ANALYST SEPTEMBER 1986 VOL. 111 1001 Analytical Applications of the Catalysed Iodine - Azide Reaction A Review G. Ramis Ramos M. C. Garcia Alvarez-Coque and R. M. Villanueva Camaiias Departamento de Quimica Analitica Facultad de Quimica Universidad de Valencia Valencia Spain Summary of Contents Introduction Mechanism of reaction and catalytic activity Induction coefficient and sensitivity Qua I it at ive a na I ysis Quantitative analysis Fixed time methods Fixed signal methods Initial slope methods Open system methods Other catalytic methods Differential kinetic methods Masking and derivatisation Separation methods Selectivity Determination of metal ions Other applications References Keywords Review; iodine - azide reaction; kinetic methods Introduction In acidic or neutral media a solution of sodium azide and iodine remains practically unchanged for a long time but in the presence of divalent sulphur compounds the irreversible redox reaction 2N3- + I2 -+ 21- + 3N2 proceeds rapidly giving rise to bleaching of the solution and the evolution of nitrogen bubbles.This reaction was described by Raschig in 1904,’ and has since then been the object of numerous studies. In addition, many analytical procedures have been developed that allow the sensitive and selective identification and determination of the compounds that catalyse the reaction. In this paper the analytical applications of the iodine -azide reaction are reviewed and the diverse significant aspects involved are critically examined together with the methods that have been used to follow the reaction and the analytical characteristics of the procedures proposed.Mechanism of Reaction and Catalytic Activity The iodine - azide reaction is catalysed by free sulphide and diverse metal sulphides by thiocyanate thiosulphate and carbon disulphide and by thiols disulphides and thioketones, among other organic compounds that contain sulphide sulphur. In contrast the reaction is not catalysed by sulphite, sulphate and organic sulphoxy compounds.2 Crystalline or coagulated elemental sulphur does not produce the catalysis, but it does catalyse the reaction as a finely dispersed su~pension.3~4 Hydrogen selenide does not show any catalytic activity ,5 but some selenium compounds such as N-benzoylsele-noureas Ph-CO-NH-CSe-R where R = piperidino or morpholino catalyse the reaction their catalytic activity being lower than that exhibited by the analogous sulphur compounds.6 However the catalytic activity of compounds such as Na2Se(S203)2.3H20 and Na2Te(S203)2.2H20 seems to be due almost exclusively to the presence of thiosulphate ions.7 Certain charcoals and carbon blacks also show catalytic activity due to the presence of active C02 complexes in their composition .g The mechanism of the reaction has been studied by several authors.9-17 Dahl and Pardue,l6 studying diverse disulphides and mercaptans established a reaction pathway that explains the behaviour of many catalysts. In the mechanism suggested, the reaction begins with the attack of the 12N3- complex by a disulphide and consists of six reaction steps: 12N3- + RSSR F== RSI + RSN3 + I- .. . . (a) . . RSI + N3- e RSN3 + I- . . . . . . . . ( b ) RSN3 + N3- e 3N2 + RS- . . . . . . . . (c) RS- + I2N3- e RSI + I- + N3- . . . . . . (d) RSN3 + RS- e RSSR + N3- . . . . . . . . U, RSI + RS- e RSSR + I- . . . . . . ( e ) Steps (a) (e) and u> represent themselves a catalytic cycle, whereas steps (b) ( c ) and (d) are an inner cycle. The reaction pathway for sulphydryl compounds is the same but step (d) would be the starting point. In this instance an initial rapid period is observed due to the high concentration of the RS-species. The reaction slows down when this species is completely oxidised to disulphide as step (a) where the reactive intermediates are regenerated is the principal rate-determining step.16 Many other catalysts such as inor-ganic sulphides thioureas and dithiocarbamates exhibit a similar behaviour.lg20 After some time the catalysed reac-tion can stop completely owing to further oxidation of the sulphur to inactive species.The following order of decreasing catalytic activity has been o bserved:21 RSH > RR’C=S > R-S-S-R‘ > H which agrees with the proposed mechanism. The differences in activity are related to the difficulty of cleaving the differen 1002 ANALYST SEPTEMBER 1986 VOL. 111 bonds to form the RS- species. Thus the unexpected high catalytic activity exhibited by lipoic acid: may be explained by the strain existing on the five-membered ring. 16 A study performed with different dithio acids of the form RR'PS2H shows that the catalytic activity decreases when steric hindrance exists on the sulphur atom.22 The same effect is observed when the active sulphur can form intramolecular hydrogen bonds.16923 Thus for example cystamine which differs from cystine only by its lack of a carboxyl group on each extreme of the molecule exhibits a catalytic activity 2.05 times larger. 16 On the other hand electrophilic groups enhance the catalytic activity which may be attributed to the greater susceptibility of the disulphide bond to nucleophilic attack. l6 Thus dithiodiglycollic acid which differs from cystine in its lack of an amino group on each side of the disulphide bond and in the fact that carboxyl groups are on the a-carbons instead of on the @-carbons shows a catalytic activity 3.2 times larger than cystine.The increase in activity may be explained by the greater difficulty in forming intramolecular hydrogen bonds and by the closer proximity of the electrophilic carboxyl groups to the disulphide bond.16 Miiller et al.23 established correlations between the struc-ture of several N-benzoylthiourea derivatives (RR'N-CS-NH-CO-Ph) and their catalytic activity. Disubstituted com-pounds are generally ten times as active as monosubstituted compounds and among the latter compounds with