US 3119668 A
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United States Patent Ofiice Patented Jan. 23, l94
3,11%,663 SULFHYDRYL TESTS AND COMPQUNDS THEREFQR George L. Elhnan, Tiburon, Calih, assignor to The Dow Qhemical Company, Midland, Mich, a corporation of Delaware No Drawing. Filed Ilan. 26, 1959, Ser. No. 788,735 2 filaims. ((Il. 23--23t)) The present invention relates to chemical analysis and is particularly directed to novel methods for the qualitative or quantitative determination of sulfhydryl groups, and to chemical compounds to be employed as reagents in the conduct of such tests.
According to the present invention it has been discovered that a brilliant yellow color is produced when a composition containing sulfhydryl groups, that is mercaptoor thiol groups, corresponding to the formula is brought into contact with the test reagent bis-(4-nitrophenyl)-disulfide, or any of a very large number of test reagents having a bis-(substituted-4-nitrophenyl)disulfide structure. The said yellow color is of an intensity proportional to, among other things, the concentration of sulfhydryl groups present in the said composition. The compounds to be used as reagents in the conduct of the sulfhydryl test of the present invention contain a structure corresponding to the skeletal formula and include compounds having substituents at any of the positions orthoand metato the disulfide linkage, subject only to the limitations that any substituent in ring positions metato a nitro group is non-nucleophilic, except that carboxyl groups and salts thereof may ocour in such position, and any branching substituent in such position must have its branching moieties separated from the benzene nucleus by at least one bridging atom. Nucleophilic radicals which are non-admissible in the said meta-positions and are, therefore, to be avoided in such position, accord with those set forth in Introduction to Theoretical Organic Chemistry, by Hermans (first English edition), Elsevier, 1954, p. 254 and following.
It will be seen that all the compounds thus suggested to be used in the present test retain the group or nucleus corresponding to the skeletal formula hereinbefore set forth, and it has been learned that compounds which contain this characteristic group are of value for the analytical test of the present invention, subject only to the limitations above stated. Examples of substituents which may permissibly or advantageously occur in one or more of the positions orthoto the disulfide linkage, that is to say, positions 2,6,2 and/or 6 in the skeletal formula, are a straight chain alkyl radical containing from 1 to 18 carbon atoms, a branched-chain organic radical of which the branching nearest a benzene moiety of the said skeletal formula is separated therefrom by at least one atom, an alkoxy radical of which the alkyl moiety contains from 1 to 18 carbon atoms, phenyl, alkylphenyl, phenalkyl whereof the alkylene moiety is a normal alkylene moiety and contains from 1 to 18 carbon atoms, and phenoxy. Examples of electronegative, that is to say, nucleophilic, substituents which should be avoided in the positions orthoto the disulfide linkage are nitro, halo. hydroxyl, phosphate, sul
fonate, and the like. Examples of substituents which may permissibly occur in one or more of the positions etato the said disulfide linkage, that is to say, the positions numbered 3,5,3, and 5 in the said skeletal formula, include electrophilic radicals generally, and in particular, alkyl radicals containing from 1 to 18 carbons, alkoxy containing from 1 to 18 carbon atoms, carboxy, nitro, phenyl, phenylalkyl containing from 1 to 18 carbon atoms in the alkyl moiety thereof, carboxyalkyl containing from 1 to 18 carbon atoms, and hydroxy alkyl of which the alkyl moiety contains from 1 to 18 carbon atoms. Also to be understood as among the reagents to be employed in practicing the present invention are the salts including particularly the alkali metal salts, alkali earth metal salts, ammonium and substituted ammonium salts inclusive of primary, secondary, and tertiary alkyl and aryl amine salts and the alkyl and aryl esters of such of the compounds as contain carboxyl groups.
While methods of preparation of the present reagent compounds may, in some instances, lead to the production of compounds bilaterally symmetrical as to the disulfide linkage, having identical substituents in the same relative position on each of the benzene nuclei, such symmetry is not essential to the utility of the present reagent compounds in the practice of the present test.
Among the reagents specifically to be employed in the conduct of tests of the present invention are bis(4- nitrophenyl) disulfide, bis(3-lauryl-4-nitrophenyl) disulfide, bis(3-carboxy-4-nitrophenyl) disulfide, bis(3-tertiarybutyl4-nitrophenyl) disulfide, bis(3-isopropoxy-4- nitrophenyl) disulfide, bis(3 phenyl-4-nitrophenyl) disulfide, bis(2-methoxy-4-nitrophenyl) disulfide, bis(3- carboxy-S -lauryloxyl-nitrophenyl) disulfide and, expressly, the sodium salts prepared by reacting a sodiurn alkali compound such as sodium carbonate, bicarbonate, hydroxide, phosphate, or bisphosphate with each of the foregoing reagent compounds in which appears one or more carboxyl groups, whereby sodium replaces the hydrogen of at least part of the carboxyl groups in each molecule of such compound. The more preferred reagents include bis(2-carboxy-4-nitrophenyl) disulfide, bis(3carboxy-4-nitrophenyl) disulfide and bis(4-nitrophenyl) disulfide. The most preferred of all the compounds of the said class is bis(3-carboxy-4-nitrophenyl) disulfide, together with, of course, its salts and esters.
