METHOD OF ANALYSIS

Abstract: Methylenebisthiocyanate and charged detergents, both biocidal, interact to form a complex; this interaction can be used in the assay of either. The complex can be dissociated with lauryl sulphate. Another assay comprises contacting an immobilised non-ionic hydrophobic material with the sample and with an agent that binds to the thus-insolubilised surfactant but not with the material, and then with an eluant for the agent, and determining the presence of the agent in the eluate.

Description: METHOD OF ANALYSIS Field of the Invention This invention relates to assays, in particular for compounds which can be converted to aldehydes and for surfactants. Background of the Invention There appears to be no simple system for determining cationic surfactants in any field, although they are widely used in waste disposal, shampoos and, as biocides, in cooling towers. Examples of cationic surfactants that are used as biocides in cooling towers, in order to control organisms such as Legionella, are tetrakis(hydroxymethyl)- phosphoniu sulphate, tributyltetradecylphosphonium chloride, and (alkyl)quaternised dimethylbenzylammoniu chloride or dodecyldimethylammonium chloride (respectively available under the trade names Tolcide, Bellacide 350, Hyamine and Arquad 2-10/50) . It would be very desirable to monitor the level of such compounds in water, e.g. from cooling towers and also in cutting fluids, effluents and in the paper industry. Known biocides also include methylenebisthiocyanate (MBTC) . MBTC releases formaldehyde under alkaline conditions such as those that prevail in cooling towers. The Purpald reaction is clearly unsuitable for the pruposes of an assay, in these circumstances, since it cannot distinguish between MBTC and any free formaldehyde with which such a compound may be in equilibrium or with any formaldehyde from another source. WO-A-9115761 describes an assay in which MBTC or another aldehyde-releaser is distinguished from free aldehyde in an aqueous sample. The assay comprises: in first and second sub-samples, respectively converting the aldehyde-releaser to aldehyde and separating the aldehyde- releaser and aldehyde; and determining the difference between the respective amounts of aldehyde obtained. The amount of aldehyde obtained from the first sub-sample corresponds to the total amount of aldehyde-releaser and free aldehyde (which may include any aldehyde already present in the sub-sample) . The amount of aldehyde obtained from the second sample corresponds to free aldehyde only, and the difference between the two amounts therefore corresponds to the amount of aldehyde-releaser. In O-9115761, the most preferred separation procedure, i.e. to separate the aldehyde-releaser and the aldehyde, comprises bringing the sample into contact with a suitably immobilised reagent, e.g. an anion-exchange resin which binds/retards the aldehyde-releaser. The bound species can subsequently be released by elution: for example, aqueous NaCl will serve to elute MBTC from an anion-exchange resin. The releaser can then be assayed. Alternatively, the aldehyde that is not immobilised may be compared with total aldehyde that can be generated by the sample. Formaldehyde that is released can of course be assayed by the Sawicki or Purpald reaction. It is also suggested that aldehyde may be removed by forming a water-insoluble complex. Summary of the Invention According to a first aspect of the present invention, in an assay of the general type described in O-9115761 for aldehyde-releasers, the aldehyde-releaser and aldehyde are effectively separated by forming a complex of the aldehyde- releaser that does not release aldehyde. According to a second aspect of the present invention, a method of testing an aqueous sample for the presence of a charged surfactant comprises contacting an immobilised non-ionic hydrophobic material with the sample and with a substance that binds to the thus-insolubilised detergent but not with the material, and then with an eluant for the substance, and determining the presence of the substance in the eluate. Description of the Invention The second aspect of the present invention is based on the principle of measuring the charged surfactant indirectly, relying on the fact that its molecular structure comprises hydrophilic and hydrophobic ends. When the surfactant contacts the immobilised non-ionic hydrophobic material, suitably