Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/805—Test papers

Definitions

• ABSTRACT A process for semiquantitative colorimetric detection of organic peroxides, especially ether peroxides, with the aid of peroxide indicating means comprising an absorbent carrier impregnated with peroxides, a color-imetric indicator, optionally a buffer, and optionally other additives.
• the indicating means is immersed in a solution of peroxides in a volatile organic solvent having a boiling point of below 80 C., preferably of below 50 C., and withdrawn.
• the indicating means is placed in a water vapor atmosphere having a relatively constant temperature between about 20 and 100 C. to evaporate theorganic solvent and deposit moisture on the surface thereof, and then examined for color change.
• colorimetric indicating means used for the determination of hydrogen peroxide are e.g., test strips for detecting glucose in the urine, or indicating strips for the determination of H in foodstuffs.
• the indicating means normally comprise an absorbent carrier containing peroxidase, an oxidation-sensitive colori'metric indicator such as a chromogen, and optionally a buffer.
• the absorbent carrier is normally paper, preferably filter paper.
• Common colorimetric indicators for this purpose include but are not limited to otolidine, benzidine, o-dianisidine, and 2,7- diaminofluorene.
• As the buffer substances alkali acetates or citrates are commonly employed, but borate, tartrate, succinate, orphosphate buffers are also suitable.
• the test strips also contain additional conventional additives, e.g. albumen, gelatin,'polyvinyl alcohol, and/or polyvinylpyrrolidone.
• the absorbent carrier is generally impregnated with the reagents, preferably by successive impregnation with two or more solutions. Usually, the first impregnating solution contains the colorimetric indicator dissolved in a volatile organic solvent.
• the carrier is next dried and then impregnated with an aqueous solution containing the buffer, the peroxidase, and optionally any further additives.
• the absorbent carrier is then generally glued or heat-sealed onto a metallic foil or plastic film strip to form the reaction zone proper of the thus-produced test strips.
• Another object of this invention is to provide a method for developing colorimetric test strips used to detect organic peroxides.
• a further object of this invention is to provide a process which improves the sensitivity of colorimetric peroxide test strips.
• An additional object of this invention is to provide a semi-quantitative colorimetric method for the analysis of organic peroxides.
• the present invention provides a process for the colorimetric detection of a peroxide which is soluble in a volatile organic solvent having a boiling point below about C., which comprises: (a) moistening peroxide indicating means comprising an absorbent carrier impregnated with peroxidase, at least one colorimetric redox indicator, and optionally a buffer, with a volatile organic solvent having a boiling point below about 80 C.; (b) removing said moistened indicating means from said volatile organic solvent and, without completely evaporating said solvent from said indicating means; (c) passing a saturating amount of moist gas having arelative humidity-of about 80 percent and a relatively constant temperature of between 20 100 C., onto said indicating means to evaporate said solvent from 'said indicating means and condense a water film onto the surface of said indicating means; and (d) observing'any resultant color change of said peroxide indicating means.
• the sensitivity of conventional colorimetric test strips can easily be increased sufficiently to conduct semiquantitative determinations of organic peroxides, especially ether peroxides.
• the sensitivity of conventional peroxide colorimetric indicating means can be raised by an order of magnitude so that even minor amounts of peroxides can be easily detected with excellent accuracy.
• the indicating means is moistened, preferably by immersion in a solution of the peroxide in a readily volatile organic solvent having a boiling point of below 80 C., preferably of 10 to 50 C.
• the peroxide indicating means is introduced into a water vapor atmosphere having a relatively constant temperature of about 20 100 C., preferably 36 100 C. before the volatile organic solvent has evaporated.
• the water vapor atmosphere preferably is at a temperature of at least 20 C.
• the use of a water vapor atmosphere having a temperature of about 36 C. and a relative humidity of about 90 percent is particularly advantageous with an ehter solvent and can be achieved in an extremely simple manner by forced breathing, preferably several times, over the reagent zone of the test strip while the organic solvent is still evaporating.
• the amount of the water condensing on the indicating reagent'surface is a function of the amount of absorbed and evaporating organic solvent as long as a sufficient amount of water vapor at a constant temperature is provided.
• the heat of condensation of the water vapor compensatesfor the evaporative cooling of the volatile.
• a water vapor atmosphere of constant temperature and uniform moisture can be produced with surprising constancy by human breath which is fairly constant at 36 C. and 90 percent relative humidity, even under fluctuating ambient conditions, and this can be used to provide optimal conditions for a semiquantitative colorimetric determination of organic peroxides, especially ether peroxides.
• test strip can be introduced into the water vapor rising from boiling water or .a water bath.
• Suitable volatile organic solvents are those having a boiling point of below 80 C., preferably below 50 C.
• the volatile organic solvent is a peroxidefree ether, especially diethyl ether.
• Also suitable as an organic solvent are lower aliphatic hydrocarbons having this boiling range, especially petroleum ether; acetone; ethyl acetate; and methyl ethyl ketone.
• the only criticality is that the solvent selected possesses a sufficient latent heat of evaporation to provide condensation of a water film, generally from 4 to 10 kcal/moL, preferably from 5 to 8 kcal/mol.
• any organic peroxides which are soluble in the volatile organic solvents used can be detected by the process of this invention.
• the present procedure can also be used for the detection or titration of other organic peroxides, e.g. for diacyl peroxides, aldehyde peroxides, ketone peroxides, alkyl hydroperoxides, alkyl peresters and peracides (e.g. perbenzoic acid, performic acid, peracetic acid, perphthalic acid, permaleic acid).
• Routine laboratory determinations can be conducted for the determination of acyl and/or diacyl peroxides, e.g.
• the peroxides of higher ethers which are not readily volatile, e.g. those boiling at 60 to 120 C., can likewise be easily detected according to the process of the present invention.
• the higherboiling ether to be examined is diluted with a peroxidefree, volatile ether, (e.g. diethyl ether) or with a volatile organic solvent; in this manner, precise semiquantitative peroxide determinations can be conducted.
• all indicating means can be employed which are suitable for the colorimetric determination of hydrogen peroxide.
• the content of peroxidase in these indicating means should preferably be relatively high.
• the content of peroxidase in the impregnating solution used to prepare the indicating means is preferably at least 20 mg./ ml., especially about 30-50 mg./l00 ml.
• the process of this invention is preferably conducted by first immersing the test strip in the solution to be tested until the reaction zone is fully moistened. After withdrawal from the solution, the test strip is immediately (i.e. preferably within up to 4, in particular 0.5 1 seconds) breathed upon several times (e.g. three to four times) for several seconds, until the maximum color depth has been attained. The thus-produced coloring is then compared with a standard color scale. Additional breathing on the indicator means does not alter the color. The only function which must be achieved by breathing is complete wetting of the reaction zone with a water film; any excess water vapor does not have a disadvantageous effect. If the ether is allowed to evaporate before the reaction zone is moistened with water, only a very weak color change is obtained. Even evaporation of the organic solvent in normal, moist laboratory air, results in only a slight color change which is difficult or impossible to reproduce.
• the process of this invention is sufficiently senstiive to reliably detect 2 p.p.m. of ether peroxides.
• a suitable color scale Using a suitable color scale, a marked distinction can be observed visually between concentrations of 5, 10, 30, 60, 100, 250, and 500 p.p.m. of ether peroxide, and even intermediate concentrations can be interpreted. Similar results are attained with color scales calibrated for other organic peroxides.
• EXAMPLE 1 Filter paper having a thickness of 0.35 mm. (Schleicher Schuell No. 1450 CV) is impregnated with a solution of l g. of o-tolidine in 100 ml. of methanol. After drying at 50i80 C., the paper is impregnated with a second solution containing, in 100 ml.'of water, 50mg. of-peroxidase, 5 g. of sodium citrate, and 1.5g. of albumen (pH 5). After drying, the papers are cut into 6 mm. strips and heat-sealed onto a plastic film backing. By cutting at right angles to the direction of the strips, test strips having a square reaction zone with a width of 6 mm. are prepared.
• test strip is immersed in a solution of 50 mg. of diethyl ether hydroperoxide in 1 liter of diethyl ether for about one second, so that the reaction zone is fully moistened. After withdrawal, the teststrip is immediately breathed upon 24 times for 3-5 seconds.
• the thus-produced blue coloring is compared with a standardized color scale and indicates a peroxide content of 50 p.p.m. H
• EXAMPLE 3 2 mg. of commercially obtained stabilized dibenzoyl peroxide to be tested for peroxide content is mixed with 100 ml. of peroxide-free diethyl ether. A test strip described in Example 1 is immersed therein and breathed upon after being withdrawn from the solution. Comparison with a standardized color scale showed a .color depth equivalent to an ether peroxide concentration of 5 mg./l. which, empirically determined, corresponds to a dibenzoyl peroxide content of mg./l.
• acyl peroxides e.g. lauroyl peroxide
• hydroperoxides e.g. cumene hydroperoxide
• a process for the colorimetric detection of a pcroxide which is soluble in a volatile organic solvent having a boiling point below about C. which comprises:
• a moistening peroxide indicating means comprising an absorbent carrier impregnated with peroxidase, at least one colorimetric redox indicator, and optionally a buffer, with a volatile organic solvent having a boiling point below about 80 C.;
• said moist gas is air having a relative humidity of about per cent.
• organic solvent is a peroxide-free ether.