electron-withdrawing groups are more active than those with electron-donating groups. Induction Coefficient and Sensitivity Because of the oxidation side reactions of the catalysts it is convenient to distinguish between catalytic activity and the induction coefficient.The catalytic activity is a value propor-tional to the rate constant of the catalysed reaction and to its initial rate whereas the induction coefficient24 or reactivity number25 has been defined as moles of iodine consumed per mole of active sulphur initially present. Therefore it depends not only on the activity of the catalyst but also on its resistance to oxidation and on reaction time. Relative catalytic activities are obtained from the measure-ments of initial reaction rates whereas the induction coeffi-cient is calculated from the extent of the catalysed reaction after a certain time period.26 Not always enough attention has been paid to this distinction ambiguous expressions such as effectiveness catalytic effect or reactivity being used.21927Jg The induction coefficient of a substance is a measure of the sensitivity that can be reached in its determination.24.29 Its value depends on the relative rates of the different competitive reactions involved and therefore it changes considerably with the experimental conditions.The induction coefficient usually increases with increasing azide and iodine concentra-tions19.30; however it can decrease when the concentration of iodine exceeds a certain value as observed for sulphide,31,32 and dithiocarbarnates.19727 An increasing iodide concentration usually causes an increase of the induction coefficient as observed for cysteine30 and free sulphide ,31-33 although for metal sulphides20 and thiocyanate ,34 a diminuition of the coefficient is produced above a certain iodide concentration.Optimum conditions are not always utilised. Thus for example moderate concentrations of azide and iodide much lower than the optimum are often used in order to achieve cheaper determinations.19,2OV*4 The induction coefficient usually decreases rapidly above pH 8 owing to the dismutation of iodine. It is inconvenient to work below pH 5 owing to the volatility and toxicity of hydrazoic acid (log KH = 4.7 at 25 "C boiling-point 37 "C).35 In the pH range 5-8 the reaction may be independent of pH as occurs with ZnS20 or it may show a more or less marked dependence as observed for substituted thioureas.28J6 Some compounds show a maximum and a minimum near pH 5-6 and 7-8 respe~tively.16~3~ Most procedures recommend a pH in the range 5.5-6.5 where the azide - hydrazoic system shows some buffer capacity.The induction coefficients may change considerably with the order of addition and even with the rate of mixing of the reagents.l9Jg This could be due to different local iodine concentrations during the process. Thus for cysteine a maximum value is obtained when the order of mixing is I-, N3- HC1 cysteine and 12 being only half as much if cysteine is added last.39 Dependences on the dielectric constant, visc0sity2~ and total saline content40 have also been reported. The induction coefficients of a variety of substances in different experimental conditions have been established.41.42 Most of the catalysts have values below 600 but in some instances they are much higher.Thus thioammeline (4,6-diamino-l,3,5-triazine-2-thiol) shows a value of 4400 in a 2% azide solution.40 Qualitative Analysis The identification of diverse organic and inorganic sulphur compounds making use of the catalysis of the iodine - azide reaction was suggested by Feigl and Anger.43.44 The test may be performed either on filter-paper impregnated with the iodine - azide reagent or on a watch-glass where the formation of nitrogen bubbles is also observed together with the loss of iodine colour. The limits of detection are in the range 0.02-0.5 pg for sulphide thiosulphate and carbon disulphide being about 2 pg for thiocyanate.2.35 Extremely low values have been obtained for some organic compounds such as thioacetic acid (0.3 ng) rhodanine (3 ng) and thiourea (5 ng).M Some qualitative uses are the detection of mercaptans in bacterial cultures45 and sulphur compounds in urine which is used for the diagnosis of intoxication by dithiocarbamate fungicides46 and by accelerators of rubber polymeri~ation.~7 A method has been proposed to differentiate between animal and vegetable fibres ( e .g . wool and cotton). After melting with sodium or potassium only animal fibres give positive results.48 Finally an iodine - azide solution is employed to develop sulphur compounds in column adsorption49 and paper chromatography.50 Quantitative Analysis The reaction has been widely used to determine small amounts of sulphur compounds and has also been applied to the determination of metal ions that form stable complexes with sulphur-containing ligands.The diverse procedures proposed for the determination of non-metallic inorganic and organic compounds are examined in this section and their detection limits and other characteristics are shown in Table 1. The analytical applications of the reaction are determined by the oxidation side reactions of the catalysts. In only a few instances under restricted conditions and during short periods may it be considered that the concentration of the catalyst is constant and that the reaction proceeds according to definite simple kinetics. For this reason most of the pro-cedures described have been developed on a purely empirical basis without establishing the corresponding kinetic -equa-tions.In most of these procedures an excess of iodine is rapidly added to the stirred mixture of azide and catalyst, some control over addition and stirring rates24 and tempera-ture being necessary. Many analytical techniques based on the measurement of unreacted iodine evolved nitrogen or heat produced have been used to monitor or establish the advance of the reaction ANALYST SEPTEMBER 1986 VOL. 111 1003 Table 1. Determination of non-metallic inorganic and organic compounds Substance determined Method Characteristics of the procedures samples and references s2- . . . . . . . . . . Titration with arsenite after a After 15 min of reaction determination of 0.03-3 p.p.m. and 3-300 fixed time. Visual end-point detection in the presence of starch p.p.b.using 0.02 ~10.01 M and 5 m ~ / 2 . 