in the form of a column, it is retained quantitatively, by hydrophobic bonding. A cationic surfactant added to a non-ionic exchange medium gives an anion-exchange medium, while an anionic surfactant gives a cation-exchange medium. Subsequently or, preferably, simultaneously, a substance that binds to the thus-insolubilised surfactant, i.e. to the anionic/cationic medium, is added. This substance (which should not bind to the immobilised material) binds quantitatively to the insolubilised surfactant via its hydrophilic portion. If necessary, the complex is now washed or otherwise treated to remove the resultant non-specific binding, but this may be unnecessary. The substance is then eluted, and its presence in the eluate is determined. The amount of the substance in the eluate is a direct measure of the amount of the detergent in the original sample. The detergent is, for example, a cationic phosphonium or ammonium compound, e.g. a quaternary alkylphosphoniu or alkylammonium compound. Specific examples of such compounds are the four biocides given above (from which it will be understood that "alkyl'' includes substituents) . The hydrophobic material may be a polymer comprising, say, C 18 or other long-chain groups, and retains such surfactants. The substance which binds to the detergent will usually be acidic. Preferably, its presence in the eluate can be determined by colorimetry, using conventional procedures. The substance should then be capable of generating a colour, e.g. on the addition of a suitable reagent, as in a standard phenol test. The preferred substance is vanillic acid, which not only gives a good colour on reaction with aminoantipyrine, but is also non- toxic. The complex or reaction product of oxidised 4-aminoantipyrine and vanillic acid absorbs at 490 nm. The vanillic acid may be added together with the detergent. It is usually desirable then to add further vanillic acid, in order to saturate the available binding sites. A suitable eluant for vanillic acid is sodium acetat . In the first aspect of the invention, the complexing material is preferably a surfactant, e.g. a quaternary compound. In a particularly preferred embodiment of the first aspect of the invention, this material is, or has the characteristicsof, tributyltetradecylphosphoniumchloride, i.e. Bellacide 350, which has been found to interact, perhaps sterically or physically, quantitatively with MBTC. The resultant complex can be in the form of micelles, so that the MBTC is resistant to hydrolysis, i.e. unavailable for conversion to aldehyde. The aldehyde-releaser is thus "immobilised" .in situ. According to a further aspect of the present invention, the total aldehyde is measured after first disassociating the aldehyde-releaser from any complex of the type described above. This is based on a realisation that formation of the complex is reversible. For example, lauryl sulphate, perhaps owing to its charges being complementary to that of an anionic surfactant, releases MBTC from a Bellacide-MBTC complex. More generally, therefore, the compound that releases that aldehyde- releaser from the complex may be any that preferentially complexes with the surfactant or other complexing material. For any given aqueous system, it cannot necessarily be assumed that the aldehyde-releaser is non-complexed. The user will therefore usually wish to employ both of the techniques described herein. This first aspect of the invention has been described primarily with a view to assaying MBTC. In view of the interaction that has been found, it will be appreciated that a derived technique may be used to assay for Bellacide or other charged surfactants, e.g. quaternary biocides. The complex can be retained while removing other substances; the surfactant can then be released by disassociation as required, and then assayed. If necessary or desired, before conducting the assay, the MBTC can be converted to formaldehyde. For use in the invention, a kit may be provided comprising Bellacide or another material that complexes the aldehyde-releaser, lauryl sulphate or another substance that releases the aldehyde-releaser from such a complex, and alkali or another agent that will release aldehyde from the aldehyde-releaser. The novel assay is simply used, because of the colorimetric reaction. The colour obtained can be read by the eye, quite satisf ctorily. Alternatively, it may be matched against a standard, e.g. in