such as <NUM>-chlorophenol, thymol or salicylates, or <NUM>-hydroxy benzyl alcohol. It is also possible to use the method described in DIN <NUM>/<NUM>, in which dichloroisocyanuric acid and sodium salicylate is used.

Various known methods can be used to provide the blue indole dye mixture of the aqueous sample. The blue indole dye mixture can, for example, be provided by providing the aqueous sample, adding a base to the aqueous sample so as to provide the aqueous sample with an alkaline pH, and adding to the aqueous sample with the alkaline pH a chlorinating agent, a phenol derivative, and nitroprusside, so as to obtain the blue indole dye mixture.

The blue indole dye mixture of the aqueous sample can alternatively be provided by providing the aqueous sample, mixing a chlorinating agent and an alkaline buffer to obtain a first mixture, mixing the aqueous sample and the fist mixture to obtain a second mixture, and adding a phenol derivative and a nitroprusside to the second mixture to obtain the blue indole dye mixture. A person skilled in the art would also know that all of the above can be added together at the same time. A person skilled in the art can also directly add all of the above into, for example, a cuvette for subsequent analysis.

The phenol derivative can, for example, be <NUM>-hydroxy benzyl alcohol. The nitroprusside can, for example, be sodium nitroprusside. The chlorinating agent can, for example, be sodium hypochlorite. The alkaline buffer can, for example, be an alkaline citrate buffer.

After the chlorinating agent, the phenol derivative, and nitroprusside as a catalyst are added to the aqueous sample with an alkaline pH, a certain time, which is generally between <NUM> and <NUM> minutes, preferably between <NUM> and <NUM> minutes, should typically elapse in order to allow the detection reaction to take place. A blue-colored indophenol (i.e., the blue indole dye mixture) thereby forms over several reaction steps. The extinction determined in the absorption region of the indophenol correlates with the ammonium content of the sample in certain measurement ranges known to the person skilled in the art. The measurement of the ammonium content can be carried out in accordance with the present invention using any variant of the Berthelot method in which nitroprusside is used as catalyst.

Five aqueous samples containing Ammonium nitrogen (NH<NUM>-N) were previously determined using conventional methods. These aqueous samples contained the following amounts of Ammonium nitrogen (NH<NUM>-N):.

<NUM> of each of the aqueous samples <NUM>-<NUM> to be tested were provided. To each <NUM> aqueous sample was added <NUM> of a <NUM>:<NUM> mixture of:.

The resulting mixture was then stirred for <NUM> minutes at room temperature (i.e., approximately <NUM>);.

A graph of the absorption data of each of aqueous samples <NUM>-<NUM> at various wavelengths from <NUM> to <NUM> is shown in <FIG>. The data for <FIG> for the wavelengths <NUM> to <NUM> is set forth in Tables <NUM>-<NUM> above.

A comparison of the quotients of Tables <NUM>-<NUM> makes clear that the quotients of each of aqueous samples <NUM>-<NUM> are for the most part remarkably close to each other. For Table <NUM>, for example, the quotient is approximately <NUM>. Starting from Table <NUM>, where a wavelength of <NUM> was used, shows that choosing a wavelength > <NUM> causes the quotient to increase, while a choice of a wavelength between approximately <NUM> and <NUM> would cause the quotient to decrease. The reason for this variance is due to the degree of extinction as is shown in <FIG>. Aqueous sample <NUM> exhibits an earlier absorption maximum of approximately <NUM> (see <FIG>) and a corresponding steeper decline in the area of measurement of the second absorption region. A measurement of the second absorption region anywhere from <NUM> to <NUM> will therefore always result in a quotient which is significantly different from those of aqueous samples <NUM>-<NUM>. Based solely on the absorbance of the aqueous solution <NUM> at the first absorption region of <NUM>, i.e., <NUM>, a person skilled in the art would normally reasonably conclude that the fifth aqueous sample has a ppm NH<NUM>-N of approximately <NUM> in that it clearly lies between the curves for <NUM> ppm NH<NUM>-N and <NUM> ppm NH<NUM>-N. However, the high quotient of <NUM> based on the measurement at the second absorption region of <NUM> clearly shows this result to not be plausible. The data for the aqueous sample <NUM> can thereby be discarded as not plausible by measuring the same sample twice. A separate measurement to determine the amount of nitroprusside remaining is not thereby required.

Claim 1:
A method for determining of a plausibility of a measurement result of a determination of an ammonium content of an aqueous sample, the method comprising:
• providing a blue indole dye mixture of the aqueous sample with an alkaline pH by,
providing the aqueous sample;
adding a base, a chlorinating agent, a phenol derivative, and nitroprusside, so as to obtain the blue indole dye mixture, whereas
the method further comprises:
• determining a first extinction value of the blue indole dye mixture in a first absorption region from <NUM> to <NUM>, the first extinction value being the measurement result,
• determining a second extinction value of the blue indole dye mixture in a second absorption region from <NUM> to <NUM>, wherein the second absorption region is distant from the first absorption region,
• dividing the first extinction value by the second extinction value so as to obtain a quotient; and
• determining the measurement result to not be plausible if the quotient > <NUM>.