5 mM iodine - azide solutions respectively; CV* < 6%. Previous separation as H,S by Titration with arsenite after a fixed time with amperometric end-point detection Gasometry Spectrophotometric determina-tion of iodine in the presence of starch after a fixed time Turbidimetry of nitrogen and enthalpimetry Appearance of fluorescence up to Direct injection enthalpimetry a fixed value Potentiometric sensor with two platinum microelectrodes Coulometric generation of iodine Addition of iodine in the presence of ascorbic acid. Biampero-metric end-point detection FIA method with biamperometric determination of iodine Elemental sulphur . . . . Titration with arsenite after a fixed time. Visual end-point detection in the presence of starch As above As above with amperometric end-point detection Effects derived from nitrogen bubble formation (see text) Turbidimetry of nitrogen and enthalpimetry Appearance of fluorescence up to a fixed value Coulometric generation of iodine FIA method with biamperometric determination of iodine s4062- .. . . . . . Gasometry SCN- . . . . . . . . . . Titration with arsenite after a fixed time. Visual end-point detection in the presence of starch point detection As above with amperometric end-Gasometry Spectrophotometric determina-tion of iodine at 350 nm after fixed time Appearance of fluorescence up to a fixed value Coulometric generation of iodine. Biamperometric end-point detection Amperometric determination of iodine Turbidimetry of nitrogen and en t halpime t ry sweeping with nitrogen; volatile mercaptans interfere.51 Similar -procedures applied to industrial waste waters sewage52 and steel31 with LODt 0.5 p.p.b.; air (LOD 1.5 pg)53-54 and bile55 Determination of 0.2-200 p.p.m.56 Determination of 1-200 pg.Water and water - organic solvent After 10 min of reaction. Applied to Ti and Zr.58 Similar procedure mi~tures38.5~ for steel32 Determination of 300-3OOO p.p.m. in 100 p1 and 0.1-100 p.p.m. if H2S is recovered from a nitrogen stream. Applied to the measurement of bacterial activity of E. coli on cysteine59 indicator60 CV < 3%. Previous separation as H2S by sweeping with nitrogen; applied to commercial copper with a CV < 5%33 Determination of 8-1000 ng CV 10-2.7% LOD 5 ng.For H2S in a gas stream; considerable amounts of SO2 HCHO CS2 and methylmercaptan can be tolerated61 Determination of 0.5-1 p.p.m. in 1 ml. Rhodamine B recommended as After 2 rnin of reaction determination of 1.2-32 p.p.m. in 0.5 ml, Determination of 4-40 p.p.b. in 25 ml CV2%62 Determination of 0.3-1.6 p.p.m. in 50 p1 CV 2.5% for 1.3 p.p.m., LOD 10 ng63 LOD 0.2 p.p.m. in 10 p164 Determination of 0.1-3.5 p.p.m. in 50 ml CV < 0.24%. Previous extraction in a miscible organic solvent. Applied to metallic suIphides,65,66 metals and alloys67 with CV < 4% ointments,4 rubber and polymers with CV < 6%6+70 Determination of 5-80 pg in 1-10 ml of extract CV < 4.9% in the range 6.6-31.8 pg. Previous extraction in DMF and oxidation to S2O32- with nitrite.Applied to carbonates71 Determination of 2 pg-0.3 g CV <2.5%. Thiocyanate is masked with Determination of 2-800 p.p.m.56 KI34,72 Determination of 4-40 p.p.m. in 12 m173 Determination of 320-3240 p.p.m. in 100 pl (batch method) and Determination of 0.5-3.5 p.p.m. in 1 ml. Rhodamine B recommended Determination of 20-280 p.p.b. in 25 ml CV < 2% ,62 112-1120p.p.m. (flow method)59 as indicator" LOD 0.1 p.p.m. in 10 p164 LOD 0.01 pg CV 2-3% .74 Similar procedure for mixtures with thiosulphate previous separation by paper chromatography75 After 30 s of reaction determination of 2.5 p.p.b.-70 p.p.m. CV 2.5 Yo 76 Determination of 2-3500 p. p. m .56 - 57,77,78 After 3 rnin of reaction determination of 5-80 p.p.b.79 Determination of 0.2-6 p.p.m.Rhodamine B recommended as Determination of 20-120 p.p.b. CV < 3%. In mixtures with thiouream indicator" Determination of 10-100 ng81 Determination of 0.7-7 g 1- in 100 pI5 1004 Table l-continued Substance determined (CH2SCN)* and PhCHzSCN . I 3-Butenyl isothiocyanate cs2 . . I . . . . . Cysteine . . . . . . Glutathione . . . . . . Ergothioneine . . . . 2,6-Thio-4-pyrimidine . . 2-Mercaptobenzimidazole 6Mercaptopurine, 2-thiouracil and 2-mercaptopyrimidine 2- 6- and 8-mercaptopurine Thioammeline . . . . BismuthiolI . . . . . . . . . , . . . . . . . . . . . . . . . . Method Titration with arsenite after a fixed time. Visual end-point detection in the presence of starch As above As above As above Fixed signal method.Bleaching time is measured Titration with arsenite after a fixed time. Visual end-point detection in the presence of starch As above Gasometric measurement after a fixed time Spectrophotometric determi-nation of iodine in the presence of starch after a fixed time Turbidimetry of nitrogen and enthalpimetry FIA method with biamperometric determination of iodine Coulometric generation of iodine Addition of iodine in the presence of ascorbic acid. Potentiometric end-point detection Titration with arsenite after a fixed time. Visual end-point detectiun in the presence of starch FIA method with biamperometric Coulometric generation of iodine determination of iodine Titration with arsenite after a fixed time.Visual end-point detection in the presence of starch Addition of an iodine - azide solution at a constant rate Spectrophotometric determina-tion of iodine after a fixed time FIA method with biamperometric determination of iodine Titration with arsenite after a fixed