a colour comparator (of the type produced by Lovibond) in which different shades are provided on a disc and correspond to calibrated concentrations of the compound being assayed. The disc is rotated until the one colour showing matches that provided by the adjacent sample. The following Examples illustrate the respective aspects of the invention. Example 1 (MBTC Assay) The following steps were conducted: Place 1.5 ml lauryl sulphate (Test) /l.5 ml Bellacide (Blank) in a 13.5 mm cell. The lauryl sulphate is provided as 100 μg/ml (sodium salt) (Sigma) in RO water. Fill each cell to the 5 ml mark with sample. To each cell add 3.5 ml NaOH/EDTA. Mix. To each cell add 1.75 ml Purpald (10 mg/ml, Sigma, in 0.5 M hydrochloric acid). Wait 5 ins. To each cell add 1 ml sodium periodate. Mix. Wait for 1-2 mins and match blank to Ml disc. Match test to colour comparator disc. Test-blank = ppm MBTC. Comparator: Blank and test matched in comparator against cooling water only blank. Example 2 (MBTC Assay) The following steps were conducted: 1. Pipette into two plastic test tubes, separately, 1 ml of the water sample (or the required volume of MBTC working solution, i.e. 50 mg/ml in DMF, made up to 1 ml with de- ionized water) . 2. To one tube, add 0.5 ml of 0.1 mg/ml lauryl sulphate (sodium salt) in RO water and mix well (termed solution A) . 3. To the other tube, add 0.5 ml of 4 mg/ml Bellacide in RO water and mix well (termed solution B) . The following steps 4 - 6 were followed for both solutions A and B (separately) . 4. Add 1.5 ml of 0.15M ethylenediaminetetraacetic acid in 3.33M sodium hydroxide and mix thoroughly. 5. Add 0.5 ml of 10 mg/ml Purpald in 0.5M hydrochloric acid. Mix and wait for 5 minutes. 6. Add 0.3 ml of 21 mg/ml sodium periodate. Mix and read the optical density (O.D.) of the deep purple solution after 30 seconds at 550 nm in a WPA CO 210 Λ Colorimeter. 7. OD (solution A) - (solution B) = OD (MBTC) Example 3 (MBTC Assay) The procedure of Example 2 was repeated but, instead of using a colorimeter, was adapted for use in a Lovibond comparator. NaOH was used at 5M and EDTA at 0.2M. MBTC is made up in N-methylpyrrolidone and diethylene glycol and diluted with RO water. The following steps were conducted: 1. To a 3.5 mm cell, add 1.5 ml Bellacide (4 mg/ml in RO water) , and fill the cell up to the 5 ml mark with sample. This is the Blank. 2. To a 13.5 mm cell, add 1.5 ml lauryl sulphate (0.1 mg/ml lauryl sulphate in RO water) , and fill the cell up to the 5 ml mark with sample. This is the Test sample. To both samples: 3. Add 3.5 ml 5M NaOH in 0.2M EDTA. Mix thoroughly. 4. Add 1.75 ml Purpald (10 mg/ml in 0.5M hydrochloric acid) . Wait 5 minutes. 5. Add 1 ml sodium periodate (21 mg/ml in RO water) . Mix and wait for 1-2 minutes. The colour is stable for this period. 6. Place blank cell in left-hand compartment and test cell in right-hand compartment and rotate disc until colour match is obtained. This procedure provides a good test for 0-5 ppm biocide (OD range 0-0.55). The same results are obtained in tap water and RO water. No problem has been encountered with cooling tower water samples. Example 4 Bellacide 350 is made up in a solution of 0.5 mM vanillic acid in 0.5 mM NaOH in 10% methanol in water. A column of Poly-prep Bio-beads SM-2 Absorbent is washed twice with 10% methanol in water, and then 500 μl of the Bellacide standard is applied to the column, followed by 5 ml 10% methanol. The eluant is collected in 700 μl samples. Each sample is tested using 70 μl of 4-aminoantipyrine in a mixture of 12.1 ml of 0.5 M aqueous NaH 2 P0 4 and 129 ml 0.5 M Na 2 PO ; . 70 μl of 5 mg/ml aqueous K 3 Fe(CN) 6 is then added. Samples are diluted with 1.96 ml water, and read ' at 490 nm using a WPA Colorimeter. The same sequence is repeated, without the initial washes and without Bellacide, in the sample. The column is then stripped using approx. 5-6 ml 0.5 mM sodium acetate in 10% methanol, and the eluant collection-colori etry steps are again repeated. The total amount of vanillic acid collected from the column was determined. It was shown that the amount of vanillic acid retained by and subsequently eluted from the column was proportional to the amount of Bellacide in the original sample. A calibration curve, i.e. a plot of vanillic acid against Bellacide, showed a linear relation from 10 to 30 μg vanillic acid with respect to 0 to 75 ppm Bellacide (vol. 0.5 ml) .