In carrying out the sulfhydryl tests, such as the qualitative tests of the present invention, at least one of the compounds of the group herein set forth to be used as reagents is brought into contact with a material of which the sulfhydryl content is unknown, thus forming a sulfhydryl test composition, wherein, when sulfhydryl groups are present, a reaction characteristic of the present test takes place, and produces a yellow coloration in the resulting reaction mixture. The fact of the development of such color constitutes the qualitative positive response of the present test. When under the said conditions, test reagent is present in such amount that addition to the test composition of further test reagent occasions no change in color, the intensity of the yellow coloration is proportional to the concentration of sulfhydryl groups, as the quantitative response to be estimated in the conduct of quantitative tests of the present invention. Depending upon such factors as sample size, foreign colored substances present if any, the manner of exhibition of the test compound according to the present invention, thickness of sample as measured along an optical axis under conditions under which the test is to be read, and like factors, the novel disulfide reagent and sulfhydryl bearing material to be tested may be present within very wide limits of concentration. The reagents according to the present invention may be employed by dissolving them, with the substance to be tested, in a mutual solvent. Alternatively, a reagent according to the present invention may be dispersed on a member which provides mechanical support, such as absorbent paper, as by the evaporation thereupon of a solution of the present compounds in a volatile solvent such as a chlorinated hydrocarbon, or precipitation in such paper of an insoluble salt or the like by reaction therein, with, for example, an alkaline earth compound and a carboxyl-substituted reagent compound of the present invention, whereby to prepare sulfhydryl test papers. These papers may then be employed in the conduct of a test according to the present invention, for example by dipping, exposure to vapors, dusts, or gases, or in chromatographic techniques, whereby they are contacted with a sulfhydryl-containing unknown. It is contemplated that such paper will usually be white before employment in such test, and will, correspondingly, be yellow upon completion of the test. However, such paper may be colored, either uniformly in in small discrete colored areas, as, for example, a pale red whereby the develop ment of the present test coloration causes it to turn to an apparent orange color; or, alternatively, such paper may be blue, whereby the development of the coloration according to the present test causes it to appear to be green. Many other such color combinations will be apparent. Moreover, the test compounds according to the present invention may be dispersed in or on a white, colored, or colorless substance which may be a solid or a granulated or finely subdivided material, such as silica gel, crushed fired ceramic material, and the like, which may then be employed in a column. Any or all of these exhibitions of the test compound according to the present invention may be estimated by, for example, visual comparison of a test preparation against a known standard which known standard may be a comparable test preparation to which has been added compositions or a composition containing a known quantity of sulfhydryl groups, whereby to develop a yellow color of intensity representing the said known. Alternatively, a color comparison standard may be prepared by printing with printing ink of selected color on paper or the like; or such standard may be of colored glass, ceramic, or plastic or the like substance, 21 color of which is selected to match or closely approach the color of a known sulfhydryl test color. It is not necessary in the conduct of the tests according to the present invention that human visual response to the color yellow be observed, or measured; satisfactory estimations either qualitative or quantitative of the response of the reagents of the present test to sulfhydryl groups may be had by measurement of transmission of light at a specified wave length or, alternatively, the absorption of light of complementary wavelengths. Such absorption may be measured as a diminution of the total amount of light transmitted, preferably in those wavelengths which are, in the chromatic sense, complementary to the frequency of the transmission maximum; or in alternative methods such as the micro-measurement of heating of preparations according to the tests, as a result of the absorption therein of the energy of incident, non-transmitted light. In other procedures the coloration characteristic of the practice of the test according to the present invention may be assayed, estimated, or made of reference by spectrophotometric and like means. Such mechanical optical means may rely upon the fact that the color developing in the test solution according to the present invention presents a light absorption maximum at the wavelength of 412 millimicrons. The results of mechanicooptical measurement of the test response may be presented as an absorbance or transmittance factor, as a spectrogram, or, in equipment which refers the test coloration to a known standard and, by optical means, makes a comparison, may be expressed directly in concentration. Such concentration may be expressed variously as percentage,
parts per million, or, in the case of single sources of sulfhydryl groups, as a quantitative analytical result in terms of the compound source.
The test according to the present invention may be carried out in the presence of substances having extraneous or anomalous color. When the test is to be carried out in the presence of one or more of such substances, 1t is often possible to prepare a known standard or standards for reference wherein the resulting yellow sulfhydryl test coloration is developed in the presence of the same anomalous or extraneous colorant as is present in the unknown so that reference thereto makes possible the direct sulfhydryl estimation of the unknown in the presence of the non-test color. Alternatively, the anomalous colorant in a separate transparent container may be placed in the optical system with a standard which does not contain the anomalous color, whereby the algebraically additive optical resultant exhibits the approxb mate absorbance and appearance of the test standard as developed in the presence of such colorant.