time. Visual end-point detection in the presence of starch As above . Asabove ANALYST SEPTEMBER 1986 VOL. 111 Characteristics of the procedures samples and references Determination of 2-20 p.p.m. and 4-40p.p.m. in 1 ml respectively. Applied to contaminated air53.54 After 30 s of reaction. Microdetermination in a half rape seed, After 30 min of reaction determination of 20-120 p.p.m. in 5 ml. Determination of mixtures with ethyl xanthate in sewage83384 After 30 rnin of reaction determination of 0.05-1 pg CV 5 % .Industrial wastes.85 Similar procedure for air53,54 Bleaching times are in the range 6-100 min. Determination of previous synthesis of the corresponding thiourea with methylamine82 0.005-1.5% CS2 in 1 ml of organic solvent. Mercaptans and H2S interfere.86 Similar procedures for 7.5 X 10-4-4 X in 1 ml of benzene and for 0.1-2.25% in 1 ml of benzene or chl0roform5~ After 4 h of reaction determination of 0.4-2.4 p.p.m. (with 1 g of NaN at pH 6) and 0.1-0.8 p.p.m. (with 2.5 g of NaN at pH 5.2) in 50 ml CV < 5%. In mixtures with glutathione29 and erg0thioneine.2’3~~ In erythrocytes previous separation by gel chromatography5 After 20-30 s of reaction determination of 13 p.p.b.-2.4 p.p.m. in 75 ml CV 4%.Cystine and methionine up to 0.8 and 0.2 mg, respectively can be tolerated. Applied to milk beer and wheat.88 Similar procedure in ref. 30. After 10-60 rnin of reaction determination of 24-120p.p.m. in 2 ml, LOD 8.6 p.p.b. in 7 mP9 CV 8%15 Determination of 350-3500 p.p.m. in 100 $59 LOD 0.2 p.p.m. in 10 p1@ Determination of 2-16 p.p.b. in 25 ml CV <2%. In albuminw CV 4.6% for 28 ng of S as cysteine2, After 5 min of reaction determination of 2-12 p.p.m. (with 1 g of NaN at pH 6) and 1-5 p.p.m. (with 2.5 g of NaN3 at pH 5.2) in 50 ml CV < 5%. In mixtures with erg0thioneine,8~ and cysteine and ergothioneine.29 In erythrocytes previous separation by gel chromatography.5 Similar procedure after 15-20 s of reaction in 0.5-3 ml of lemon and orange juices91 LOD 0.2 p.p.m.in 10 ~1~ Determination of 40-240 p.p.m. in 25 ml CV < 2%w After 5 rnin of reaction determination of 0.02-8 p.p.m. in 50 ml, CV 3%. Cysteine and glutathione are blocked in 5 min with N-ethylmaleimide.29.87 In erythrocytes previous separation by gel chromatography5 Determination of 10-100 p.p.m. in 10 ml LOD 2 p.p.m.92 Determination of 50-200 p.p.b. CV 1.9-5.6%. In zinc electroplating baths. Zn2+ is masked with EDTAg3 LOD 0.1 p.p.m. in 10 ~ 1 6 4 Determination of 3-12,l-12 and 5-80 pg of the three compounds, with CV 2.5,2.0 and 2.0% respectively” Determination of 1CL150 p.p.b. in 50 ml CV2%. Other mercaptans are blocked with N-ethylmaleimide and Cuz+ is masked with oxalic acid95 Determination of 0.06-1 p.p.m. in 50 m ANALYST SEPTEMBER 1986 VOL.111 Table l-continued 1005 Substance determined Methionine . . . . . . . . As above Ethionine . . . . . . . . As above Sulphathiazole . . . . . . Asabove Penicillin G . . . . . . . . As above Thiamine . . . . . . . Promazine chloropromazine andpromethazine . . , Cystine Enerbol Lipoic acid Thiourea Method As above with biamperometric end-point detection As above visual end-point detection in the presence of starch As above Gasome try Spectrophotometric determi-nation of iodine in chloroform extracts at 525 nm after a fixed time. Also gasometry Fixed signal with potentiometric monitoring of iodine Initial slope method with potentiometric monitoring of iodine enthalpimetry Turbidimetry of nitrogen and Titration with arsenite after a fixed time.Visual end-point detection in the presence of starch As above As above Gasometry Addition of an iodine - azide solution at a constant rate Spectrophotometric monitoring of iodine in the presence of starch FIA method with biamperornetric determination of iodine Coulometric generation of iodine. Biamperometric end-point detection Turbidimetry of nitrogen and enthalpimetry Effects derived from nitrogen bubbles formation (see text) Characteristics of the procedures samples and references After 30 min of reaction determination of 7-230 p.p.m. in 50 ml, CV 3%. Cystine up to 20 p.p.m. can be tolerated97398 Determination of 10-700 p.p.rn. in 50 rnl CV 1-3%oY9 After 2 h of reaction determination of 0.3-10 p.p.m.in 50 ml, CV ~ 2 % . Drugslm Determination of 0.2-6 p.p.m. of S in 5 ml equivalent to 10-350 pg of potassium salt or 15-500 pg of procaine salt CV 5% for 1 p.p.m. S. The compound is previously hydrolysed in basic medium. Streptomycin and aureomycin do not interfere1o1 After 10 min of reaction determination of 0.15-2.2 g 1-1 in 10 ml, CV 2% for 1 g 1-1. In drugs ascorbic acid is titrated separately in absence of a ~ i d e 3 ~ After 1 h of reaction. Drugs102 After 30 min of reaction determination of 1-20 p.p.m. in 50 ml, CV 3%. Insulin hair wool and beer.103 Similar procedure applied to beer; cysteine up to 1.6 p.p.m- can be determined in the same sample; methionine up to 2.7 p.p.rn. is tolerated.88 Similarly after 2 h of reaction 0.05-5 p.p.m.(CV 2-3%0) can be determined in 100 ml The procedure is applied to insulin wool and hair104 LOD 1 ng c v 2 . 