CS273302B2 - Quick tests for demonstration and determination of hydrogen peroxide and substances reacting during hydrogen peroxide or oxidatively active substances formation - Google Patents

Abstract

The solution concerns the quick tests for detection and determination of hydrogen peroxide and substances that react in the process of formation of hydrogen peroxide and peroxidically active substances and lies in the fact that they contain 3,3', 5,5'- tetraalkyl benzidine of the general formula 9 as an active chromogenic substance where R1, R2, R3 and R4 are identical or non-identical and stand for lower alkyl groups with 1 to 6 carbon atoms and with a linear or branched chain.

Description

The subject of the invention is rapid tests for the detection and determination of hydrogen peroxide and substances reacting to form hydrogen peroxide or peroxidically active substances.

The reaction of hydrogen peroxide with oxidation indicators under catalysis by peroxidase or peroxidically active compounds to form colored compounds plays a special role in analytical chemistry, as it allows, in addition to the detection of hydrogen and peroxidases, a number of substances which react with oxygen and oxidase to form hydrogen peroxide. The following are examples of some of these substances and in brackets the corresponding oxidases:

glucose (glucose oxidase), galactose (galactoxidase), L-amino acids (L-amino acid oxidase), cholesterin (cholesterino oxidase) and uric acid (uricase). These are mainly substances whose evidence is of great importance in medical diagnostics and food chemistry.

In particular, a method for determining hemoglobin is suitable as a reaction for the detection of peroxidases, whereby organo-hydroperoxides can also be used as oxidizing agents instead of hydrogen peroxide.

Consistent with their great importance in medical diagnostics, so-called rapid tests are described for almost all of the above-mentioned reactions, which allow qualitative or quantitative determination of individual reactants. In this case, absorbent carriers or films are used which contain all the reagents and, upon contact with the liquid to be examined, a color reaction occurs if the substance of interest is present. The resulting coloration is determined using color comparison tables or is measured with remission photometers so that the concentration of the reactant can be inferred from the color intensity.

A number of compounds have been described as oxidation indicators for these rapid tests in the literature and patents, but in practice only a few have succeeded. These are exclusively benzidine series compounds, and in particular of these are o-tolidine. Despite the wide spread of this indicator and its undoubted advantage over other known indicators, o-tolidine also has drawbacks. First, this substance is not always optimally stable in fast-test formulations, which can lead to undesirable discoloration and loss of sensitivity when stored for a long time. Second, the blue-green radical cation resulting from the reaction of the indicator is not very stable, since it is relatively easily converted to brown quinondiimine by further oxidation. The reaction colors therefore have a partly dirty character due to the brown secondary color, and they change rapidly and therefore cannot be evaluated correctly most often shortly after the prescribed reading time. This is a crucial drawback in serial analyzes and documentation. While it is possible to stabilize the radical cation with anionic surfactants to some extent, they are not always useful, as they can destabilize the oxidases in the primary reaction.

Surprisingly, it has now been found that rapid tests of the above type are obtained which are more durable and provide more stable dye radicals when 3,3 ', 6'-tetraalkylbenzidine is used instead of o-tolidine.

SUMMARY OF THE INVENTION This invention relates to rapid tests for the detection and determination of hydrogen peroxide and hydrogen peroxide-reactive substances or peroxidically active substances, consisting of an absorbent carrier or a swollen synthetic film impregnated with an impregnating solution containing 100 ml per 100 ml. 01 to 0.5 g of peroxidase, optionally 0.1 to 2 g of hydroperoxide, to 20 g of a buffer, for example or morpholinetane sulfonic acid, to 5 g of a wetting agent, for example dioctyl sodium sulfosuccinate, lauroylsarcosine, polyoxyethylene sorbitan monolaurate -2,5-dihydroperoxide and 0.1 to 1 g of chromogen consist in that they contain as active chromogenic substance 3,3 ', 5,5 ^z -tetraalkylbenzidine of the general formula I

CS 273 302 B2 / 1 / if>.

wherein R 6, R 8, R 8 and R 8 are the same or different and are lower alkyl groups having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and straight or branched chain.