It is among the desirable and useful properties of the reagents according to the present invention, particularly but not exclusively as they are dispersed on a chemically inert supporting solid, that the test response coloration developed over a period of time as, for example, by exposure to gases or dusts, represents the total cumulative or additive value of the sulfhydryl groups to which such test substance is exposed. Therefore, not only can the present tests be carried out in relatively short periods of time as in determining the composition of definite unknown mixtures, but also, if desired, a sulfhydryl test preparation may be exposed continuously over an extended period of time to continuing and cumulative contact with sulfhydryl groups or compositions containing such groups, whereby the resulting color which develops over such extended period of time accurately indicates the amount of sulfhydryl groups contacted in such cumulative exposure.
Yet another advantage of at least many of the reagents to be employed in the test of the present invention is their limited toxicity to warm blooded animals whereby, if desired, such compounds together with desired excipients and additives and the like may be administered orally or by injection in useful amounts to such animals, whereby sulfhydryl tests of, for example, the contents of the alimentary canal may be conducted in vivo. In such application, a compound may be selected for low solu bility in both water and lipid substances and the resulting yellow coloration may, thereafter, be extracted and exhibited for estimation quantitatively or qualitatively from the excrement of such animal, employing as solvent, for example, diethyl ether. Alternatively, a highly watersoluble compound according to the present invention may be ingested for the conduct of in vivo tests of sulfhydryl concentrations of body fluids, by the subsequent examinations of such fluids.
When the sulfhydryl group to be tested is present in' an ionized or easily ionized form, the test according to the present invention may be carried out in either the presence or absence of solvent. When the sulfhydryl group to be tested is present in a non-ionized form, provision must be made for it to enter ionically into the reaction of the present test. This may easily be accomplished by presenting the test reagent in a basic state as, for example, by the presence intimately admixed with it of at least a small amount of slightly basic solvent, such as perhaps, an amount of such solvent equimolecular with the said test reagent; or, the reagent, if a carboxyl substituted reagent, may be presented as the salt such as the alkali metal or the amine or substituted amine salt thereof. The water-free or practically Water-free testmaterials such as solutions, columns, and papers as de-- scribed, wherein are included intimately admixed with the test reagent small amounts of basic solvents, are consid-' ered to be part of the present invention; Suitable slightly" basic solvents include, among others, liquid amines, pyridine, and the like.
When either the reagent or the sulfhydryl-bearing composition is present in a liquid phase, and especially when present in an aqueous phase, it is desirable for the eiiicient conduct of the present test that the liquid phase be maintained in a pH within the range of 5 to 9 and preferably within the range of 7 to 8 during the time the test is to be carried out and estimated. At a pH below about 5, the color-producing reaction of the present test is inhibited, and the color development takes place so slowly as to render the test inefficient. At a pH much below 5 the development of the yellow coloration of the present test may take place so slowly as to be imperceptible and worthless in laboratory procedures. At a pH maintained continuously in the range of 7 to 8 during and after the conduct of the present test, the resulting yellow coloration develops quickly, accurately, and is sufficiently stable for all ordinary laboratory uses. At a pH much greater than 9, the test reagent decomposes under the influence of hydroxide ion at a rate which increases as the pH increases much above 9. Thus, for example, yellow solutions prepared in the conduct of the present test in the pH range of 7 to 8 have proved to be completely stable in color for at least as long as a year, whereas similar solutions wherein the test is carried out at a pH in the range of 11 to 12 give the desired yellow coloration promptly, but the resulting coloration disappears through decomposition or gives way to anomalous and meaningless color changes within a short time, such as an hour or less. When the present test reagents are to be employed in solution such as aqueous solution, preferably such solution should contain buffering agents whereby to maintain the pH in the preferred range of 7 to 8. This is especially true if such solutions are to be held in storage for a period of time prior to their use. If such buffering is not to be employed, fresh reagent solutions should be prepared for each test, and the test carried out preferably with continuous measurement of pH which pH is continuously maintained in appropriate manners in the preferred range of at least 5 to 9 and preferably 7 to 8.
When carrying out the test of the present invention in non-aqueous solution, water-free pyridine of such purity as to be essentially colorless is a satisfactory solvent. Alternatively, a colorless hydrocarbon or halogenated hydrocaroon solvent may be employed in the presence of at least a minimal amount, such as an amount equimolecular with the test reagent, of an organic base such as a nitrogen-bearing compound which may be pyridine or a primary, secondary, or preferably tertiary amine, free from water.
