FIELD

The present invention relates to a method to measure phosphonates in the aid of lanthanide(lll) ions and buffering agents.

BACKGROUND Phosphonates are chelating agents that contain one or more phosphonic acid groups [C- PO(OH)2].They are widely employed as chemical additives to function as threshold antiscalants, corrosion inhibitors, chelants, sludge conditioners, deflocculants, dispersants and crystal growth modifiers in various industrial water treatment processes. They are used predominantly as scale and corrosion preventatives for boiled and cooling water towers and in oil and gas industry. They are also used in industrial cleaning and in laundry detergents. Phosphonates are also used in the medical industry to treat for example bone and calcium metabolism related illnesesses.

A standard method for determining the presence of phosphonates is ion chromatography with post-column reaction with Fe(lll), and detection of the Fe(lll) complexes at 300-330 nm [Anal. Chem. 328, 1987, 46]. Phosphonates have also been determined by ion-pair HPLC as Fe(lll) complexes using an eluent consisting of a bicarbonate solution at 8.3, tetrabutylammonium bromide as a counter ion an 14% acetonitrile on a reversed-phase polymer column, and detecting the complexes at 260 nm [J. Chromatography A, 773, 1997, 139]. Other methods include post column oxidation of the phosphonate to phosphate and detection of the phosphate with molybdenum blue method [Anal. chem. 329, 1987, 584]. UV photochemical oxidation method for phosphonate analysis involves a photochemical oxidation of phosphonate followed by conventional colorimetric determination of the liberated orthophosphate by ascorbic acid method. Phosphonates have also been determined by ICP spectrometry and by capillary electrophoresis. The prior art methods have several drawbacks. They either suffer from high detection limit, or they require the use of expensive instrumentation or laborious sample preparation.

SUMMARY

The following presents a simplified summary in order to provide a basic understanding of some aspects of various invention embodiments. The summary is not an extensive overview of the invention. It is neither intended to identify key or critical elements of the invention nor to delineate the scope of the invention. The following summary merely presents some concepts of the invention in a simplified form as a prelude to a more detailed description of exemplifying embodiments of the invention.

In the present invention it was observed that phosphonates can be analyzed with low detection limit simply by admixing a sample including one or more phosphonates with lanthanide(lll) ions in a buffer solution including certain buffering agents.

In accordance with the invention, there is provided a new method to determine quantity of one or more phosphonates in a sample, the method including:

- obtaining a sample including one or more phosphonates,

- admixing the sample with lanthanide ion in a form of lanthanide salt, and a buffer solution including a buffering agent, wherein pK _a value of the buffering agent is from 6 to 10, and wherein the buffering agent does not include two or more COOH groups but includes one or more OH groups,

- allowing the lanthanide ion to chelate with the one or more phosphonates,

- detecting signal derived from the lanthanide(lll) ion with luminescence measurement, and

- determining the quantity of the one or more phosphonates in the sample based on the signal derived from the lanthanide(lll) ion.

According to another aspect the present invention concerns a computer program product including program code means stored on a computer-readable medium, which code means are arranged to perform all the steps of the method claims 1 -14 when the program is run on a computer.

Various exemplifying and non-limiting embodiments of the invention both as to constructions and to methods of operation, together with additional objects and advantages thereof, will be best understood from the following description of specific exemplifying embodiments when read in connection with the accompanying drawings.

The verbs "to comprise" and "to include" are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows photon counts as a function of DTPMP concentration (ppm) according to Example 1 ,

Figure 2 shows photon counts as a function of DTPMP concentration (ppm) according to Example 2, Figure 3 shows photon counts as a function of DTPMP concentration (ppm) according to Example 3,

Figure 4 shows photon counts as a function of DTPMP concentration (ppm) according to Comparative Example 4,

Figure 5 shows the effect of a buffering agent at a constant DTPMP concentration (10 ppm) to the Signal to Noise ratio,

Figure 6 shows photon counts as a function of DTPMP concentration (ppm) in various concentrations of HEPES; 0.1 mM (gray line), 5 mM (dashed black line) and 10 mM (solid black line),