5 % 0 ~ ~ After 30-60 min determination of 36-144 p.p.m. in 0.5 ml confidence limits f 6 p.p.m. ; determination of 0.2 k 0.04 g cystine in samples with 1 g of protein nitrogen (99% confidence level). Applied to human serum protein hydrolysates14 Measured times are in the range 20-200 s; determination of 0.25-2 p.p.m. and5-25 p.p.m. in 2 ml CV 1 and 2% respectively. Alanine, leucine hystidine and glycine up to 500 p.p.m. and Ca2+ Zn*+, Co2+ and Cd2+ up to Measurements taken in less than 30 s determination of 0.25-2 p.p.m. and 2-25 p.p.m. in 1 ml CV 1 and 2%0 respectively106 M do not interfere105 Determination of 1.5-15 g I-' in 100 p159 CV 1 % in drugs107 Determination of 10-700 pg CV 1.5%.lo8 Similar procedure applied to urine4 Determination of 10-30 p.p.m. in 2 ml. Acid copper electroplating Determination of 2-200 pg in water and water - organic solvent Determination of 7-80 p.p.m. in 10 ml LOD 2 p.p.m. ,92CV5%110 baths.109 Similar procedure for 5-150 pg in citric fruits4 mixtures38 Determination of 10-100 p.p.b.111 LOD 0.2 p.p.m. in 10 p P CV < 3% for 16 p.p.b. In mixtures with thiocyanateso Determination of 0.38-3.8 g 1-1 in 100 pP9 Determination of 1-10 p.p.m. in 12 m17 1006 Table l-continued Substance determined Thiourea and substituted thioureas . . . . . . ANALYST SEPTEMBER 1986 VOL. 111 Method Characteristics of the procedures samples and references Titration with arsenite after a fixed time.Visual end-point detection in the presence of starch Spec tropho tometric de termina-tion of iodine in the presence of starch after a fixed time Direct injection enthalpimetry Thioureas substituted thioureas and tetrarnethylthiuram sulphide Biamperostatic method 2-Thiobarbituric acid . . 2-Thiobarbituric acid and derivatives . . . . Rubeanic acid and derivatives . . . . Dithiocarbamates . . . . Ethyl xanthate Merthiolate . , h i d e . . . . . . . . . . . . . . . . . . . . * Coefficient of variation. t Limit of detection. After 30 s of reaction determination of 2-300 pg of S . 1 l 2 Mixture of thioureas previous separation by paper chromatography5O LOD 3 p.p.b. thiourea and 7 p.p.b. phenylthiourea in 7 m P After 30 s of reaction determination of 0.05-1 pmol in 100 p1, CV < 3% .18 Similar procedure for 50-500 pmol in 5 pl LOD 5 pmol CV < 2%; applied to the determination of mixtures of thioureas in the presence of mercaptans isothiocyanates, thiosulphate and sulphide after separation by TLC CV 6% referred to the sample36 Determination of 2.6-26 nmol thiourea and phenylthiourea 69-624 nmol benzoylthiourea and 0.6-6.1 nmol tetramethylthiuram sulphide in 5 ml CV 2.5% for 13 nmol thiourea25 FIA method with biamperometric LOD 0.1 p.p.m.in 10 1.1164 determination of iodine Titration with arsenite after a fixed time. Visual end-point detection in the presence of starch Determination of 0.02-7 p.p.m. in 100 ml previous separation by paper chromatography113 As above LODO.l-0.2p.p.m.in50m1 CV3-6Y0114 As above After 2 min of reaction determination of 0.1-3.2 p.p.m. sodium diethyldithiocarbamate in 50 ml CV < 3% ; Fe3+ and AP+ can be masked with fluoride.24 Similar procedures for other derivatives,1*5J16 and mixtures of N-monoalkyl derivatives, previous separation by high-voltage electrophoresis1~7 in water and water - organic solvent mixtures38 CV 2% at a 30 p~ levelly Gasome try Direct injection enthalpimetry Determination of 0.5-100 pg sodium tetramethylenedithiocarbamate After 10 s of reaction determination in the 5-65 p~ range in 5 ml, Titration with arsenite after a fixed time. Visual end-point detection in the presence of starch After 15 s of reaction determination of 20-55 pg of S.In mixtures with CS2 in sewage83 Spectrophotometric determina- LOD 1 p.p.m. in 7 mlgy tion of iodine in the presence of starch after a fixed time Back titration with thiosulphate Determination of 0.1-700 mg of N3-.118A 15-20 min reaction time after total consumption of azide in the presence of thiocyanate is recommended119 Fixed Time Methods The selection of an optimum reaction time implies a compro-mise between the current rate and extent of the reaction. When the rate slows down excessively a longer time of reaction does not yield any valuable gain in sensitivity. The most suitable reaction time can be easily established when a signal - time graph is recorded. The extent of the reaction is obtained either by titrimetry of the remaining iodine with sodium arsenite3.56,65,71,98 o r hydrazine sulphate,51 or by a spectrophotornetri~,79~93J~~J~~ enthalpimetri~19933~36 or gasometric method.57.77.A flow injection analysis method (FIA) has been described, in which 10-pl samples are injected into an iodine and azide solution stream. Measurement of the concentration of iodine is carried out biamperometrically .64 In enthalpimetric methods the temperature rise produced by the reaction is proportional t o the amount of iodine and azide consumed and therefore t o the amount of catalyst. These methods are very sensitive as the iodine - azide reaction is highly exothermic. Methods based on the measurement of released nitrogen are less sensitive although together with enthalpimetric methods they offer the advantage over other instrumental methods of not being affected by turbidity or precipitates in the sample ANALYST SEPTEMBER 1986 VOL.111 1007 Fixed Signal Methods Pardue and Shepherd105 proposed a fixed signal method for the determination of cystine. A potentiometric concentration cell sensitive to iodine is used and the time required for the cell voltage to reach a given value is measured. The concentrations of azide and iodide are large compared to that of iodine and remain essentially constant. Under these conditions and at cystine concentrations below 2 p.p.m. the rate of the reaction is proportional to the instantaneous concentrations of cystine C and iodine: where kl is a rate constant. When these pseudo-first-order kinetics are obeyed the time interval At required for the voltage interval AE to be overcome is inversely proportional to cystine concentration which may be expressed by AE 1 kkl At .. . . . . ' * (2) c=- X-where k is a temperature-dependent constant from the Nernst equation. However above 2 p.p.m. of cystine the reaction order n with respect to cystine increases gradually leading to positive errors. Equation (1) should be rewritten in the form -- d[121r - kl C" [I2It . . . . . . . . dt where n b 1. Equation (2) then becomes (3) (4) which states that C is proportional to the nth root of the reciprocal of the time interval. Under the conditions used and for the range 5-25 p.p.m. when the average value used is n = 1.24 the errors are within 2%. A method has been proposed for the determination of CS2 based on