When the present test is carried out upon a sulfhydrylbearing composition in a solid phase, which may be dispersed in a non-solvent liquid system, the resulting test coloration determines only those surficial sulfhydryl groups available to make contact with the present reagent. In certain biochemical applications this may be a preferred manner of carrying out the present tests. When it has been desired to estimate the total sulfhydryl content of a composition upon the basis of a quantitative determination of sur'ficial sulfhydryls, it has often been possible to calculate a factor greater than unity which may be called a form factor expressing the ratio between surficial sulfhydryl groups and total sulfhydryl groups in such solids. The product from multiplication of determined surficial sulfhydryl content by the said form factor represents tota sulfhydryl content subject, of course, to procedural error.
In carrying out the tests according to the present invention, the test reaction may be carried out at any temperature over a very wide range. In practice, conduct of the test at temperatures lower than 0 C. results in a very slow development of the test color; whereas temperatures much above about 50 C. may result in the thermal decomposition of the test compounds. A preferred temperature range for the conduct of the present tests is, therefore, from room temperature, that is to say about 20 (3., to about 50 C.
In carrying out quantitative sulfhydryl estimations in the tests of the present invention, it is to be understood that preliminary or range-finding qualitative or quantitative estimations may be carried out to ascertain the fact of the presence of, and the approximate concentration in an unknown composition of the sulfhydryl groups therein. For example, a dilute solution of a reagent of the present invention may be employed whereby to determine whether the unknown contains sulfhydryl groups in quantity sufficient to saturate the color response of such dilute test solution. Similarly, a more concentrated solution of reagent of the present invention may be employed whereby to determine whether the unknown contains sulfhydryl groups in quantities sufiicient to saturate the color response of such concentrated test solutions. In such manners, a general range of sulfhydryl concentration may quickly be ascertained, and thereafter, the present test may be employed in any degree of precision desired in the measurement of sulfhydryl groups within the range thus established. As but one embodiment of the rangefinding use of the present tests, a test paper of the sort described may be prepared containing a relatively high concentration of at least one of the reagents of the present invention, intended to be used for rough estimation and range-finding purposes preliminary to the conduct of more precise estimations of sulfhydryl groups if such more precise estimations are desired.
The sulfhydryl test of the present invention is illustrated by the following examples which are not to be considered as limiting.
EXAMPLE 1 Bis(4-nitrophenyl) disulfide (melting at 183-184" C.) was dissolved in acetone to prepare a solution containing 30.8 milligrams of the disulfide compound per 100 milliliters. This solution was 1/ 1000 molar and is referred to in the present example as the reagent solution. A 0.1 molar aqueous solution of disodiurn hydrogen phosphate and a 0.1 molar aqueous solution of sodium dihydrogen phosphate were mixed together to form an aqueous solution having a pH value of 8.0. This solution is referred to in the present example as the buifer solution. In carrying out the sulfhydryl estimation, a measured amount of the sulfhydryl-bearing unknown was dissolved in 4 milliliters acetone. One milliliter each of the reagent solution and of the butler solution were added to the said unknown. Thereupon a color change took place, whereby the resulting mixture appeared to be yellow. The light absorbance of the yellow mixture as referred to a corresponding blank solution is measured at 412 millimicrons wave length of transmitted light. The concentration of the sulfhydryl group in the final solution is calculated from the formula wherein C is the concentration of the sulfhydryl group and A=2 log T when T is the transmission of light through the colored unknown expressed as a percentage of light transmitted by the corresponding blank. Such values of A are commonly read directly from photometers of known kinds. When such test is carried out with simple mercaptans such as the lower alkyl mercaptans, the 'test reaction goes to completion virtually instantaneously and the resulting color remains stable for at least 0.5 hour. For cysteine and related mercaptoethylarnines, full intensity is developed after 60 to minutes. To the eye, the development of the test appears as a change from a colorless or nearly colorless solution to a vivid and intense yellow to yellow-orange solution which may be Table I Known Amount Amount Meas- Amount Measof Tested Comured by the med as Percent- Compound Tested pound Added Test of the Known (Micrograms Present In- Amount Added per 4 ml.) vention 20. 1 20. 2 101 20.1 20.1 20.1 20. 2 2-Mereaptoethane 1005 10. 2 ml 10.05 10.0 99. 10.05 9.9 98. 5 5.03 5. 1 101 5.03 5.3 9799 r 5. 03 4. 5 ys L .9 82 1.10 1. 0 91 1. 10 1. 2 100 limit of concentration of tested compounds in test solutions proved to be approximately 2 to 4X10 molar, in which range the resulting color characteristic of the test was of such dilution as to reach the practicable lower limit of the sensitivity of the photometer. Qualitative sulfhydryl tests have proved to be much more sensitive.
EXAMPLE 2 LHL'LSUREMENT OF SULFHYDRYL 'COl\".l?EZ\"ll IN \VHOLE BLOOD This example illustrates the conduct of the test of the present invention in the presence of materials which alter optical properties of the test coloration, as, by the presence of non-test colorants.
