ORGANIC POLAROGRAPHY STANLEY WAWZONEK S t a t e University of Iowa, Iowa City,Iowa
S
amino acids (bog), and indicates that quinones react with or are destroyed by glycolytic enzymatic medium (92). In the study of substituted anthraquinones only ammonia-ammonium chloride buffers gave results that were comparable to potentiometric measurements. Phosphate and borate buffers formed complexes and stabilized semiquinones; tartrates and acetates interacted in only a few cases (186). The method has been used to estimate aloins and aloe-emodin in Curacao aloes. The latter, which is a hydroxyanthraquinone, is best determined spectrophotometrically, as fewer steps are involved (185, 187). The reversibility of the catechol-o-benzoquinone, 4-methylcatechol-4methyl-o-benzoquinone, and 2-naphthoquinone-l,2dihydroxynaphthalene systems has been established a t the dropping mercury electrode. The behavior of pyrogallol apparently is irreversible and difficult to interpret, as definite oxidation products could not be prepared ( 5 0 ) . The only conditions reported to give reducible products were air oxidation in aqueous solution for 8 hours (207). Sharply defined polarographic anodic waves can be obtained with a platinum microelectrode for hydroquinone, catechol, or ascorbic acid. Substances that undergo destructive oxidation, such as resorcinol, give curves with a sharp maximum peak followed by a continuous rise ( 1 7 5 ) . The polarographic method has been used to follow the decomposition of o-benzoquinone in water into catechol and an unknown compound which is neither 2-hydroxybenzoquinone nor purpurogallin (51), to investigate the nature of freshly prepared solutions of tannins (24))and to study the reaction of o-quinones with a-amino acids and with hydrogen peroxide. The product in the last reaction from its polarographic behavior is apparently a peroxidic o-benzoquinone (51). p-iiminophenols give anodic waves a t the dropping mercury electrode a t more positive potentials than the corresponding hydroquinones. The values for the oxidation of 2,6-polymethylene-4-aminophenols, for example, are about 0.06 volt more positive than the value for the oxidation of the corresponding 2,6polymethylenehydroquinones (148). The oxidation yields the quinoneimine and represents a reversible reaction;
TUDIES in the field of organic polarography in the past two years have extended the use of this method to a variety of new groups. Contributing to this increase has been the greater use of solid electrodes. More electrode reactions have been verified by large scale electrolysis under controlled conditions with the isolation of products. In general, conclusions drawn from such experiments are in good agreement with the reactions a t the electrode. Occasionally the products are different, as the large scale experiments involve rapid stirring and are not dependent solely upon a diffusion process. Intermediates, therefore, may be removed from the electrode surface to the solution where dimerization, disproportionation, or rearrangement may occur. The possibilities of the cathode-ray polarograph in the organic and reaction kinetics fields have been indicated (178). Bn extensive list of half-wave potentials of organic compounds with the conditions used has been published (218). As in the past, most of the studies have been carried out in mixtures of organic solvents and water. The number of anhydrous solvents employed has been small and consists of methyl Cellosolve ( I @ ) , methanol (152), ethanol, 1-propanol, butanol, ethylene glycol, glycerol, and so'lutions of some of these with benzene or dioxane (46). The diffusion current was found to be inversely proportional to the square root of the viscosity of the solvents, and the waves became more drawn out as the amount of alcohol was decreased in dioxane mixtures with methanol, ethanol, and 1-propanol (46). As supporting electrolytes the tetraalkylammonium salts have continued to be useful. Their range has been found to be dependent upon their purity. It has been suggested that catalytic hydrogen waves are responsible for the nave observed with these salts and not the reduction of the cation (180). Directions are given for the preparation of tetramethylammonium chloride for polarographic analysis ( 7 ) . REVERSIBLE SYSTEMS
The polarographic method has been used to determine the oxidation-reduction potentials of a number of quinones and related compounds. Values have been obtained for a number of polymethylene quinones having the following structures and compared with that of 2,6-diethylbenzoquinone in order to determine whether or not the rings were strain-free. Oxidation of the corresponding hydroquinones a t the same point established the reversibility of the system (146,148,149).
polarographically a value of +0.739 volt is found for EO of p aminophenol, whereas potentiometrically a value of f0.733 volt is reported for the reduction of pquinoneimine. This value is 0.028 volt more positive than that for hydroquinone. The study of the system is not possible in all buffers, since the quinoneimine is hydrolyzed very easily to the quinone. The rate of this decomposition varies with the substituents present and is slow enough with the oxidation product of 4-amino-2-methyl-lnaphthol (vitamin Ks)so that the reaction can be followed polarographically. m-Aminophenol gives no reproducible wave and o-aminophenol gives in a borate buffer (pH = 9 ) a drawn-out anodic wave. The polarographic method has demonstrated that the oxidation
I11 The
EO for bis-(2,4,5-trimethylquinonyl)methanewas found
to be 0.457 volt and very similar to that of duroquinone (177). The polarographic method has been used to study qualitatively the reaction of benzoquinone with proteins, polypeptides, and 32
V O L U M E 2 4 , NO. 1, J A N U A R Y 1 9 5 2
33
products of vitamin Kb formed by oxygen and by human red blood cells are 2-methyI-l,Qnaphthoquinone and 2-methyl-1,4naphthoquinone oxide (IV) and that the violet dye formed from this vitamin is an indophenol (V) (108).