the measurement of the time needed for iodine to disappear which may be carried out by visual observation.86 Rhodamine B has been used as a fluorescent indicator in the determination of sulphide thiosulphate and thiocyanate.The fluorescence of the indicator quenched by iodine appears gradually as the reaction proceeds. The reciprocal of the time required for the fluorescence to reach a certain intensity is proportional to the catalyst concentration .60 Initial Slope Methods Pardue106 proposed an initial slope method to determine cystine. The initial rate of the reaction is measured by the decrease in iodine concentration which is monitored poten-tiometrically. A linear response with a slope proportional to the cystine concentration is obtained.The relationship is given by The method suffers from the same drawbacks of the corre-sponding fixed signal method,lOs leading to high values above 2 p.p.m. of cystine. Open System Methods Some procedures have been described in which an iodine -azide solution is added at a constant rate to the sample solution. When the catalyst has been completely destroyed an increase in the iodine concentration is observed. The volume of reagent added is proportional to the initial concentration of the catalyst.92.110,122.123 The iodine concentration must be kept low and constant in order to achieve reproducible results. Therefore the addition and stirring rates must be carefully controlled. A precise and convenient control of the concentration of iodine may be attained by using competitive reactions, coulometric iodine generation or stat methods.Muller et al.23 proposed the addition of iodine in the presence of a given amount of ascorbic acid which competes with the catalysed reaction giving rise to a larger consumption of iodine. The time required for iodine to appear in the solution is propor-tional to the initial concentration of the catalyst. A similar method for microsamples has been described.63 Jedrzejewski and Ciesielski62~80~90 used the anodic gener-ation of iodine. The end-point was detected biamperometric-ally. In the biamperostatic method proposed by Pantel,25 the iodine concentration is measured continuously and a biamper-ometric signal is used to control the rate of addition from an automatic burette.In this way the iodine concentration is kept constant at a value below 0.2 mM. The determinations are accomplished in a short time. Other Catalytic Met hods Weisz and Meiners73 described an unconventional method, where drops of a chloroformic iodine solution are introduced with a capillary into a long and narrow vertical glass tube containing the mixture of azide and catalyst. The nitrogen produced in the catalytic reaction is retained on the falling drops and their downward movement gradually stops. The depth of fall and the time needed for the drops to return to the upper end of the tube are non-linearly related to the concentration of the catalyst. In the turbidimetric method described by Weisz et al. ,59 the measurements are carried out in a 3 + 1 glycerol - water medium which regularises the formation of nitrogen bubbles.During a given time and owing to light scattering the absorbance increases and then decreases giving rise to a maximum. The height of the maximum and the time required to reach it are both a measure of the concentration of the catalyst although the relationships are not linear. The ratio of the two parameters gives more precise results than either used separately. The authors employed this method and direct injection enthalpimetry simultaneously to determine several catalysts in batch and flow-through systems (double indi-cation). Enthalpimetric detection provides a very precise indication of the zero time point. A sensor for determining hydrogen sulphide in a gas stream has been designed.61 The end of the sensor is a sintered-glass ball which exudes an iodine - azide solution at a constant flow-rate.Hydrogen sulphide transported by the carrier gas catalyses the reaction giving rise to a potential difference measured by two platinum electrodes one inside and the other outside the ball. Selectivity The selectivity of most of the procedures is given by the selectivity of the iodine - azide reaction itself. Iodine and iodide oxidants and iodine reductants interfere. However, these interferences may usually be overcome by means of blank determinations in the absence of azide.37.52 Inter-ferences due to cations that precipitate or complex iodide or the catalyst may be avoided in some instances with masking agents.24352.95 The amount of iodine consumed by the oxi-dation of the catalyst is always negligible.Procedures allowing the selective determination of different catalysts found together in the sample make use of differen-tial kinetic methods of analysis other selective reactions to block or destroy certain catalysts and previous separation 1008 ANALYST SEPTEMBER 1986 VOL. 111 Differential Kinetic Methods Differential kinetic methods can only be applied in some favourable instances when the behaviour of the catalysts is sufficiently different. A procedure for the simultaneous determination of xanthates and CS2 in sewage based on the difference in their induction coefficients has been described. The extent of the reaction 15 s and 3 min after the introduc-tion of iodine is measured in parallel experiments.The amount of reacted iodine is proportional to the initial concentration of xanthates and to the sum of xanthates and CS2 respectively.83 Mixtures of cysteine and cystine can be resolved by a similar procedure. Cysteine is determined 20-30 s after the beginning of the reaction whereas the sum of both compounds is obtained after 30 min. The procedure was applied to their determination in milk beer and wheat.88 Jedrzejewski and Ciesielskiso applied their coulometric titration method to the selective determination of mixtures