A mixture is prepared containing 0.01 milliliter whole blood, 5 milliliters water, and 2.7 milliliters acetone. To this is added 2 milliliters of the phosphate buffer solution described in Example 1. The resulting buffered blood solution is allowed to stand at room temperature for 1 hour. Thereafter, 0.3 milliliter of the reagent solution of bis(4-nitrophenyl) disulfide as described in Example 1 is added to the buttered blood solution and thoroughly stirred and the absorbance at the 412 millimicron wave length is photometrically determined, wherefrom is calculated the sulfhydryl content of the blood. As a blank to be employed in the photometer, for the absorbance of the blood solution alone, the following preparation is employed; 0.010 milliliter blood, 5.0 milliliters water, 2.0 milliliters butter and 3 milliliters acetone. This preparation is allowed to stand at room temperature, along with the test preparation to which the reagent is to be added.
Of the blood samples tested in this method, most reached equilibrium response in 0.5 minute to 1 minute after addition of the reagent solution to the buffered blood sample. However, in occasional samples as long as 40 minutes were required to reach equilibrium. Therefore, in such work, it is desirable to measure absorbance successively at several different times noting any change. When two or more successive readings are alike, equilibrium may be assumed to have been reached. Samples of blood tested and results of such tests are indicated in the following table:
BLOOD lVlERCAPTAN CONCENTRATIONS Individual [torn Whom Sample was Taken The individuals represented in the above test were male except for the last two who were females.
When it is desired to estimate the sulfhydryl concentration of blood serum, the following preparation may be employed; the first absorbance measurement should be taken immediately upon completion of the preparation. As a test solution, 0.1 milliliter serum, 1.0 milliliter of the phosphate buffer described in Example 1, and 0.3 milliliter of the disulfide reagent solution described in Example 1, together with 2.7 milliliters acetone and 5.9 milliliters Water. As a blank preparation for comparison, 0.1 milliliter blood serum, 1.0 milliliter buffer solution, 5.9 milliliters Water, 3.0 milliliters acetone.
EXAMPLE 3 Bis(3-carboxy-4-nitrophenyl) disulfide, sodium dihydrogen phosphate and disodium hydrogen phosphate were dissolved in water to form a phosphate buffered aqueous solution thereof containing one one-thousandth grammole of phosphate and one ten-thousandth gram-mole of disulfide compound (39.6 milligrams) in milliliters of the solution, the ultimate solution having a pH of 8.0 This solution will be referred to in the present example, and in others carried out in similar manner, as the reagent solution.
A supplementary solution was prepared by mixing together a 0.1 molar aqueous solution of disodium hydrolgBH phosphate and a 0.1 molar aqueous solution of sodium dihydrogen phosphate to form an aqueous solution thereof having a pH of 8. This solution will be referred to in the present example and others carried out in similar manner as the butter solution.
In carrying out the sulfhydryl estimation, :1 measured amount of the sul fhydryl-bearing unknown is initially dissolved in 8 milliliters of Water or in a waterrniscible solvent. One milliliter each of the reagent and of the buffer are added to the unknown solution. If a water miscible solvent is used to dissolve the unknown, it may be necessary, according to this test procedure, to add further Water so that the resulting test mixture has a volume of 10 milliliters. The resulting mixture is thoroughly agitated and allowed to stand at room temperature for at least one minute and not more than four hours. A yellow color having a light absorption maximum at 412 millimicrons wave length develops in the test mixture, which color is of an intensity that is directly proportional to the amount of sulfhydryl groups present over the range of from approximately 3.7 millionths to 73 millionths gram-mole per liter. The color can be measured in any standard colorimeter or photometer or may be visually estimated in comparison with standards containing known concentrations of sulfhydryl groups and reagent, prepared in similar manner. When sulfhydryl estimation is to be carried out in the presence of colored materials whose colors alter the yellow appearance or the characteristic absorbance of the test, viewing or measurement against a known standard may be made with a suitable concentration of the colored unknown in the optical axis of and before such standard. Thus, the test has proved to be useful in the presence of such colored materials as tissue extractives, urine, blood, and the like.
Tests conducted on tissue extracts which were made by extracting the tissues with hot 80 percent ethanol gave the results set forth in the following table.
TISSUE SULFHYDRYL ANALYSIS Sull'hydryl Content as Millimoles per Gram of 80 percent Hot Ethanol Tissue Extract Time to Develop Full Tissue (Rabbit) Color (minutes) 1. Less than one minute. 2.
EXAMPLE 4 EXAMPLE 5 A solution of crude soap is adjusted to pH 8 and the sul'fhydryl content thereof determined using as test reagent a buffered solution of bis(3-lauryl-4-nitrophenyl) disulfide. This reagent is chosen for the similarity of its solubility performance to that of the said soap.
EXAMPLE 6 A sample of coal is crushed and then ground in pyridine to yield a paste suspension containing the said coal as an impalpable powder. The sulfhydryl concentration in the resulting suspension is determined in manners similar to the determination in the foregoing experiments, employing a photometer cell designed to exhibit the test suspension in one tenth millimeter section, and employing, as reagent, the compound bis(3-phenyl-4-nitrophenyl) disulfide, dissolved in pyridine.