Accordingly, the present invention provides rapid tests for the detection and determination of hydrogen peroxide and substances which react to form hydrogen peroxide or peroxidically active substances consisting of a absorptive carrier or a swellable synthetic film comprising either a peroxidically active substance or a hydroperoxide and an a coloring with a hydroperoxide or peroxidically active substance, and optionally a buffer, wetting agent, thickening agent and activators, characterized in that they contain, as an active ohromogenic substance, 3,3 *, 5,5 * -tetraalkylbenzidine of the formula I

Wherein R ^, R ^, R R and R ^ are the same or different and are lower alkyl groups having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and straight or branched chain.

The process for the preparation of the compounds of the formula I is known, but to date only 3-3 *, 5-5 ^of -tetramethylbenzidine is described (VR Holland et al. Tetrahedron 30, 3299-3302 (1974)) and 3,3 ', 5'. , 5 ^from -tetraethylbenzidine (R. Stroh et al. Angew. Chem. 69, 124-131 (1957)) · Production of tetramethylbenzidine according to VR Holland et al. loo. cit., was conducted in a known manner by reducing the known 2,2 ', 6,6 -tetrametylazobenzenu ^of zinc dust and benzidinovým following rearrangement.

The hitherto unwritten compounds 3,3'-diraethyl-5,5'-diethylbenzidine and 3,3 ', 5,5'-tetraisopropylbenzidine can be prepared, for example, according to Stroha et al. loo. cit., from o-tolidine and ethylene, optionally from benzldine and propylene.

Other methods which should in principle be accessible for higher alkyl derivatives not yet described are as follows:

CS 273 302 B2

a) Oxidation of 2,6-dialkylaniline by alkali fericyanide (arová V.R. Holland, loc.cit.)

G. Schulz, Ber. dtsch., chem. · Ges. 17. 476-478 (1884)?

B. Haselbaoh, Helv. Chem., Acta. 53, 1526-1543 (1970).

b) oxidation of 2,6-dialkylaniline with silver oxide (cf. B. Ortiz et al., J. Org. Chem. 3Z, 2748-2750 (1972)).

c) reduction of 2,6-dialkylnitrobenzene with lithium aluminum hydride (cf. R. R. Nystrom; and

H. C. Brown, J. Amer. Soc. 70, 3738-3740 (1948)).

d) reaction of 2,6-dialkylbenzenediazonium salts with an ammoniacal solution of monovalent copper (cf. D. Vorlander and R, Mayer, Liebigs Ann. Chem. 320, 122-144 (1902)).

Further reduction of the azobenzene thus obtained to the hydrazobenzenes and their (partially instantaneous) benzidine rearrangement is carried out by known methods (for example Houben-Weyl X / 2, 705-724 and X / 1, 839-848).

The essential ability of 3,3 ', < 5 > -tetramethyrbenzidine, which can be expected based on the structure for evidence of occult bleeding in stool and urine, is described in V.R. Ilollanda et al., With lower canoeogenicity over tolidine being preferred. However, the unexpectedly greater stability of the amine and its radical cations at velocities is not described by Holland, since this did not carry out corresponding experiments. There was also no impulse for such experiments, as the rapid test user did not manipulate the reagents directly and therefore the question of canoeogenicity plays virtually no role for him.

The compounds of the formula I are also described for rapid tests for the detection of halides. optionally pseudohalides in body fluids (Can. Pat. Nr. 818 344). These rapid tests also contain divalent copper compounds that react with halides to form monovalent copper halides and free halides that oxidize the bezidine compounds.

This oxidation appears to result in the same color radical cations as the peroxidase-catalyzed hydroperoxide oxidation. In said patent, among the numerous derivatives claimed, o-tolidine (3,3'-dimethylbenzidine) is preferred, and it can be concluded that tetraalkylbenzidines are less suitable for rapid tests.

The greater stability of the compounds of formula (I) and their free-radical cations is further surprising that, due to the electron pressure of the additional alkyl groups, an increased oxidisability and thus a further reaction to the brown quinondiimine could be expected.