of thiourea and thiocyanate. The method differs from those cited above because the concentration of the generated iodine is controlled instead of the induction time. Thiourea catalyses the reaction at a lower iodine concentration and at a greater rate than thiocyanate.Finally the biamperometric sensor of Kiba and Furusawa61 allows the determination of hydrogen sulphide in the presence of substantial amounts of carbon disulphide and methylmer-captan owing to their different catalytic activities and the transient nature of the signal. Carbon disulphide and methyl-mercaptan react more slowly so that a 100-fold amount with respect to hydrogen sulphide merely broadens the peaks, leaving their heights unaffected. Masking and Derivatisation The resolution of mixtures of diverse sulphydryl compounds has been performed making use of their reactions with a$-unsaturated carbonyl compounds such as N-ethylmale-imide and with aldehydes. Mercaptans are added to the N-ethylmaleimide double bond giving thioethers which have very low catalytic activities : O G O + R’SH - 0 xo I R N-Ethylmaleimide blocks cysteine and glutathione in less than 5 min whereas no reaction is observed with ergo-thioneine even after a few hours.Analytical procedures based on these differences have been described for determin-ing mixtures of the two former compounds with the latter.g7 Similarly thioammeline can be determined in the presence of other sulphydryl compounds.95 Aldehydes also block sulphydryl groups giving thioacetals: Mixtures of cysteine glutathione and ergothioneine have been resolved making use of the different rates of reaction with formaldehyde.29 The percentages of blocked compounds in a 0.3% formaldehyde solution at pH 8-9.5 and after 30 s reaction time are 92,5 and 27% respectively the reproducibi-lity of the blocking percentages being within 2%.To determine mixtures of cysteine (x) and glutathione (y) a first experiment is carried out after 4 h of reaction time giving rise to a result A proportional to the sum of both substances ( A = C + Cy). In a second experiment the sample reacts with formaldehyde during the 30 s before the introduction of azide. RCHO + 2 R ’ S H j RCH(SR’)2 + H20 The second result obtained B is related to the concentration of the compounds according to B = 0.O8Cx + O.95Cy The concentrations are found by solving the equation system. Similarly a series of three parallel experiments using both reagents formaldehyde and N-ethylmaleimide permits the resolution of mixtures of cysteine glutathione and ergo-thioneine .29 Sulphite although it does not catalyse the reaction reduces iodine and therefore interferes with the determination of sulphide.This interference may be avoided in a similar way by blocking sulphite with formaldehyde. 121 The selective determination of isothiocyanates in the presence of other catalysts of the iodine - azide reaction may be performed by treating the sample with an amine. The corresponding thiourea which is a much more active catalyst, is formed. The micro-determination of 3-butenyl isothio-cyanate in a half rape seed has been carried out in this way, after prior extraction of the compound with ethanol. An aliquot of the extract is treated with methylamine to give N-methyl-N’-(3-butenyl)thiourea the induction coefficient of which is much greater than the coefficients of the other sulphur-containing compounds present in the seed.A blank determination is also made with an aliquot of the extract not treated with me thylamine .82 Mixtures of thiocyanate with sulphide or thiosulphate have been resolved making use of the inhibitory action of iodide on the activity of thiocyanate.34 Separation Methods Among the compounds that exert a catalytic action only H2S, CS2 and lower alkyl mercaptans volatilise at room tempera-ture. The determination of sulphide can be performed by sweeping H2S with a nitrogen stream and absorbing it in dilute NaOH33>5* or in a zinc(I1) or cadmium(I1) solution.51 It is, however faster and simpler to absorb it directly in the iodine -azide solution.54 Interference from thiosulphate and thiocyanate in the identification of the sulphide ion is overcome by precipitation of sulphide with zinc or cadmium carbonate.2 Similarly, sulphide and thiosulphate are eliminated by precipitation with HgC12 for the identification of thiocyanate.35 Small amounts of elemental sulphur have been determined in metals and alloys ,67 metal sulphides,65,66 vulcanised rub-ber,68@ sulphur-containing polymers68370 and ointments,4 after extraction with an organic solvent.Elemental sulphur must be finely dispersed to act as a catalyst which is achieved by using a miscible organic solvent such as dimethylformam-ide and adding an aliquot of the extract into the iodine - azide aqueous solution .3,65770 Thin-layer chromatography has been used to resolve mixtures of substituted thioureas.36 The separation is per-formed on silica gel layers with dioxane - benzene - acetic acid (90 + 80 + 1) as the eluent.The spots are located by spraying chromatograms of standard solutions with an iodine - azide solution. The corresponding areas on the chromatogram of the sample solution are scraped off and suspended in an azide solution and the components are determined by direct injection enthalpimetry . Substituted thioureas50 and mixtures of thiosulphate and tetrathionate75 have been determined after separation by paper chromatography and high-voltage electrophoresis in a pH 9.2 borax buffer has been used for substituted dithiocarba-mates.117 The determination of cysteine glutathione and ergo-thioneine in haemolysate of erythrocytes has been performed after separation in a Sephadex G-10 column.By elution with a pH 6.8 phosphate buffer two fractions are obtained the first corresponding to the mixture of cysteine and glutathione ANALYST SEPTEMBER 1986 VOL. 111 1009 Table 2. Determination of metal ions Element Hg(1) and Hg(I1) . . Co(I1) * Ni(I1) . Cu(I1) . Zn(I1) . . Pd(I1) . . Fe(II1) . . Rh(II1) . . Au(II1) . . Bi(II1) . . Ir(1V) . . Pt(1V) . . . . . . . . * . . . . . . . . . . . . . . I Ru(VII1) and Os(VII1) . I . . . . . . . . . . . . . . . . . . . . . . . . Ligand* Characteristics of the procedures samples and references Ethylenediamine- Determination of 0.5-8 pg115 dithiocarbamate 6-Mercaptopurine Determination of 1-30 pgl24 Pyrrolidinedithio- After 10 min of reaction determination of 0.5-15 pg, car bamate CV < 2.570 previous separation from Ni Mn Cu Fe and Zn by ion exchangel25 5-100 ml CV 5% (9'/0 at 10 p.p.b.level) Fe(II1) and AI(II1) can be masked with fluoride. 