EXAMPLE 7 The sulfhydryl content of an unrefined sour, that is to say, sulfur-bearing, crude petroleum is determined by diluting the crude petroleum with petroleum ether and employing, as reagent, bis(3-carboxy-5-lauryloxy-4- nitrophenyl) disulfide; and using a similar dilution as a color-reference blank.
EXAMPLE 8 The dilution of sulfhydryl-bearing waste outflow from a waste disposal plant into a natural body of water is automatically controlled by the simultaneous photometric scanning of, on the one hand, a continuously replaced sample from the said natural body of water containing the diluted waste, into which sample is continuously metered and intimately mixed a reagent according to the present invention together with buffering agents and, on the other hand, a color standard corresponding to an acceptable level of sulfhyclryl content. The electrical outputs of the photometric systems are connected in opposition one to another through a resistance bridge and the system so balanced that a current flow in the output of the bridge network corresponds to a sulfhydryl concentration at about a maximum tolerable level in the diluted output. Such current flow is caused to actuate a reversible solenoid-controlled valve and alarm system by the action of which sulfhydryl-bearing waste outflow is interrupted and an alarm is energized until, responsive ot a reduced sulfhydryl content, the bridge output current ceases to flow and the solenoid-controlled valve operates in reverse whereby the outflow is resumed.
EXAMPLE 9 A wet-strength absorbent paper is impregnated in a moist condition with calcium bicarbonate and there is subsequently introduced into the same paper an excess of bis(3-carboxy-4-nitrophenyl) disulfide whereby the waterinsoluble calcium salt of the said disulfide compound is formed in situ within the said paper. Thereafter, excess disulfide and calcium bicarbonate materials are removed by washing with water and the resulting paper dried.
Another test paper is prepared by soaking a wet-strength absorbent paper in a buifered solution of bis(2-methoxy-4- nitrophenyl) disulfide, and the paper is thereafter evaporated to dryness.
In similar manner, a granular apparently dry silica gel is immersed in an isopropanol solution of bis(3-isopropoxy-4-nitrophenyl) disulfide, the excess is removed, and the silica gel bearing a surface application of the said disulfide is gently warmed to vaporize and remove solvent.
In each of the foregoing preparations, the evaporation to dryness of the resulting test agent is carried out in an atmosphere essentially free from sulfhydryl groups and at temperatures not exceeding 56 C. The products of the said preparations are subsequently used for quantitative sulfhydryl estimation in gases and liquids, by comparison of a test paper brought into contact with the unknown with a similar test paper brought into contact with a known sulfhydryl solution. Each of the test materials is essentially white in color before being used in such test and during the course of the said test develops a yellow color whose intensity depends upon the concentration of sulihydryl groups.
In each of the foregoing examples, and in other similar preparations of the compounds to be used in tests according to the present invention, the preparation of a reagent solution containing one ten-thousandth gram-mole of disulfide reagent compound in milliliters of solution obtains a reagent solution wherein the yellow color developing in the conduct or" the sulfhydryl test is directly proportional to the amount of the sulfhydryl groups present over approximately the range of from 3.7 millionths to 73 millionths gram-mole per liter. The test reaction and the development of yellow coloration having a light absorption maximum at a wave length of 412 millimicrons proceeds reliably and quantitatively over a wide range of concentrations of reagent and reagent solution, being limited only by, at the lower extreme of concentration, the limit of sensitivity of the eye of the observer, or alternatively, of the mechanico-optical system wherein the light absorbance is measured; and at the other extreme of concentration, by the limits of the powers of the observer, or the development of a coloration so intense as to exceed the limits which can be measured in the photometric equipment employed. In practice, however, these limits do not essentially restrict the range of sulfhydryl group concentrations which can be tested, inasmuch as sulfhydryl-bearing materials may be diluted or concentrated by dissolving or dispersing them in a suitable liquid in known ratio, such ratio being chosen to exhibit the resulting sulfhydryl concentration in a range which can conveniently be estimated in the quantitative method according to the present invention, the sulfhydryl concentration of the original being thereafter easily calculated. Also, the colored test solution may be viewed in comparison with a standard, both test and standard being controlled as to depth of viewed section.
One of the compounds to be employed as reagent in conduct of the present test is known, although its use in such test is novel. Bis(4-nitrophenyl) disulfide is set forth in Beilstein, Handbuch der organischen Chemie at 6, 340; I, 160; and II, 312. This compound may be dissolved in acetone, and in such solvent has been successfully employed in the conduct of the qualitative test according to the present invention. However, in quantitative tests, especially those conducted with the assistance of a photometer, the results obtained by the use of the said compound proved to be unreliable. Attempts to replicate test results successively by the testing of aliquot portions of the same test solution gave anomalous results. This was especially noted in the conduct of sulfhydryl tests on substances naturally occurring in living organisms, including, for example, proteins. Unexpectedly, certain other compounds of the class set forth to be used as reagents according to the present tests have been used successfully, overcoming the said ditficulties and other difficulties encountered in the use of the bis(4-nitrophenyl disulfide. The novel compounds bis(3-carboxy-4-nitrophenyi) disulfide and bis(2-carboxy-4-nitrophenyl) disulfide were found to give reliable and replicable test results.