The tetraalkylbenzidines according to the invention can be used in all cases in which o-tolidine has been used hitherto, whereby the known formulations can possibly be modified somewhat. Optionally, the anionic surfactants used can be replaced by less active or completely omitted to stabilize the color. Thus, for example, tetraalkylbenzidines can be used in test papers to detect glucose in urine, blood in urine, cholesterol in serum, hydrogen peroxide in milk, or test films for the determination of blood and serum glucose and in many other tests. All these tests are characterized in that the color reactions are clearer, more stable and in part better graded than when using o-tolidine, since the blue-green to green colorations of the radical cations of tetraalkylbenzidines are not stained with brown quinondiimine fractions.

In the rapid tests described in the following examples, the properties of the tetraalkylbenzidines according to the invention are particularly prominent and individual representatives of the class of compounds may possibly have particularly advantageous effects: this shows an increased stability of the tetraalkylbenzidines of formula I over o-tolidine. in power. The increased stability of the free radical cations leads to the possibility of measurement in glucose test films

CS 273 302 B2 in a relatively long period of time. The use of tetraalkylbenzidine in urine glucose testing results in test papers in which the reaction color remains almost unchanged for days, which is of particular importance for early diagnoses in which the test papers used are sent to the central evaluation sites for diagnosis. In further examples, the invention is to be explained in more detail:

Example 1

Test paper for evidence of blood in the sea

Filter paper (Sohleider and Schull # 23SL) is impregnated with the following solutions and dried at 40 ° 0:

Solution 1

1.2 molar citrate buffer pH 5.25 35, P ml ethylenediaminotetraootic acid, disodium salt 0.1 g dioctyl sodium sulfosuccinate 0.5 g

2,5-dimethylhexane-2,5-dihydroperoxide (about 70%) 1.6 g phosphoric acid trlmorpholide 12.7 g ethanol 30.0 ml distilled water ad 100.0 ml

Solution 2

3,3 *, 5,5 * -tetramethylbenzidine phenanthridine toluene

0.3 g 0.2 g and 100.0 ml

A white test paper is obtained which, when soaked in urine containing blood, turns blue-green after about 5 to 20 seconds. If the erythrocytes are intact, the papers are blue-green spotted. If hemolysis occurs, or if free hemoglobin is found in the urine in advance, the paper stains uniformly blue-green. The sensitivity is about 5 erythrooyts / mm or the corresponding amount of hemoglobin. A lower number of intact erythroocytes may, under certain circumstances, induce individual blue-green spots on the test paper. The sensitivity to hemoglobin corresponds to that of hemoglobin.

After a three-day temperature load at 60 ° C, the test papers are completely slightly yellowish colored, their sensitivity remained essentially the same, only the reaction times were prolonged to about 60 to 90 seconds. Test paper of the same composition with o-tolidine instead of tetramethylbenzidine is yellowish brown after the same load, the sensitivity is about 20 erythrooyts / mn.

Papers with the same sensitivity and stability, which react only slightly more slowly and with a pure green color, are obtained using equimolar amounts of 3,3 *, 5,5 * -tetraethylbenzidine, 3,3 * -dimethyl-5,5 * -diethylbenzidine or 3 *. , 3 *, 5,5 * -tetraisopropylbenzidine.

CS 273 302 B2

Example 2

Test paper for determination of blood in power

If dioctyl sodium sulfosuccinate is replaced with the same amount of lauroyl sarcosine in the recipe of Example 1, omitting the meso-active agent, the test papers are less sensitive than those of Example 1, but are still more sensitive than the corresponding papers. -tolidine. The color reactions with power containing 50 heraolyzed erythrooyls / mm 2 are summarized in the table below.

tetraraethylbenzidine o-tolidine without wetting agent light green light gray-brown lauroylsarcosine green light brown-green

Example 3

Test paper for urine glucose

Filter paper (Schleioher and Schull 597 NF) is impregnated with solutions of the following composition and dried at 50 ° 0:

Solution 1

Glucose oxidase (70 U / mg) 222 mg peroxidase (70 U / mg) 28 mg morpholinoethanesulfone buffer (pH 5, 1.0 IU); 50 ml tartrazine 80 mg polyvinylpyrrolidone 300 mg Distilled water ad 100 ml

Solution 2 3,3 ', 5,5 ^z- tetramethylbenzidine 600 mg ethanol ad 100 ml

Test papers are prepared in the same manner containing equimolar amounts of 3,3 ' 5,5 ' -tetraethylbenzidine or o-tolidine. The reaction colors of these test papers with urine containing glucose after about 60 to 120 seconds after soaking are summarized in the table below. For tetramethylbenzidine, these colors will remain constant for about 10 to 15 minutes, become more bluish on drying and remain stable in this form. oelé dny. Coloring with tetraethylbenzidine somewhat fades on drying somewhat without changing the color value and is then stable for many days.