Figure 7 shows effect of various concentrations of admixed potassium and calcium ion to the Signal to Noise ratio while DTPMP concentration is 10 ppm; 0.5 M KCI (1 ), no salts (2), 1 .0 M CaCI ₂ (3) and 0.5 M CaCI ₂ (4), and

Figure 8 shows effect of various concentrations of admixed barium ions to the Signal to Nose ration while DTPMP concentration is 10 ppm; 1 .0 M BaCI ₂ (1 ), 0.75 M BaCI ₂ (2), 0.5 M BaCI ₂ (3) and 0.25 M BaCI ₂ (4). DESCRIPTION

According to one embodiment the invention of the present disclosure concerns a method to determine quantity of one or more phosphonates in a sample, the method including:

- obtaining a sample including one or more phosphonates,

- admixing the sample with lanthanide ion and a buffer solution including a buffering agent, wherein pK _a value of the buffering agent is from 6 to 10, and wherein the buffering agent does not include two or more COOH groups, and wherein the buffering agent includes one or more OH groups,

- allowing the lanthanide ion to chelate with the one or more phosphonates, - detecting signal derived from the lanthanide(lll) ion with luminescence measurement, preferably with time-gated luminescence measurement, and

- measuring the quantity of the one or more phosphonates in the sample based on the signal derived from the lanthanide(lll) ion.

The method of the present disclosure can be used for determination of any phosphonates. According to a preferable embodiment, the one of more phosphonates have two or more phosphonic acid groups in its/their structure. This is to achieve better lanthanide chelate stability. This may be important if the sample includes interfering components such as competing ions and/or chelating agents, like carboxylic acids, amines, phosphates and soaps, and the interfering components cannot be removed or diluted prior to analysis. Exemplary phosphonates that can be quantified with the method of the present disclosure are aminotri(methylene phosphonic acid), 1 -hydroxyethylene-(1 ,1 -diphosphonic acid) (HEDP), aminotris(methylenephosphonic acid) (ATMP), ethylenediaminetetra- (methylenephosphonic acid) (EDTMP) and diethylenetriaminepenta(methylenephosphonic acid) (DTPMP). A preferable phosphonic acid is DTPMP which is widely used in oil and gas industry as scale and corrosion inhibitor, wherein the concentration of DTPMP typically varies from 0 to 50 ppm.

The buffering agent according to the present disclosure has pK _a value from 6 to 10. The buffering agent does not include two or more COOH groups, but it includes one or more OH groups. According to a preferable embodiment the buffering agent does not include any COOH groups. Exemplary buffering agent suitable for the method of the present disclosure are 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES), 2-(bis(2- hydroxyethyl)amino)-2-(hydroxymethyl)propane-1 ,3-diol, bis(2-hydroxyethyl)amino- tris(hydroxymethyl)methane (Bis-Tris), 1 ,3-bis(tris(hydroxymethyl)methylamino)propane (Bis-Tris-propane), 3-[[1 ,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxy- propane-1 -sulfonic acid (TAPSO), tris(hydroxymethyl)aminomethane (Tris), and 2-(bis(2- hydroxyethyl)amino)acetic acid (bicine). Preferable buffering agents are HEPES, Bis-Tris, TAPSO and TRIS. A particular buffering agent are TAPSO and Bis-Tris. Also mixtures of the buffering agents can be used.

Concentration of the buffering agent wherein concentration is preferably from 0.1 mM to 100 mM, more preferably 0.1 to 50 mM and most preferably 0.1 to 10 mM. Figures 1 - 3 show photon counts as a function of DTPMP concentration (ppm) measured in according to the method of the present disclosure using HEPES and Bis-Tris as a buffering agent and europium(lll) as a lanthanide ion. The concentration of the phosphonate can be easily quantified, since the photon counts (i.e. the europium signal) are dependent on the phosphonate concentration. By contrast, when the buffering agent was changed to W-cyclohexyl-3-aminopropanesulfonic acid (CAPS), the phosphonate concentration cannot be analyzed, since the increase of phosphonate concentration does not have any effect on the europium signal (Figure 4).