The following example, which is not to be considered as limiting, sets forth one method by which may be prepared the bis(3-carboxy-4-nitrophenyl) disulfide.
EXAMPLE In the first step of the process, six grams (0.0335 mole) of 5-chloro-2-nitro-benzoic acid was suspended in 150 milliliters of water to which concentrated aqueous sodium hydroxide was added with constant stirring to bring the mixture to a pH of 7.2 and dissolve the 5-chloro-2- nitro-benzoic acid as a sodium salt. Sodium sulfide (Na SBH O, 12 grams: 0.050 mole) dissolved in milliliters of water was added portionwise thereto with continuous stirring. The reaction mixture thus formed was heated at 50 C. for 1.25 hours with continuous stirring. Stirring was then discontinued and the reaction mixture allowed to cool. When it had cooled to approximately room temperature the reaction mixture was acidified by the addition of concentrated hydrochloric acid to a pH of approximately 1 whereupon an oily intermediate product separated. The oily product was removed in a separatory funnel. In the second step of the process, the said oily intermediate product was dissolved in 50 milliliters of water by the addition of concentrated aqueous sodium hydroxide to a pH of 7.2. To this solution approximately 30 milliliters of an aqueous solution of iodine in 5 weight percent concentration and potassium iodide in a 2 weight percent concentration was added dropwise and with continuous stirring over a period of 15 minutes at room temperature. The quantity of the aqueous solution of iodine and potassium iodide to be added was determined by color change in the resulting mixture. When addition of the iodine solution began, the mixture to which it was added was of a deep orange-yellow color. During the course of the addition this color became li hter and eventually changed to a pale yellow. Further additions of the iodine solution tinged the reaction mixture with the reddish-brown color of iodine. At this point, addition of iodine solution was discontinued. The pale yellow reaction mixture thus formed was then acidified to a pH of approximately 1 by the addition of concentrated hydrochloric acid, to produce the desired bis(3-carboxy-4-nitrophenyl) disulfide product. The product was separated from the reaction mixture by repeated extractions with diethyl ether. Ether was evaporated from the extract and the residual product was purified by recrystallization from glacial acetic acid to obtain a bis(3-carboxy-4-nitrophenyl) disulfide product as a pale yellow crystalline solid which was slightly soluble in each of the solvents ether, alcohol, acetone, and toluene and was insoluble in water, but readily soluble in dilute aqueous alkali. The product 12 melted with decomposition at 237 to 238 C. Product solutions exhibited a characteristic ultraviolet absorption spectrum with a peak at 315 millimicrons. Titration of one gram-mole of the purified product required 2 gram chemical equivalents of base. Bis(3-carboxy-4-nitrophenyl) disulfide has a molecular weight of 396.5.
In similar preparation there is prepared a novel bis(2- carboxy-4-nitrophenyl) disulfide, by employing as starting reactant 2-chloro-5-nitrobenzoic acid. The bis(2-carboxy-4-nitrophenyl) disulfide is useful in a manner similar to the bis(3-carboxy-4-nitrophenyl) disulfide but exhibits numerous unexpected distinctive and different properties in use. As but one example of such distinctive properties, the 2-carboxy-4-nitrophenyl compound reacts at a much slower rate, requiring approximately a half hour to develop color responses which the 3-carboxy compound usually develops in less than a minute.
The said novel bis(2-carboxy-4-nitrophenyl) disulfide is especially useful in the conduct of automatically controlled operations such as that set forth in Example 8, in use of apparatus wherein a sensing apparatus is employed, whereby the activation of a response such as the operation of a solenoid-controlled device is delayed until a replicate number such as two or more sensings of the contents of a sulfhydryl test cell are found lie within predetermined limits of equivalents. By the employment of the slowreacting Z-carboxy compound it is possible to achieve, as results of a small number of tests, control responses which approximate an integration of a larger number of shorttime tests and avoid sudden and short-cycle operations of the controlled device. Alternatively, when it is desired to operate such equipment in a manner to obtain very quick response to sulfhydryl content as measured, the 2-carboxy substituted compound should be avoided, and, for example, the 3-carboxy compound employed instead. Also, mixtures of the two such compounds may be prepared, which mixtures react, in a desired degree of approach to completeness, in known times, with the result that the operative cycle of automatic equipment may be adjusted by such chemical means, within wide limits.