The dyes obtained by 3-o-tolidine start to turn yellow and fade after 5 to 10 minutes and are completely discolored or slightly olive-brown, depending on the glucose concentration, after 2 to 4 hours.

CS 273 302 B2

mg% glucose o-tolidine tetramethylbenzidine t e tra ethylbenzidine 0 yellow yellow yellow 50 light green light green light green 100 ALIGN! green blue-green Medium green 250 blue-green green-blue green 500 green-blue blue-black dark green 1000 olive black black deep green Test papers, which contains equimolar amount 3.3 ^from -dimethyl-5,5'-diethylbenz:

dinu. they behave in the same way as tetraethylbenzidine papers with regard to the nature and stability of the reaction colors.

Example 4

Test papers for the detection of hydrogen peroxide

The filter paper was impregnated with the following solutions and dried at 40 ° C.

Solution 1

tri-sodium citric acid dihydrate 225 mg citric acid monohydrate 175 mg peroxidase (70 U / mg) 50 mg natriumalginat 150 mg polyoxyethylene3-organo monolaurate (Tween 20) 1300 mg mixed vinylpyrrolidine polymer with vinyl acetate 375 mg Distilled water ad 100 mg

Solution 2

3,3 *, 5 »5 * -tetramethylbenzidine 500 mg acetone ad 100 ml

The test paper thus produced gives a blue-green reaction coloring up to a concentration of 0.1 mg% with hydrogen peroxide in aqueous solutions such as milk.

Example 5

Test paper for evidence of serum cholesterol and esters

The filter paper (Schleicher and Schull 597 NP Ind.) Is impregnated with solutions of the following composition and dried at 40 ° C:

Solution 1 molar citrate buffer pH 7 cholesterin oxidase (60 U / mg) cholesteriesterase (18 U / mg)

20.0 ml

0.1 g 0.25 g

OS 273 302 B2 peroxidase (70 U / mg) distilled water

Solution 2

3,3 ', 5,5 “tetraraethylbenzidine polyethylenoctylphenol ether (Triton X 100) methylene chloride

0.05 ε ad 100.0 ml

0.28 g

0.5 g and 100.0 ml

The test papers thus obtained react with sera containing cholesterin and / or cholesterinester to produce green color tones whose intensity corresponds to the concentration of cholesterin. Corresponding test papers, which contain o-tolidine instead of tetramethylbenzidine, also react to produce green tones which are less intense.

Example 6

Test film to determine lower blood or serum glucose levels

Components of polyvinyl acetate propionate (Propiophan 70 D) dispersion 45.0 g

1,85% solution of sodium alginate in 0,5 m phosphate buffer pH 5,5 '35,0 g natriumnonyl sulphate dissolved in 5,0 ml water 0,75 g glucose oxidase (62,7 U / mg) ·) 0,189 gy dissolved i in 10 ml water peroxidase (68.8 U / mg) 0.235 g

3,3 ', 5,5-tetramethylbenzidine dissolved in 5 ml of acetone 0.68 g

The ingredients are mixed well, coated at 200 / U film thickness and dried at 60 ° C for 35 minutes. Dropping the glucose-containing blood and wiping it for 1 minute after a further 2 minutes, with a glucose content ranging from 5 to about 100 mg%, gives a blue-green reaction color of increasing intensity, which can be measured with a commercially available remission photometer. The following table shows the color development kinetics at 60 mg% glucose compared to a test film in which 500 mg o-tolidine is contained instead of tetramethylbenzidine. If the test film was heated to 50 ° C for 12 days, virtually no coloring could be detected. On the other hand, the test film with o-tolidine is strongly yellowish-brown.

time (min) 1 23 5 10 tetramethylbenzidine (TMB) intensity 88 87 o-tolidine intensity 73 68

CS 273 302 B2

glucose o-tolidine tetramethylbenzidine tetraethylbenzidine 0 yellow yellow yellow 50 light green light green light green 100 ALIGN! green blue-green Medium green 250 blue-green green-blue green 500 green-blue blue-black dark green 1000 olive black black deep green

Test papers containing equimolar amounts of 3,3 ^z- dimethyl-5,5 ^z- diethylbenzidine behave in the same way as tetraethylbenzidine papers with respect to the nature and stability of the reaction dyes.