0
v
IV
wave which is dependent upon the amount of ammonia present and which together with the aldehyde wave decreases on standing (217). The polarographic determination of formaldehyde has been carried out in the presence of acetone and alkali using the cathode ray polarograph (178) and has been used to study the reaction between formaldehyde and urea (40, 41), acetamide and benzamide (42). The polarographic determination of acetaldehyde is used indirectly for determining lactic acid. The latter is oxidized to acetaldehyde by a mixture of potassium permanganate, phosphoric acid, and manganous sulfate (47). Formaldehyde and acetaldehyde sulfoxylates, RCHS02Ka, are
I
OH The oxidation of p-phenylenediamines a t the dropping mercury electrode is limited to only a few examples. Substitution of a platinum microelectrode, however, gives half-wave potentials which differ by not more than 3 to 7 mv. from those obtained by the dropping mercury electrode and increases the number of possibilities that can be studied (103). By this technique values for 55 different p-phenylenediamines were determined and found to be related to the development rate in photography and in part to the high allergenic potency of these compounds. Reversibility of this system is indicated by a two-electron slope for the curves but was not established definitely because the quinonediimines were too unstable to study (10). Extensive work has been done on the oxidation of ene-diol systems to determine whether the irreversible behavior observed for ascorbic acid a t the dropping mercury electrode is typical of this system. Dihydroxymaleic acid (VI) ($IO), reductic acid (VII) (262), reductone (VIII), and coumarindiol ( I X ) ( 2 2 ) all give irreversible anodic waves and behaviors resembling that of ascorbic acid. Only reductone (VIII) gave a cathodic wave that appeared close to the deposition of sodium ions. It has been suggested that the irreversibility of this system is caused by the hydration of the oxidation product (22). OH OH
HOCCOOH
I/
I
C=C
/
I I HOCCOOH H?C C=O \/
CH=CCHO
I
1
OH
OH OH
CH,
VI
i-I1
VI11
IS
Ascorbic acid gives an oxidation wave a t a platinized platinum electrode (124). In other reversible systems the polarographic method has been concerned mainly with the quantitative aspect. The method has been used to determine the amount of methylene blue adsorbed by charcoal (95) and the amount of alloxan in blood (204), and has shown that p-dimethylaminoasobenzene (dimethyl Pontachrome Violet S mi, and Pontachrome Blue yellow) (i44), Black R (2i1)give waves suitable for their determination. 'IRREVERSIBLE SYSTEMS
Aldehydes. The polarographic behavior of acetaldehyde in various buffers has been verified and the degree of hydration calculated by comparing the diffusion current with that of a n indicator wave (48, 49). The half-wave potential of this compound differs very little from propionaldehyde and n-butyraldehyde, but by making use of a differential distribution between two immiscible solvents it has been possible to analyze binary mixtures and ternary mixtures of these compounds polarographically (74). Addition of ammonia to an acetaldehyde solution produces a
reported to b- identifiable polarographically (66). In the carbohydrate field the polarographic method has been used in two ways. lldvantage has been taken of the effect of impurities (organic nonsugars) in refined sugars on suppressing the oxygen maximum in devising an empirical method for determining these substances (202). The determination has to be made under uniform conditions, as the effect is accentuated very easily by external conditions such as heat (203). Directly, the polarographic method has been used to determine fructose in various fruits without correcting for the glucose present (212). The aldehyde group in the streptose unit of streptomycin must be responsible for the two reduction waves observed with streptomycin. This reduction makes the polarographic method a suitable one to follow the alkaline decomposition of streptomycin and mannosidostreptomycin, as the products are not reducible. The rate dependence upon pH is similar to that found polarographically for the rearrangement of a-hydrosyisobu tyraldeh) de t o acetoin by alkali and is offered as evidence that the same general reaction is responsible for the rate-determining step in each example ( 2 3 ) . The polarographic behavior of the unsaturated aldehydes, citral, jasmine aldehyde, and citronellal, is reported in various media (69). Values are also given for benzaldehyde, p-dimethylaminobenzaldehyde, and cinnamaldehyde (114). Addition of ammonia is found to shift the benzaldehyde wave t o more positive values by 0.210 volt (217). The polarographic method has been used to determine benzaldehyde, anisaldehyde, cuminaldehyde, cinnamaldehyde, and citral in essential oils ( I S ) , and cuminaldehyde in cumin seed
(164). Ketones. The simple' aliphatic ketones, acetone and cyclohexanone, are reduced in a 0.05 N tetraethylammonium iodide75y0 dioxane solution a t -2.46 and -2.45 volts (S.C.E.), respectively (180). S o wave is observed in 0.1 N lithium hydroxide solution, but if ammonia or glycine is present a t this pH a reduction wave occurs a t - 1.5 and - 1.54 volts, respectively, for acetone (217). The height of the wave is influenced by the concentration of both the acetone and the ammonia and reaches a maximum height of 20% of what would be expected for the same concentration of pyruvic acid. The current is diffusion-controlled and reaches its maximum value after 15 seconds. It has been suggested that acetoneimine is responsible for this early reduction (219). Waves at approximately the same point, -1.56 volts (S.C.E.), are obtained in a buffer of p H 8.2 for the Girard derivatives of acetone, methyl ethyl ketone, diethyl ketone, methyl n-decyl ketone, and di-n-octyl ketone. Cyclic ketone derivatives give values which vary between -1.50 and -1.63 volts, depending upon the size of the ring. The stability of the Girard derivative towards hydrolysis is influenced by the pH of the medium and is least for the cyclohexanone derivative (141). This instability explains the previous reported failure to reduce this compound and
34
ANALYTICAL CHEMISTRY methoxide upon colchicine ( X I I ) produces colchiciene (XIII) and colchicic acid (XIV) (160)
substituted cyclohexanone derivatives (214). The polarographic data point t o the following electrode reactions (147).