EXAMPLE 11 The novel compound bis(3-lauryl-4-nitrophenyl) disulfide is prepared. In one manner of preparation, laurylbenzene is nitrated by the action of nitric acid in the presence of sulfuric acid, whereby there are produced mixed isomers of ring-nitrated laurylbenzene. Such mixture is resolved into its components from which the o-nitrolauryl benzene is separated by crystallization from hot alkanol solution as such solution cools. The o-nitrolaurylbenzene is then dissolved in carbon tetrachloride and, in the resulting solution, chlorinated by the addition thereto of gaseous chlorine in an amount approximately equimolecular with the o-nitrolauryl benzene. Upon completion of the chlorination, the chlorinated mixture is purged with nitrogen, to remove unreacted chlorine and hydrogen chloride of reaction and the resulting solution is thereafter dried over anhydrous magnesium sulfate and treated by the addition thereto of sodium sulfide and sulfur as finely divided solids. Such addition is carried out slowly, portionwise, and with vigorous stirring at room temperature. Stirring is thereafter continued for a period of time to carry the reaction to completion and form the desired bis(3-lauryl- -nitrophenyl) disulfide reagent which may thereafter be extracted from the resulting reaction mixture by evaporat ing the chlorinated hydrocarbon solvent nearly to dryness under reduced pressure and with gentle heating to temperatures not in excess of 50 C., and thereafter extracting the desired disulfide compound with petroleum other as extractive solvent. Bis(3-lauryl-4-nitrophenyl) disulfide has a molecular weight of 644.8. In manner similar to the foregoing there is prepared a bis(3-phenyl-4-nitrophenyl) disulfide compound by the employment, as starting material, of biphenyl. Bis(3-phenyl-4-nitrophenyl) disulfide has a molecular weight of 461.5.
1? EXAMPLE 12 The novel compound bis(3-isopropoxy-4-nitrophenyl) disulfide is prepared by a two-step process. The first such step consists of the chlorination of bis(4-nitrophenyl) disulfide by the direct addition of gaseous chlorine to the said disulfide compound dissolved in a chlorinated hydrocarbon solvent, purging the resulting reaction mixture with nitrogen to remove unreacted chlorine and hydrogen chlorine of reaction, drying the resulting chlorinated mixture. The second step consists of adding to the dried first step product a quantity of sodium isopropoxide approximately equimolecular with the starting disulfide compound. The isopropoxide is added slowly, portionwise, and with stirring over a period of time to carry the reaction to completion. The resulting compound may thereafter readily be separated in such manners as fractional crystallization, and the like. Bis(3-isopropoxy-4-nitrophenyl) disulfide has a molecular weight of 424.9.
EXAMPLE 13 The compound bis(2-methoxy-4-nitrophenyl) disulfide is novel. in its preparation, there is employed, as starting reaction the compound 3-nitroani-sole which is set forth in Beilstein, Handbuch, at 6, 224; I, 116; and II, 214. The anisole compound is directly chlorinated, the chlorination products purged with an inert gas and subsequently dried and treated with a sulfur-sodium sulfide mixture. Thereafter, the desired bis(2-methoxy-4-nitrophenyl) disuifide compound is separated by solvent extraction employing methanol as solvent. The bis(2-methoxy-4-nitrophenyl)- disulfide has a molecular weight of 368.3.
EXAMPLE 14 The novel compound bis(3-carboxy-5-lauryloxy-4-nitrophenyl) disulfide is prepared. In such preparation there is employed, as starting reactant, the bis(3-carboxynitrophenyl) disulfide whose preparation is set forth in Example 11. The said starting reactant is chlorinated by direct addition of chlorine in an amount equimolecular with the disulfide compound, in a chlorinated hydrocarbon solvent. The resulting mixture is purged with an inert gas and dried and thereafter reacted with sodium lauryloxide and the desired product thereafter separated by fractional crystallization, from a mixture of petroleum ether and chlorinated hydrocarbon as solvents. Bis(3-carboxy- 5-lauryloxy-4-nitrophenyl) disulfide has a molecular weight of 733.0.
In manners similar to the foregoing preparations, other reagent compounds of the present invention are prepared.
The present application is a continuation-in-part of application Serial No. 712,667, filed February 3, 1958, now abandoned.
1. A method of testing for su'lfhydryl groups in an unknown suspected of containing sulfhydryl groups which comprises the step of bringing the said unknown into contact with a test reagent compound containing the structure corresponding to the skeletal formula substituents thereupon being limited in that any substituent in a position metato a nitro group is non-nucleophilic, except that carboxyl groups and salts thereof may occur in such position, and any branched substituent in such position must have its branching moieties separated from the benzene nucleus by at least one atom; and thereafter comparing the resulting yellow coloration in the resulting test reaction mixture with at least one color standard.
2. The method of claim 1, modified by the limitation that the test reagent compound is employed in such amount that bringing the test reaction mixture of claim 1 into contact with further test reagent compound does not increase the depth of the resulting reagent mixture color.
References Cited in the file of this patent UNXTED STATES PATENTS Haddock Jan. 7, 1936 Gowberg Dec. 2, 1958 OTHER REFERENCES