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• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
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• Proteomics, Peptides & Aminoacids (AREA)
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• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
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• Genetics & Genomics (AREA)
• Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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Cited By (6)

Citations (2)

• 1971
  • 1971-03-26 DE DE2114638A patent/DE2114638C3/de not_active Expired
• 1972
  • 1972-02-28 GB GB901272A patent/GB1341364A/en not_active Expired
  • 1972-02-28 ZA ZA721309A patent/ZA721309B/xx unknown
  • 1972-03-02 NL NL7202770A patent/NL7202770A/xx unknown
  • 1972-03-03 IL IL38883A patent/IL38883A/xx unknown
  • 1972-03-13 CS CS7200001672A patent/CS179378B2/cs unknown
  • 1972-03-14 SE SE7203247A patent/SE395964B/xx unknown
  • 1972-03-21 US US00236791A patent/US3770381A/en not_active Expired - Lifetime
  • 1972-03-22 IT IT22302/72A patent/IT965674B/it active
  • 1972-03-24 FR FR7210389A patent/FR2131595A5/fr not_active Expired
  • 1972-03-24 CH CH446772A patent/CH571488A5/xx not_active IP Right Cessation
  • 1972-03-24 BE BE781213A patent/BE781213A/xx not_active IP Right Cessation
  • 1972-03-25 JP JP3023672A patent/JPS5527798B1/ja active Pending

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