+ +H
>C=NXHCOCHrk( CH3)3 e
+
H
+
C H E O ~ ' ~ \ L S H C O C H ,SaOCH3
-+
)CKHNHCOCH2%(CH3),
)C--SHNHCOCH,?(CH,), + e + H * --+ >CHSHNHCOCH&( CH3)3
Ketones and aldehydes may likewise be determined indirectly by measuring their ability to decrease the wave of sulfur dioxide in a sodium bisulfite solution. This technique gives results which differ by about 2% from the values obtained by titrating the hydrogen chloride liberated in the oxime method (189). Polarographic data are given for methylionone and pseudomethylionone (69). The method has been used to determine carvone, pulegone, and diosphenol in essential oils ( I f ? ) , diosphenol in oleum bucco ( f 4 ) , C and carvone in drugs (15). In the steroid field the method has used the Girard derivatives t o determine 3-ketosteroids in buffers with higher pH to prevent hydrolysis (147) and in combination with digitonin precipitation to estimate amounts of a- and p-17-ketosteroids ( 2 5 ) . Steroids without reducible groups have been determined indirectly. Cholesterol has been estimated in both the free and bound form in blood serum by being precipitated with excess digitonin and determining the excess digitonin polarographically. This determination is dependent upon finding the amount of suppression of the cobalt maximum in an ammonia-ammonium chloride solution (196). Estradiol may be determined by converting it into estrone by means of a Streptomyces ( 8 8 ) or into a reducible nitroso compound. The latter reaction has also been used with estrone and estriol to give polarographically reducible compounds (78, 87). Diacetyl has been reinvestigated and found to give only one wave a t -0.67 volt in a tetramethylammonium bromide solution. The previously reported wave a t - 1.9 volts is apparently due to an impurity. Addition of ammonia gives a second wave a t -0.53 volt which increases a t the expense of the wave a t -0.67 volt (84). In the aromatic ketones series polarographic studies are reported for benzophenone, acetophenone, benzoylacetone (f 12), phenyl 2-naphthyl ketone ( 4 ) , fluorelione, anthrone, benzophenone (as), and p-aminoacetophenone ( 2 ) . The last compound, which reduces a t - 1.2 volts, gave the best yield of pinacol when large scale controlled electrolysis was carried out a t - 1.5 volts. Compounds with more than one ketone group such as benzil (67') and ninhydrin (206) can be determined polarographically. The latter, owing to the occurrence of hydration and adsorption a t the electrode, gives five waves in phosphate buffers and three waves in Britton buffers. It is best determined a t a pH of 8.3 in buffers containing boric acid or borates. I n the aromatic unsaturated ketone series the effect of the substitution of hydroxyl groups in chslcones ( X ) upon the amount of bimolecular reduction and normal reduction a t the dropping mercury electrode has been studied for 16 chalcones (X) (68).
X
0 SI
The method has been used to follow the formation of chalcones and to distinguish between these compounds and the related flavanones ( X I ) (169), to show that the action of sodium
/I
XII(R=CHs) XIII(Fb=H)
I
\/ COOH XIV
and that Brilliant Green (XV) and 2-chloro-5-hydroxy-4', 4"-bisdimethylaminotriphenylmethyl bisulfate (XVI) have similar structures (I%?).
CI & C = D = G (
h-
XF'
ClH5)2
CH3)2
SVI
The polarographic method has been useful in determining ketones and aldehydes in a study of their relative strengths as oxidizing agents. Values are given for the reduction of 43 of these compounds ( 1 ) . Acids and Their Derivatives. Simple aliphatic acids give hydrogen waves a t the dropping mercury electrode with halfwave potentials that become more negative with an increase in concentration of the acid ( 1 1 6 ) ; the ethyl esters give no wave8 (180). Polybasic acids, aromatic carboxylic acids, and sulfonic acids likewise give hydrogen waves, as do some of the substituted sulfanilamides having a first acid dissociation constant above Compounds having values below this, such as sulfidine, sulfanilamide, and boric acid, do not give a wave (116). This method can be used quantitatively (115). Derivatives of benzoic acid differ from those of aliphatic acids and give reduction waves (180). In the hydroxy acid series ascorbic acid or vitamin C gives an anodic wave which can be used t o determine this compound in plants (85) using metaphosphoric acid, citric acid, or potassium citrate as an extracting medium (140). The oxidation product if prepared using air is not reducible unless ammonia is present (217 ) . Polymeric lactic acid may be determined quantitatively by using its effect upon the oxygen maximum ( 9 9 ) . Glucuronic acid, which is an aldehydohydroxy acid, is not reduced polarographically but upon standing gives reducible decomposition products ( 9 7 ) . Pyruvic acid, the simplest keto acid, in the presence of ammonia gives two waves, the first of which is dependent upon the concentration of the ammonia and is more positive than the second wave. A similar behavior is observed with glycine, alanine, cotarnine ( S l y ) , histidine, and histamine (2f6). KO effect occurs with dimethylamine, trimethylamine, tetramethylammonium chloride or hydroxide, di- and triethanolamine, and diphenylamine ( 2 1 7 ) . A similar production of two waves is observed with sodium fluoride but not with the other halides (121).