Claims: CLAIMS 1. A method of assaying an aqueous sample for a compound capable of releasing an aldehyde under given conditions, which comprises: in a first sub-sample, converting the compound to aldehyde in a second sub-sample, separating the compound and aldehyde; and determining the difference between the respective amounts of aldehyde obtained from the first and second sub- samples; characterised in that the separation comprises contacting the second sub-sample with a material that complexes the compound. 2. A method of assaying an aqueous sample for a compound capable of releasing an aldehyde under given conditions, which comprises: separating the compound and aldehyde; converting the aldehyde-free compound to aldehyde; and determining the amount of aldehyde obtained by conversion; characterised in that the separation comprises contacting the sample with a material that complexes the compound. 3. A method according to claim 1 or claim 2, wherein the material is a cationic surfactant. 4. A method according to claim 3, wherein the surfactant is a quaternary ammonium or phosphonium compound. 5. A method according to claim 4, wherein the surfactant is selected from tetrakis(hydroxymethyl)phosphonium sulphate, tributyltetradecylphosphoniumchloride andalkyl- quaternised di ethylbenzylammonium chloride or dodecyldi ethylammonium chloride. 6. A method according to any preceding claim, wherein the aldehyde is formaldehyde. 7. A method according to claim 6, ' wherein the formaldehyde is determined by the Purpald or Sawicki reaction. 8. A method according to any preceding claim, wherein the compound releases aldehyde under alkaline conditions. 9. A method according to claim 8, wherein the compound is methylenebisthiocyanate. 10. A method of assaying an aqueous sample for a compound capable of releasing an aldehyde under given conditions, which comprises: in a first sub-sample, converting the compound to aldehyde; in a second sub-sample, separating the compound and aldehyde; and determining the difference between the respective amounts of aldehyde obtained from the first and second sub- samples; characterised in that the or each amount of aldehyde is measured after the addition of a substance capable of disassociating a complex of the compound with tributyltetradecylphosphonium chloride. 11. A method of assaying an aqueous sample for a compound capable of releasing an aldehyde under given conditions, which comprises: separating the compound and aldehyde; converting the aldehyde-free compound to aldehyde; and determining the amount of aldehyde obtained by conversion; characterised in that the amount of aldehyde is measured after the addition of a substance capable of disassociating a complex of the compound with tributyltetradecylphosphonium chloride. 12. A method according to claim 10 or claim 11, wherein the substance is lauryl sulphate. 13. A method according to any of claims 1 to 9, which additionally comprises the characteristic step defined in any of claims 10 to 12. 14. A kit, suitable for use in a method according to claim 13, which comprises the material, the substance, and alkali or other reagent that will release aldehyde from the compound. 15. A method of assaying an aqueous sample for a charged surfactant, which comprises contacting an immobilised non- ionic hydrophobic material with the sample and with an agent that binds to the thus-insolubilised surfactant but not with the material, and then with an eluant for the agent, and determining the presence of the agent in the eluate. 16. A method according to claim 15, wherein the surfactant is as defined in claim 4 or claim 5. 17. A method according to claim 15 or claim 16, wherein the agent is capable of generating a colour, and its presence is determined by colori etry. 18. A method according to claim 17, wherein the colour is generated in a phenol reaction. 19. A method according to any of claims 15 to 18, wherein the agent is vanillic acid and the eluant is sodium acetate. 20. A kit, suitable for use in a method according to any of claims 17 to 19, which comprises an agent capable of binding to a charged surfactant but not to a hydrophobic material, and capable also of generating a colour on reaction with a suitable reagent; the reagent; and an eluant for the agent. 21. A kit according to claim 20, which additionally comprises a non-ionic hydrophobic material. 22. A process for separating a charged surfactant from an aqueous sample, which comprises contacting the sample with methylenebisthiocyanate, disassociating the resultant complex and, if desired, converting the methylenebisthio- cyanate to formaldehyde. 23. A process according to claim 22, which additionally comprises assaying the separated surfactant by a method according to any of claims 15 to 19.

Inventors: HILL MARTYN WILLIAM, RECTON PETER DARIO, SHARMAN DENNIS FRANK, HILL, MARTYN, WILLIAM, RECTON, PETER, DARIO, SHARMAN, DENNIS, FRANK
Applicants: CTS BIOCIDES LTD, CTS BIOCIDES LTD.
Application year: 1992
Main classification: G01N31
IPC codes: G01N21, G01N31