Example 4

Test papers for the detection of hydrogen peroxide

The filter paper was impregnated with the following solutions and dried at 40 ° C.

Solution 1

tri-sodium citric acid dihydrate 225 mg citric acid, monohydrate 175 mg peroxidase (70 U / mg) 50 mg natriumalginat 150 mg polyoxyethylene sorbitan monolaurate (Tween 20) 1300 mg mixed vinylpyrrolidine polymer with vinyl acetate 375 mg Distilled water ad 100 mg

Solution 2

3.3 ^Z , 5.5 ^z- tetramethylbenzidine 500 mg acetone ad 100 ml

The test paper thus produced gives a blue-green reaction coloring up to a concentration of 0.1 mg% with hydrogen peroxide in aqueous solutions such as milk.

Example 5

Test paper to detect serum cholesterol and esters of oholesterin

Filter paper (Sohleioher and Schull 597 NF Ind.) Was impregnated with solutions of the following composition and dried at 40 ° C.

Solution 1 molar citrate buffer pH 7 cholesterin oxidase (60 U / mg) cholesteriesterase (18 U / mg)

20.0 ml 0.1 g

0.25 g

Peroxidase (70 N / mg) distilled water

Solution 2

3,3 ', ^5,5, -tetrametyl'benzidin polyetylehoktylfenoleter (Triton X 100), methylene chloride

0.05 g and 100.0 ml

0.28 g

0.5 g and 100.0 ml

The test papers thus obtained react with sera containing oholesterin and / or cholesterinester to produce green color tones corresponding to the oholesterin concentration. The corresponding test papers, which contain o-tolidine instead of tetramethylbenzidine, also react to produce green color tones, but these are intense Siena.

Example 6

Test film to determine lower blood or serum glucose levels

Components of polyvinyl acetate propionate (Propiophan 70 D) dispersion 45.0 g

A 1,85% solution of sodium alginate in 0,5 m phosphorephonate buffer, pH 5,5 35,0 g natriumnonyl sulphate dissolved in 5,0 ml water 0,75 g glucose oxidase (62,7 U / mg) γ - 0,189 gy dissolved, _r (in 10 ml water peroxidase (68.8 U / mg) ^J 0.235 g

3,3 ', 5,5'-tetramethylbenzidine dissolved in 5 ml of acetone 0.68 g

The ingredients are mixed well, coated at 200 / U film thickness and dried at 60 ° C for 35 minutes. Dropping the glucose-containing blood and wiping it for 1 minute after a further 2 minutes, with a glucose content ranging from 5 to about 100 mg%, gives a blue-green reaction color of increasing intensity, which can be measured with a commercially available remission photometer. The following table shows the color development kinetics at 60 mg% glucose compared to a test film in which 500 mg o-tolidine is contained instead of tetramethylbenzidine. If the test film was heated to 50 ° C for 12 days, virtually no coloring could be detected. On the other hand, the test film with o-tolidine is strongly yellowish-brown.

time (min) 1 2 3 5 10 tetramethylbenzidine intensity (TMB) 88 o-tolidine intensity 73

CS 273 302 B2

Example 7

Test film for determination of normal and elevated blood and serum glucose content

A test film was prepared according to Example 6, which additionally contained 0.050 g of 9 - (. Beta.-aminopropyl) -3-aminocarbazole dihydrochloride. Dropping the blood containing glucose and wiping it off after 1 minute gives the following reaction coloring after another minute:

mg% glucose - ocher

120 mg. % glucose - olive

180 mg% glucose - blue-green with variable color depth

These colors do not change on their own for two days in a laboratory atmosphere.

For an analogous test film containing 0.600 g o-tolidine and 0.025 g 9- (4-aminopropyl) -3-aminocarbazole dihydrochloride, the blue-green color changes towards the yellow-green tones.