V O L U M E 2 4 , N O . 1, J A N U A R Y 1 9 5 2 The method may be used to detect pyruvic acid in a fermentation mixture (167). Acetoacetic ester is reported not to be reducible a t the dropping mercury electrode (21). In the unsaturated acid series extensive work with fumaric and maleic acids and their ethyl esters in various buffers indicates that the reduction of the acids requires a buffering capacity of 0.9 M t o obtain a regular behavior (60). Equations have been derived t o account for the effect of ionic recombination on the polarogrsphic limiting current caused by the reduction of the undissociated molecules or anions belonging to dibasic acids and have been verified by the experimental data obtained with maleic, fumaric, and citraconic acids (82). The polarographic technique has been used to demonstrate the existence of unsaturation in 3,5-diiodo-4-(4’-hydroxyphenoxy)cinnamic acid and 3,5-diiodo-4(4’-hydroxy-3’,5’-diiodophenoxy)cinnamic acid (208) and to show that 6,y-angelica lactone and a,P-angelica lactone form a reducible peroxide uith air. Only a,&angelica lactone is reducible a t the dropping mercury electrode (105). Organic Halogen Compounds. A wide variety of organic halogen compounds has been studied polarographically. From a study of alkyl halides, aryl halides, and polyhalogen and unsaturated halogen compounds the following observations have been made. 1. Iodides reduce more easily than bromides and these more easily than chlorides 2. The more halogens there are on a carbon atom the more positive is the potential and the greater is the number of waves c are more easily reduced than vinyl halides. 3. A U y l ~halides Controlled large scale electrolysis indicates that the unsaturation is not affected. 4. The longer the aliphatic chain the more difficult the reduction. 5 . The ease of reduction of a halogen attached t o an aromatic ring is similar to that of a halogen attached to a large alkyl gioup. 6. The half-wave potential IS independent of pH
In the majority of cases the waves represent reduction of the halide to the hydrocarbon.
RS
+ 2e + H + +
RH
+ S-
This mechanism has been confirmed by controlled electrolvsis on a large scale for methyl iodide, ethyl bromide, a-bromonaphthalene, allyl bromide, 1,2-diiodoethene,and diiodoacetylene. Vicinal dihalogen compounds are exceptions in that they give intermediates nhich form olefins. Thus, ethylene bromide gives 80% ethylene and 20% ethane and 2,3-dibromobutane gives butylene (180). Factors such as ionic strength, alcohol content, and the nature of the cation of the supporting electrolyte have been found to be important in the reduction of aromatic iodides (71 ). Substitution of a fritted-glass disk electrode for the dropping mercury electrode gives similar results with organic halogen compounds (137). Introduction of another functional group into the organic halide molecule changes the behavior only with respect to the effect of p H upon the half -wave potential. In the a-halogenated acid series, for example, the half-wave potential is independent of pH in regions where only one species of the acid is present. In the intermediate region the half-wave potential becomes more negative with an increase in p H (155). Values are reported for a-bromoacetic (57, 166), a-bromopropionic (155), a-bromobutyric ( 5 6 ) , dibromoacetic, tribromoacetic, iodoacetic (57), dichloroacetic, and trichloroacetic acids. Chloroacetic acid gives no reduction in the range +0.4 t o -2.00 volts ( 5 9 ) . The reduction of meso-a,a’-dibromosuccinic acid parallels the behavior of the simple 1,2-dibromo compounds and forms fumaric acid by a trans elimination (153). Results are also reported for bromoacetic acid (136) and chloroacetamide a t the fritted-glass disk electrode (137), and for the
35 iodobenzoic acids a t the dropping mercury electrode. The mechanism in the latter series has been checked for p-iodobenzoic acid by a large scale electrolysis a t controlled potentials ( 7 1 ) . A similar dependence of half-wave potential upon pH has been observed with the iodoanilines (71 ) and halogenated acetaldehydes (63, 106). The behavior of the latter class is complicated by hydration of the aldehyde group. Reduction of the halogens is likewise observed for the acetals of these compounds. The polarographic method has been used t o determine chloroform and carbon tetrachloride separately and in mixtures of the two (110), carbon tetrachloride in mixtures with tetrachloroethylene, and carbon tetrachloride and chloroform in mixtures with methylene chloride and methyl chloride (180). The isomers of hexachloroc) clohexane have been intensely studied polarographically because of the importance of the gamma isomer as an insecticide. The reduction wave obtained for the y- isomer and a- isomer involves six electrons and represents the formation of benzene. This mechanism has been verified on a large scale (168). The reduction can be limited to the ?