Figure 5 shows signal to noise ratios (S/N) in a methods for quantifying a phosphonate in the presence of various buffering agents. While CAPS does not have any effect on S/N ratio, the buffering agents according to the present disclosure increase the S/N ratio significantly.

An exemplary optimization of buffer agent (HEPES) concentration is shown in Figure 6. The phosphonate concentration could be easily measured by using buffer agent concentration from 0.1 to 10 mM. An optimal buffer agent concentration according to this experiment was about 5 mM. As seen from Figure 6, the buffering agent concentration can be over 10 mM or below 0.1 mM, but the detection sensitivity may be lower.

According to the method disclosed herein, the lanthanide ion concentration should be preferably below 10 mM, more preferably between 10 mM and 0.0001 mM most preferably around 10 μΜ. If the initial concentration of the one or more phosphonates in the sample is over 10% by weight, the sample is preferably diluted. Exemplary diluent is water or aqueous buffer solution, preferably including HEPES, TAPSO or Bis-Tris.

The buffer of the method of the present disclosure must be selected as disclosed to obtain good detection sensitivity. When the method is performed as claimed, phosphonates in the range below 10 ppm in the sample can be quantified

Figures 1 and 2 show phosphonate analysis in the presence and absence of a divalent ion (Ca ²⁺ ), respectively. As shown admixing with a divalent ion increases the detection sensitivity significantly.

Figures 7 and 8 show results of phosphonate determination in the presence and absence of admixed ions. Divalent ions such as Ca ²⁺ and Ba ²⁺ significantly enhance the signal to noise ratio and the detection sensitivity. By contrast, a monovalent ion, such as K ⁺ did not have any effect. According to a preferable embodiment the method further includes admixing with a divalent metal ion. Exemplary divalent metal ions are Ba ²⁺ , Sr ²⁺ and Ca ²⁺ . Preferable divalent metal ions are Sr ²⁺ and Ca ²⁺ . Most preferable divalent metal ion is Ca ²⁺ . The divalent metal ions are preferably admixed as corresponding salts, such as halides. An exemplary calcium salt is CaC . An exemplary barium salt is BaC . An exemplary strontium salt is SrC . The lanthanide ion used in the present disclosure is selected from europium, terbium, samarium and dysprosium, preferably europium and terbium, even more preferably europium. A combination of lanthanide ions may be used according to the invention.

The lanthanide ion used in the present disclosure is admixed in the form of lanthanide salt, such as lanthanide(l l l) chloride or lanthanide(l l l) acetate. A preferable lanthanide(l l l) salt is lanthanide(l l l) halide, such as europium(l l l) chloride. Accordingly, the lanthanide(l l l) ion is introduced as a salt, such as europium chloride and the lanthanide(l l l) chelate is formed upon conjugation of the lanthanide(l l l) ion with the one or more phosphonates.

The analysis of the one or more phosphonates according to the method of the present disclosure is performed in a detection vessel. The vessel may be e.g. a well, a part of a fluidic device or a cuvette.

In some applications, the sample includes molecules that have to be removed or diluted prior to analysis. According to this embodiment the sample is subjected to pretreatment step prior to admixing with the lanthanide(l l l) ion. Exemplary pretreatment methods are size exclusion chromatography, ion-exchange chromatography, filtration and dilution with an appropriate solution. It should be understood that the pretreatment step means preferably removal or dilution of molecules that may disturb the examination of phosphonates of interest, not isolation of the phosphonates. According to another embodiment, the pretreatment includes collecting the one or more phosphonates to an ion-exchange resin or to an immobilized metal followed by liberating the one or more phosphonates by elution with a suitable solvent or solvent system.