The color stability time at 150 mg% glucose measured on a reflomat is shown in the following table time (hours) 0 1 2 4 8 24

TMB 61 62 62 62 62 62 o-tolidine 51 52 51 50 47 '42

If the test film was heated to 50 ° C for 12 days, virtually no staining could be detected and comparative color evaluation was undoubtedly possible. On the other hand, test strips containing o-tolidine are strongly yellowish-brown and comparison with the color range is no longer possible.

The following examples illustrate the preparation of two hitherto unknown compounds of formula I.

Example 8

3,3'-dimethyl-5,5'-diethylbenzidine

Variant I

96.5 g (0.564 mol) of 2-methyl-6-ethylaniline hydrochloride are suspended in a two-liter, three-necked flask with stirrer and dropping funnel and thermometer, mixed with 79.5 ml of concentrated hydrochloric acid (d = 1.18) and 350 ml of water. and with good ice cooling, a solution of 39 g of sodium nitrite in 150 ml of water is added dropwise at 0 to 3 ° C over 30 minutes with stirring. The mixture is then stirred for an additional 1 hour at 0-3 [deg.] C. and the diazonium salt solution is diluted with 600 ml of ice water.

Meanwhile, 210 g of copper sulfate was dissolved in 900 ml of water in a 6 liter three-necked flask with stirrer, dropping funnel and thermometer (Y-piece) and made up with 363 ml of concentrated ammonia (d = 0.88). Thereafter, 90 g of hydroxylammonium chloride are dissolved in 150 ml of water and, under ice-cooling, a solution of 95 g of potassium hydroxide in 72 ml of water is added dropwise. This well cooled solution is added at 10-15 ° C under nitrogen over 15 minutes to a stirred solution of cupric tetramine to form a light blue salt complex.

CS 273 302 B2 monovalent copper. Under ice-cooling, vigorous stirring and under nitrogen, a cooled diazonium salt solution is now added dropwise at 12 to 15 ° C (the dropping funnel is equipped with a lead-in tube which reaches the copper salt solution below the U-bend). After the addition was complete, it was stirred for an additional hour, then the reaction solution was shaken with 3 x 200 ml of ether. Shake the combined ethereal solutions with 3 x 50 ml 25% sodium hydroxide solution, 2 x 100 ml water, 3 x 100 ml 2N hydrochloric acid and 2 x 100 ml water. 55.4 g of azo compound are obtained in the form of a red oil. Chromatographic separation on diatomaceous earth (filling height 100 cm, column diameter 4.8 cm) with a 1: 4 mixture of chloroform and ligroin (quantity of diatomaceous earth 1.5 kg, diameter 0.063 to 0.2 mm) yields 33.7 g of pure 2.2 1'-dimethyl-6-diethylazobenzene as a red oil. This was dissolved in 1.6 liters of diethyl ether, 330 ml of ethanol were added, followed by a solution of 135 g of ammonium chloride in 1.5 liters of water, and 300 g of zinc dust were added portionwise at 22 ° C with vigorous stirring. Upon stirring for 5 hours, the reduction was complete. Bleached almost completely separates, «

shake the organic phase and the aqueous layer three times with 250 ml of ether each time. The combined ethereal extracts were then charged with 210 ml of 3N sulfuric acid and stirred. The sulphate is formed in the form of a colorless, initially sticky precipitate. Addition of 200 ml of ethanol gives 30.5 g of 3,3 ^, -dimethyl-5,5 ^, -diethylbenzidine sulphate as colorless crystals. This product is added to 150 ml of boiling water, cooled to room temperature with stirring, made up to 200 ml of 21 L sodium hydroxide solution and extracted with 4 x 100 ml of chloroform. The combined chloroform extracts were dried over sodium sulfate and concentrated. The residue was 22.5 g of light brown crystals. This crude product was recrystallized from methanol-water. 23 g = 30.4% of colored crystals, mp 69-70 ° C.

Dihydric chloride. 2HCl melts at 289-291 ° C (dec.).