-isomer by using either 1% potassium iodide ( 9 4 ) or 0.1 S ammonium chloride as the electrolyte (215) and will give good accuracy in a quantitative determination if the temperature is held constant (138) and a correction is made for the heptachlorocyclohexane, since this compound changes the base line (13’4). Octachlorocyclohexane if present will also give a Rave (139). Location of the -,-isomer Lyave can be facilitated b r adding zinc ions t o the electrolyte; the zinc wave ail1 come directly before the start of the wave for they- isomer (SO). The polarographic method has been used to show that a reaction occurs between the ?-isomer and cysteine (198),to follow the dehydrochlorination of the various isomers in kinetic studies (98, I % ) , and to determine the ?-isomer in commercial insecticides containing the other isomers, talcum powder, mercuric compounds (76), or DDT. The last compound gives a wave a t a more positive value which represents all the isomers of DDT (197). Better accuracy is reported for the polarographic determination of the y-isomer than for the infrared method (193). The isomers of hexachlorocyclohesane suppress the oxygen maximum in the folloxing order: y, p, 6, a , E ( 3 4 ) . Polarographically dichloroacetic acid and trichloroacetic acid can be determined in mixtures (68). I n the aromatic series polarographic investigations have mainly concerned themselves with the determination of thyroxine and related compounds. Out of 23 related substances only 3,5-diiodo4-(4’-methoxyphenoxy)benzoic acid and 3,5-diiodothyronine interfere with the determination of thyroxine. 3,5-Diiodotyrosine interferes only if its molar ratio is greater than 10 (19). This method has been used t o determine thyroxine in rlrdein (ground nut protein), thyroid powders, and casein ( I ? ‘ , $ ) , and to show that cleavage of the diphenyl ether structure of 5-(3’,5’diiodo-4‘-pmethouyphenoxybenzyl)hydantoin occurs in treatment with 57% hydriodic acid (18). Nitro Compounds. A variety of aliphatic nitro compounds have been studied polarographically. Values are reported for nitromethane, nitroethane, 1-nitropropane, and 2-nitropropane in aqueous buffers (181). The presence of hydroxyl groups does not change the mode of reduction of the nitro group in 3-nitro-2butanol, 2-nitro-1-butanol, l-nitr0-2-butano1, 2-methyl-l-nitro-2propanol, and its di- and trihydroxy derivatives but made it easier than reduction of the hydrocarbon. With all compounds a four-electron reduction to the hydroxylamine is involved (170). Conversion of 2-nitro-1-butanol into an ether or ester made it more easy to reduce polarographically. The formyl and acetyl derivatives’ behavior was complicated by the loss of formic and acetic acids, respectively, and formation of the 2-nitro-I-butene which hydrated to 2-nitro-1-butanol (171). This transformation of the acetate can be followed polarographically (172). Tetranitromethane gives polarographic waves in a variety of buffers. The wave a t pH 12 is suitable for concentration deter-
36 mination but overlaps with that of dinitrobenzene and trinitrobenzene if these are present. The tetranitromethane can be removed from such solutions by adding methanol and vacuum distilling a t 70’ c. (44). The method has been used to determine nitromethane in air using an evacuated Winchester to obtain a sample (813). I n the aromatic series more information has been found about the reduction of nitro groups. By oscillographic measurements nitrobenzene is found to reduce four times more quickly than nitromethane (90). The second wave has been found to be partially kinetically controlled and must involve in addition to the reduction of phenylhyldroxylamine to aniline the following rearrangement (182).
Previous results on o-nitrophenol could not be verified. The compound gives a four-electron reduction a t p H 6 and a six-electron reduction in more acid or alkaline region. The reason for this behavior is that the resulting hydroxylamine can form a quinoneimine which is reduced further to the aminophenol (182).
ANALYTICAL CHEMISTRY Sitroso R salt is reported to be reduced polarographically (74). Amino Acids and Their Derivatives. Amino acids and their derivatives do not give reduction waves unless some reducible group is present. They will give a hydrogen wave, however, if the dissociation constant is greater than IO-*. This wave has served as a basis of estimation in the aromatic amino acid series for anthranilic, sulfanilic, and naphthionic acids (115). Amino acids mag be determined also indirectly by forniing a metal complex and determining this substance polarographically. The copper complex has been used mcst frequently (IO@, but the nickel complex may be likewise suitable (145). This method has been used to determine amino acids separated by paper chromatography (129) and to determine the amount of ethylenediaminetetraacetic acid in the commercial product (66). The polarographic behavior of kynurenine resembles that of acetophenone, o-aminobenzaldehyde, and lobeline and favors structure ( S V I I ) for this compound (159).
~~COCH~~HCOOH
xs-I1
A similar process must be involved in the reduction of p-nitroaniline, as a six-electron wave is obtained a t all pH values studied (ITS).