The method described herein is used for measuring the quantity of the one or more phosphonates. If the phosphonate is known, the method allows the quantification of the phosphonate in the sample. The method allows also quantification of total amount of phosphonates in the sample. The analysis of the one or more phosphonates according to the method of the present disclosure can be quantitative. For quantitative analysis, typically a standard curve or standard point is first prepared, and then the concentration of the known phosphonate is calculated using the standard curve or the standard point. Alternatively the instrument used for the analysis can be pre-calibrated to support the quantification. The signal derived from lanthanide(lll) ion is detected by luminescence measurement. Typically, lanthanide is excited at excitation wavelength and measured at emission wavelength. According to a preferable embodiment the lanthanide(lll) ion is detected using time-gated luminescence. Any TRF reader can be employed. Excitation and emission wavelengths are selected so that the S/N is the best. Also the delay time can be optimized. According to a preferable embodiment the lanthanide(lll) ion is europium and the excitation and emission wavelength is 395 nm and 615 nm, respectively. A preferable delay time for europium is 400 με. When other lanthanides, i.e. terbium, samarium or dysprosium are used, the excitation and emission wavelengths and the delay time are chosen based on the requirements of the lanthanide ion. According to another embodiment the lanthanide(lll) is detected using luminescence measurement without time gating.

According to another embodiment the present disclosure includes a computer program including software modules for determining information indicative for the sample, in order to evaluate the signal derived from the method. The software modules can be e.g. subroutines of functions implemented with a suitable programming language and with a compiler suitable for the programming language and the programmable processor.

A computer program product according to an exemplifying embodiment of the present disclosure includes a computer readable medium e.g. a compact disc, encoded with a computer program according to an embodiment of the present technology. A signal according to the exemplary embodiment is encoded to carry information defining a computer program according the embodiment.

As disclosed herein, the method of the present disclosure can be used to quantify phosphonates using cheap and small instrumentation. The simple assay protocol enables performing the phosphonate test on site e.g. on oil fields, and environmental monitoring agricultural chemicals such as fertilizers, pesticides and soil conditioners, as well as healthcare monitoring of therapeutic levels in biological samples e.g. HEDP as osteoporosis medication.

Examples

Example 1

To a known volume of a sample including unknown amount of DTPMP was added CaC , EuCI ₃ and HEPES-buffer to give final concentration of Ca ²⁺ , Eu ³⁺ and HEPES 0.5 M, 10 μΜ, and 5 mM, respectively. Eu ³⁺ was excited at 395 nm, and the luminescence signal from Eu ³⁺ was monitored at emission wavelength 615 nm with a spectrofluorometer (Aqsens, Espoo, Finland). Delay time was 400 με. The concentration of DTPMP was predicted using a standard curve. Results are shown in Figure 1 . Example 2

The experiment was performed as in Example 1 but omitting CaC . Results are shown in Figure 2.

Example 3

The experiment was performed as in Example 1 but replacing HEPES buffer with Bis-Tris buffer (5 mM). Result is shown in Figure 3.

Example 4 (Comparative Example)

The experiment was performed as in Example 1 , but by replacing HEPES buffer with N- cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer. Results are shown in Figure 4.

Example 5. Effect of various buffering agents. The experiment was performed as in Example 1 but the buffering agent was varied and concentration of phosphonate was constant at 0 ppm or 10 ppm. Results are shown in Figure 5.

Example 6. Optimization of Buffer concentration.

The experiment was performed as in Example 1 but the concentration of HEPES buffer was varied from 0.1 mM to 10 mM. The results are shown in Figure 6.

Example 7. Effect of admixed salts. Calcium and Potassium. The experiment was performed as in Example 1 but concentration of phosphonate was 10 ppm or 0 ppm and the assays were performed in the presence or absence of admixed salts (0.5 M K ⁺ ; 0.5 M or 1 .0 M Ca ²⁺ ).The results are shown in Figure 7.

Example 8. Effect of admixed salts. Barium.

To a known volume of a sample including constant amount of DTPMP was added BaC , EuCb and Bis-Tris buffer to give final concentration of Ba ²⁺ , Eu ³⁺ and Bis-Tris 0.25 M -1 M, 10 μΜ, and 5 mM, respectively. Eu ³⁺ was excited at 395 nm, and the luminescence signal from Eu ³⁺ was monitored at emission wavelength 615 nm with a spectrofluorometer (Aqsens, Espoo, Finland). Delay time was 400 με. The results are shown in Figure 8.

The specific examples provided in the description given above should not be construed as limiting the scope and/or the applicability of the appended claims.