Variant II

1.5 g of aluminum and 0.1 g of mercury chloride are heated with 75 g of aniline in an autoclave at 300 ° C, whereby the aluminum dissolves under the evolution of hydrogen. After cooling, 92 g of o-tolidine and 4.5 g of anhydrous aluminum chloride are added. It is then heated to a temperature of 280 to 300 ° C and injected with 200 atm of ethylene. After about 3 hours, the ethylene uptake is complete. It is then cooled and the reaction time is shaken with dilute sodium hydroxide solution. The 2-ethylaniline and 2,6-diethylaniline (up to about 120 ° / 10 mm) are distilled off from the organic phase in vacuo.

The residue is recrystallized several times from dilute hydrochloric acid. 3.3 ^of -dimethyl-5,5'-diethylbenzidine dihydrochloride is obtained.

M.p. Mp 288-292 ° C (dec.).

Example 9

3,3 ', 5,5'-Tetraisopropylbenzidine

A solution of 100 g of 2,6-diisopropylaniline in 1280 ml of water and 102 ml of concentrated hydrochloric acid (d = 1.18) is heated to 90 ° C and then added dropwise to a 97 ° C hot solution consisting of potassium hexacyanoferrate in 5100 ml. water and 143 g of sodium hydroxide. As a result of the free hydrocyanic acid formed, a well-drawn off-take is used. The resulting red suspension was stirred for a further 15 minutes, cooled and shaken with 6 x 300 mL of ether. The ethereal extracts are dried over sodium sulfate, filtered off with suction and concentrated. 97 g of azo compound are obtained in the form of a red oil. Chromatographic separation on kieselguhr column (filling height 110 cm., Column diameter 8 om)

CS 273 302 B2 with a 1: 4 mixture of chloroform and ligroin (amount of diatomaceous earth 1.5 kg, diameter 0.05 to 0.2 mm) yields 32.45 g of brick red crystals. M.p. Mp 134-136 ° C.

The azo compound is dissolved in 500 ml of glacial acetic acid, 150 g of zinc dust are added and the mixture is heated under reflux for 30 minutes with stirring. Excess zinc powder is then aspirated and the solvent is concentrated in vacuo. 17.5 g of colorless crystals are obtained, which are mixed with 150 ml of water, 150 ml of 2N sodium hydroxide solution are added and shaken three times with 150 ml of chloroform each. 12.4 g of crude 3,3 ', 5,5 * -tetra isopropylbenzidine are obtained; make up to 90 ml with acetic anhydride, boil briefly and evaporate excess acetic anhydride on a rotary evaporator. The residue is dissolved in 20 ml of isopropanol, 180 ml of ligroin is added and the crystals formed are filtered off with suction. The obtained N, N ' -diacetyl compound is recrystallized once more from a mixture of isopropanol and ligroin 2; 1. 7.6 g of colorless crystals are isolated, m.p. 366 ° C. These are heated to 130 DEG-140 DEG C. for 6 hours in a mixture consisting of 180 ml of glacial acetic acid and 50 ml of concentrated hydrochloric acid in an autoclave for the cleavage of the N-acetyl groups.

The reaction solution is then concentrated, the residue is taken up in 200 ml of 2N sodium and the liberated base is extracted with 4 x 50 ml of chloroform. The chloroform extracts are dried over sodium sulfate, filtered off with suction and concentrated. 3.3 ', 5.5% ^of tetraisopropylbenzidine is thus obtained in the form of a brownish oil.

Claims (1)

Rapid tests for the detection and determination of hydrogen peroxide and substances reacting to form hydrogen peroxide or peroxidically active substances, consisting of an absorbent carrier swelling a synthetic film impregnated with an impregnating solution containing, per 100 ml, 0,01 to 0,5 g of peroxidase, 0.1 to 2 g of hydroperoxide, to 20 g of a buffer such as citrate or morphine olefinethanesulfonic acid, to 5 g of a wetting agent such as dioctyl sodium sulfosuccinate, lauroylsarcosine, polyoxyethylene sorbitan monolaurate, to 15 g of activator such as 2,5-dimethylhexane-2,5-dihydroperoxide , 1-1 g chromogen, characterized in that as active ingredient contain the chromogen 3,3 ', 5,5 ^r -tetraalkylbenzidin formula I. wherein R 6, R 8, R 8 and R 8 are the same or different and represent lower alkyl groups having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and straight or branched chain.