Polarographic behaviors of pnitrobenzoic acid (62), o-, m-, pnitrobenzaldehydes (118), pnitrophenol (143), 4,4’-dinitrodiphenyl ether, 4-nitrodiphenyl ether, and 4-nitro-4’-sminodiphenyl ether (130) are reported. The addition of alcohol has been found to shift the half-wave potentials to more negative values. The reduction of nitrobenzene has also been studied a t copper, silver, and amalgam spherical electrodes in 0.1 S potassium chloride (184). The polarographic method has been used t o determine nitrobenzene in small amounts in aniline (194), m-dinitrobenzene in nitrobenzene (196) and blood serum and plasma (154), nitroxylene and dinitroxylenes in nitroxylene feeds and resulting xylidines ( r 9 ) , picric acid in the presence of phenol (156), chloromycetin in broths, concentrates, and solids (86),trinitrobenzene and dinitrobenzene in the presence of each other (43, 44), and parathion [p-N02C&OPS( OC2Hj)2] in dusts and wettable powders. In the last example p-nitrophenol does not interfere but the oxygen analog gives waves a t the same point (20). The method has also been used to demonstrate that the nitro group in 5-nitro-2-furfuraldehyde semicarbazone is more easily reduced than either the semicarbazone group, or the nitro group in nitrobenzene and 5-nitropyridine (199), and that the nitro group is chelated in 4-nitrobenzimidazole (150). Using a streaming mercury electrode and “derivative” curves it is possible to differentiate between o-, m-, and pnitrophenols in the presence of nitrobenzene (69). Nitroso Compounds. The nitrosobenzene-N-phenylhydroxylamine system is reversible a t the dropping mercury electrode. The reduction wave of nitrosobenzene involves two electrons and is followed in acid solution by a further reduction of the phenylhydroxylamine to aniline (1‘76). This second step has been verified using phenylhydroxylamine (18%’). Reduction of nitrosoamines is possible in acid solution and is the basis for the analysis of dimethylamine in the presence of methjlamine and trimethylamine. Nitromethane, which is formed as a by-product in the reaction of nitrous acid on methylamine, is removed by sodium hydrosulfite (61).
Certain sulfonamides such as sulfathiazole, sulfadiazine, 3-SUIfapyridine, and sulfacetamide give hydrogen waves in 0.1 N tetramethylammonium iodide solution (1I S ) . Sulfanilamide does not give a wave up to -2.3 volts but is surface active and lowers the oxygen maximum to varying degrees. A similar effect has been observed with p-aminobenzoic acid ($06)and sulfanilic acid (165). The tripeptide glutathione fglutamylcysteinylglycine) is reported to give three anodic waves in alkaline solutions, the third of which interferes with the determination of ascorbic acid. The concentration of the former in fruits is not high enough to affect the ascorbic acid determination (39). The polarographic method has demonstrated the presence of albumins or globulins and peptones in port Kine (3). Diazonium Salts. Benzenediazonium chloride yields two waves in buffered solutions containing tetramethylammonium chloride. The first wave is independent of pH and represents the following electrode reaction.
This mechanism has been verified by coulometric analysis and the isolation of phenylmercuric chloride and diphenylmercury in a large scale electrolysis a t controlled potential. The second n-ave, which is about four times as high, is pH-dependent and probably represents the following electrode reaction:
Coulometric analysis and large scale electrolysis, however, give similar results to those observed for the first wave. One explanation for this discrepancy is that the stirring in the latter removes the intermediate from the electrode surface and prevents further reduction (6). Substituted diazonium salts behave similarly, with the exception that if acid groups are present the first wave varies with pH (5,54). The polarographic reduction of diazonium salts is the basis for the amperometric titration of couplers in the dye industry (55) and of pharmaceutical compounds containing phenolic and amino groups (101), and has been used to follow diazotization rates and decomposition rates of diazonium salts. The cathode ray polarograph is especially useful for reactions of this type, for which the rates are very fast (178). Unsaturated Hydrocarbons. Aliphatic unsaturated hydro-
V O L U M E 2 4 , N O . 1, J A N U A R Y 1 9 5 2
37
carbons such as butadiene, allene, ethynylacetylene, vinylacetylene, and divinylacetvlene are reducible polarographically, while simple ethylenes and acetylenes are not (180). I n the aromat,icseries the effect' of substituents in t,he ring and o n the a- and p-carbon atoms of stilbene upon t,he ease of reduction has been studied (75). Stilbestrol under these circumstances is not reduced hut may be determined by its suppression of the oxygen mariniuin (11 ) . Styrene has been found to give a satisfactory curve in a 7594 alcohol solution coiitaining 0.2 Jr tetrabutylamnicniuni iodide (190) and can be detrmiined in crude benzene fractions with a maximum error (Jf 5.5% (191). X straight-line relationship has heen obtained between the halfu-ave potentials of aromatic hydrocarbons and the coefficient of the molecular orbital resonance integral in the expression for the energy of the 1oxvPst unoccupied cirbital of these compounds ( 1 2 ; ) and verifies t,he previously p ~ s t u l a t ~ emeclianism d for the reduction of these compounds ( 1 2 2 ) . Including the solvation energy of the ions in the c*:ilculntione does not change the relationship (125). The polarogr:tphic, nirth(irl has I m r i used to determine carotene in plants (91). Peroxides. Single polarographic n-aves have been obtained with dihenzoyl peroxide, cuniene hydroperoxide ( T i ' ) , and ascaridol (12) and are suitable for their determinations. lIet,hyl methacrylate peroxid?, YiIiyl acetate peroxide, and styrene peroxide give two w:ives, the aecond of which resembles that for twt-butyl peroxide, Electrolytic reduction o f the styrene peroxide gave phenylglycol and indi:rtedthat these peroxides are polymers of the mononier and oxygen with a hydroperoxide (HOO-) end group
6).
Sulfur Compounds. Dialliyl disulfides and diary1 disulfides are reduced at the dropping mercury electrode in a two-electron step.
+ 2FI- + 2e -+
R-S--S-R
2RSH
The method can be used t o determine these compounds alone or in the presence of sulfur (70). The presence of groups other than carbon does not seem to change the mode of reduction in cysteine sulfonate (111)
and bis-(diethylthiocarbamyl)disulfide ( 7 7 ) ; both give waves involving tlvo electrons. I n the second example an adsorption wave is otiserved.
(C,H,),SC-S-S-C-S(C,Hj)L
'I S
l~
s
Arsonic Acids. hrsonic acids give a wave in acid solutions of a height which points t o the following electrode reaction. ArAsOaHz
+ 6 H + + 6e +AriisH? + 3HlO
By making use of the reduction of the nitro group in m-nitrophenyl arsonic acid it is possible to use this compound in amperonietric titraticns of uranyl, thorium, zirconium, and stannic tin (109).
Organometallic Compounds. Phenylmercuric salts, CeH,HgX, give tTvo waves a t the dropping mercury electrode, the half-wave potentials of n-hich are independent of the anion (X) used. The first n-ave represents the following reaction: RHgS
+ +RHg + SB
vhile the second involves the follon-ing change: RHg
T
I€+
+e
=
RH
+ Ilg
Large scale electrolysis gave diphenyl mercury, a disproportionation product of diphenyl dinierrury. blerthiolate, p-chloromercuribenzoic acid, and Salygan hehave similarly (9). I n the aliphatic series the behavior is reported to be different; the anion as well as the radical affects the half-wave potentials observed (35). By using the second wave it is possible to determine hlerthiolate in antiseptics and vaccines but not, in whole blood or in the presenceof antimony compounds (141). The behavior of other organometallic compounds has been studied and is reported for dichlorodiethyltin (151). trialkyltin halides (38), trialkyllead bromides (36))and dialkylthallium bromides ( 3 7 ) . Tetraethyllead is not reducible polarographically but can be determined indirectly by decomposing the compound with nitric acid (10;) o r n-ith hydrochloric acid (81, 1%) and determining the lead. Oxygen Heterocyclic Compounds. Heart poisons having the y-lactone structure (XVILI) are reducible polarographically only in quaternary ammonium hydroxide o r halide solution. C'ompounds with the pyrone structure (XIX) reduce more easily by 0.5 volt and can be determined in ammonium chloride, potassium chloride, potassium hydroxide, or lithium chloride solution. The substances give m-aves of two elect,rons and can be determined in the presence of each other (131, 163). The method was suitable for determining these substances in squill and various drugs (163) and has been used to determine digitoxin in blood (93).
+ 2e +B(C,H,),S-C--SI1 s
The method has been used to study the reaction of cystine and dithioglycolic acid with sulfite (188).
Coumarin (XX) gives a single wave which is independent of pH and represents a one-electron reduction to the dihydrodicouniarin
(XXI). 2 o O j = "
2-Thiohydantoins give anodic curves a t the dropping mercury electrode but no catalytic hydrogen waves in ammoniacal cobalt solutions ( 3 2 ) . The latter phenomenon is shown by degradation products of penicillin and can be used to determine pure penicillin if this antibiotic is inactivated first with alkali. The results agree nithin 10yoof the biological assay (33). p-Aminophenylthiocganide and pdimethylaminophenylthiocyanide are reported to undergo a six-electron reduction a t the dropping mercury electrode. As the wave height decreases with a n increase in pH, the possibility of a catalytic wave cannot be discounted (167).
+ 2e + 2 H + +
(pEO "=(;o 'v
XX
XXI
This mechanism has been verified on a large scale. Analysis is best carried out a t p H 6.8, because beyond this value the lactone ring starts to open and form coumarinic acid with a corresponding decrease in diffusion current. Above p H 11.2, for example, very little coumarin is present. Substituted coumarins behave in a Eimilar fashion (26, 83). Flavones as a rule give two reduction waves which may coa-
ANALYTICAL CHEMISTRY
38 leece if hydroxyl groups or derivatives of these are present. Each wave involves one electron and represents reduction to the pinacol and alcohol, respectively (68). Flavanones give only one reduction wave involving two electrons. Groups when substituted in the 5- and 7-positions will affect the half-wave potentials (68). Polarographic data are given for the naturally occurring flavones, rutin, quercetin, and morin (U), and the furochromones, khellin, visnagin, and khellol glucoside. Differentiation qualitatively of khellin and visnagin is not possible (6). Quantitative estimation of khellin and visnagin is equivalent, however, in accuracy to the ultraviolet determination but is less convenient to carry out (53). The fluorescein dyes, Uranin, Eosin, Erythrosin, and Rose Bengal are reducible a t the dropping mercury electrode (&), but the closely related Mercurochrome is reported not to reduce (66). Nitrogen Heterocyclic Compounds. Pyrroles substituted with alkyl groups in the 1-,2-, and 3- positions are not reduced polarographically (16). Pyrrolesulfonic acids are likewise inert but are repoited to be oxidized a t the dropping mercury electrode. As these anodic waves are reported in 0.5 S potassium chloride and occur from 0.015 to 0.052 volt, the actual process occurring a t the electrode is questioned (200). The polarographic behavior of isatin is complicated because of the possibility of ring opening and subsequent formation of three reducible species (192). The method can be used, however, to determine isatin in acid or alkaline medium (119), and has been employed to study the kinetics of the formation of isatinic acid from isatin (120). Polarographic studies are also reported for dihydroxyindole (the reduction product of isatin), indigocarmine (119),and phthnlimide (117). In the pyridine series pyridinium salts related to one of the components of codehydrase have been studied polarographically. Values are reported for pyridinium methobromide, trigonelline, ieonicotinamide methobromide, 1-tetraacetylglucosido-3-carbamylpyridinium bromide, cozymase, nicotinamide methobromide (1 7 9 ) , and related pyridinium chlorides, bromides, and iodides (XXIII) in which the alkyl group v-as varied from methyl through butyl ( 3 1 ) .
quinoline-8-carboxylic acid (184) is the basis for the amperometric titration of copper. The former may also be used for the determination of zinc. Molecular orbital calculations for the introduction of a methyl group into a pyridine nucleus predict a shift of -0.030 volt. The experimentally obtained half-wave potentials for 2-methyl- and 2,6-dimethylquinolinium methiodides differ by -0.039 volt (166). The significance of this agreement is questioned, since the example cited illustrates the introduction of a methyl group into a benzenoid nucleus and not into a pyridine ring. A very careful polarographic study of acridine indicates that the complex behavior is due to adsorption of the acridinium ion, because the anomalies are worse in acid solution. The complex behavior disappears in solutions containing 50% or more of alcohol and is replaced by two one-electron reduction steps. Methylacridinium salts give similar adsorption phenomena in both acidic and basic medium which are not eliminated by alcohol (104). Substituted acridines related to Mepacrine (XXVII) (x = 3)
SX\TI 5
= 1-4
x
= 2-7
give behaviors similar to acridine but show no definite correlation between antimalarial activity and the reduction potential observed (80). Rfercaptobenzothiazole (XSVIII) gives a cathodic wave only in the pH range 2.5 to 7 and an anodic mave in the range 1 to 13. Large scale electrolysis a t +0.1 volt gave a white precipitate and very little mercuric ion in solution and suggested the following reaction for the anodic wave (166).
~ C O N H Z
SXIII The polarographic method indicates that inimosine has structure XXIV, as waves resembling those of pyridones XXV and XXVT are obtained ( 1 7 )
0
0I
I1
CH~CHCOO-
I
KH: SXIV
&H~
SXV
6HZCOOII
XSVI
and has been used to follow the hydrolysis of nicotinamide (100). I n the quinoline series polarographic values are reported for 8quinolinol (96) and cinchoninic acid (quinoline-4-carboxylic acid) (29).
The polarographic reduction of quinaldinic acid (283) and of
The polarographic behavior of pterins is similar to that for pteroylglutamic acid (vitamin B,) and is good evidence for the structureof the latter (28). The method is suitable for the quantitative estimation of vitamin B, (52). Tablet constituents do not interfere but ferrous sulfate, if present, will give high results (1%). Barbituric acid is not reducihle polarographically in 0.2 M tetraethylammonium bromide (62). The polarographic method has been used to determine berberine, cotarnine, and hydrastinine in pharmaceutical preparations (158)) colchicine in seed extracts o r tinctures (161), and strychnine in viscera (201). LITERATURE CITED
(1) Adkins, H., Elofson, R. >I., Rossow, 4.C., and Robinson, C. C.. J . Am. Chem. Soc., 71,3622 (1949). ( 2 ) Allen, hl. J., and Corwin, A. H., Ibid.,72, 114 (1950). (3) Almeida, H. de, Anais inst. t i n h o Porto, KO.9,21 (1948). (4) Ansidei, R. M., and Scaramelli, G., Boll. sci facolta chim. ind. U n h . B O ~ O Q5~, C 1 (1944-47). L, (5) rltkinson, E. R., Warren, H. H., Abell, P. I., and Wing, R. E., J. Am. Chem. SOC.. 72. 915 (1950). (6) Bailey, S. D , Geary, P.'.4 , and Wald, A. E. de, J . Am. Pkarm. Assoc.', 40,280 (1951). (7) Baret, C , and Leveque, P., Bull. SOC. chim. France, 1949,832.
V O L U M E 2 4 , NO. 1, J A N U A R Y 1 9 5 2 Barnes, C. E., Elofson, R. AI., and Jones, G. D., J . Am. Chem. Soc., 72, 210 (1950). Benesch, R., and Benesch, R. E . , Ibid., 73, 3391 (1951). Bent, R. L., Bessloch, J. C., Duennebier, F. C., Fassett, D . W., Glass, D. B., James, T. H., Julian, D. E., Ruby, IT. R., Snell, J. XI., Sterner, J. H . , Thirtle, J. R., Vittum, P. IT., and Weissberger, il., Ibid., 73, 3100 (1951). Biegenheimer, L. E., J r . , and Christian, J. E., J . A m . Pharm. Assoc., 38, 117 (1949). Bitter, B., Collection Czechoslou. Chem. Communs., 15, 677 (1950). Bitter, B., Hanc, O., arid Santavy, F., Chem. Listy, 43, 137 (1949). Bitter. B., and Hobsa, J . ,I b i d . , 43, 208 (1949). Bitter, B., and Manis, J.,Ibid., 43, 206 (1949). Bonino, G . B.. Pontif. Acad. Sei. Acta, 9, 65 (1945). Bonting, S. L., Jr.. and Schepman, F. R., Rec. traz;. chim., 69, 1007 (1950). Borrows, E. T . , Clayton, .J. C., and Hems, B. 9., J . C'hem. Soc., 1949, Sl99. Borrows. E. T., Hems. R . A,, and Page, J. E.. Ibid.. 1949, S204. Bowen. c'. I-.,and Edwards, F. I., Jr., .\X.LL. CHEY.,22, 706 (1950). Brdicka. I