126 Applied to nickel chloride and drugs; Co(I1) is previously separated from Ni(I1) and Cu(I1) by ion exchange4" Diethyldithiocarbamate After 2 min of reaction determination of 10-240 p.p.b. in As above After 2 min of reaction determination of 28-280 p.p.b. in 5 ml CV < 8%. In margarine and drugs Ni(I1) is previously separated from Cu(I1) and Co(I1) by ion exchange40 After 10 s of reaction determination of 60-840 p.p.b.in 5 ml CV < 3%. In Zamack alloys previous separation by Determination of 1-30 pg124 As above? extraction with dimethylglyoxime19 6-Mercap topurine Diethyldithio- Determination of 6 X Cu in 1 g NaC1120J27 carbarnates Thioammeline Determination of 6-250 p.p.b. in 50 ml CV < 10%. In zinc salts without separation or pre-concentration of copperl28 Determination of 3-24 p,p.b. in 10 ml and 2-240 p.p.b. in 50 ml. In tap and distilled water commercial azide acetic and oxalic acids. 129 In drugs previous separation of Co(I1) and Ni(I1) by ion exchange4" As above 8-Mercaptopurine Thiopental Determination of 0.04-1.6 p.p.m. in 50 m1130 Determination of 20-200 p.p.b. in 50 ml previous separation of Zn(I1) by ion exchange131 As above Determination of 40-400 p.p.b.in 50 ml previous separation of Cu(I1) by ion exchange131 6-Mercaptopurine After 10 min of reaction determination of 0.01-3 p.p.m., CV 4% but 10% at ng level. Other Pt-group metals can be tolerated in the presence of masking agents132 As above Determination of 0.1-30 pg1Z4 2-Mercaptopurine Determination of 0.2-lOp.p.m. CV 3°/~133 As above 2- and 6-mercapto-purine Determination of 0.1-0.8 p. p.m. CV 5.1 YO 134 LOD 20 p.p. b. in 5 m1135 Bismuthiol Determination of 0.1-20 p.p.m. in 50 ml CV 2-10%; Sn(II), Pd(II) Cu(II) Fe(III) Pb(I1) and Zn(I1) interfere96 2-Mercaptopurine Determinationof 10-400p.p.b. CV8.2%133 6-Mercaptopurine Determinationof0.2-5 p.p.m. CV 1lYO136 As above Determination of 0.1-1 p.p.m. Ru(VII1) and0.02-1 p.p.m.Os(VII1) in 5 ml CV 6.1 and 6.4% respectively. Previous separation by volatilisation as Ru04 and 0s04137 2- and 6-mercapto- LOD 2 and 5 p.p.b. in 5 ml respective1yl35 purine * Except in the instances indicated the procedure involves the determination of the free ligand by titration of the unconsumed iodine with 1- Determination of the free ligand by direct injection enthalpimetry. $ Extraction of the complex displacement of the ligand and spectrophotometric determination of the unconsumed iodine after a fixed reaction arsenite after a fixed reaction time. time 1010 ANALYST SEPTEMBER 1986 VOL. 111 which in the column are oxidised to disulphides and the second corresponding to ergothioneine .5 The selective deter-mination of cysteine and glutathione is carried out as described above ,29 after reduction of the disulphides with H2Se.Determination of Metal Ions Metal complexes with sulphur-containing compounds usually exhibit a lower catalytic activity than do free ligands owing to the blocking of active sulphur atoms by coordination. More-over the catalytic activity of the complexes is often almost cancelled. This inhibition effect may be used in the indirect determination of metal ions following the extent of the iodine - azide reaction by either of the techniques described above. The characteristics of the proposed procedures are sum-marised in Table 2. The inhibition effect may also be used to establish the stoicheiometries and conditional stability constants of the As corresponds to methods based on an inhibitory effect, calibration graphs show negative slopes and the upper limit of the application range depends on the analytical concentration of the ligand and on the conditional stability constant of the complex.The smaller the constant is the shorter the calibration linear range will be.139 In several instances side-reactions of complex formation with azide are important and must be ~onsidered.~5J~~ Although most metal - azide complexes are weak the use of a large excess of azide can lead to a decrease of the conditional constant .I39 Metal ion - catalyst - azide equilibria are often attained slowly. Therefore it may be necessary first to mix these components and to wait some time before starting the catalysed reaction. In the determination of Co2+ 126 and Ni*+ 19140 with DDTC a 10-min waiting time has proved to be adequate.In addition to the methods based on the inhibitory effect a direct method for Rh(III) based on the catalytic activity of the Rh(II1) - 2-mercaptopurine complex has been described.133 A procedure for determining Cu(II) Pb(I1) and Cd(I1) on a different basis has also been proposed. The complexes of these metals with DDTC are extracted with chloroform the excess of ligand is eliminated by washing with NaOH and the extract is added to a methanolic iodine - azide solution. Metal -DDTC complexes are partially decomposed in the presence of an excess of azide there being free DDTC proportional to the amount of metal. The determination is performed by measur-ing spectrophotometrically the amount of unreacted iodine 5 min after the beginning of the catalysis.The sensitivity of the method is 50 times better than that of the usual spectropho-tometric method where a direct measurement of the Cu(I1) -DDTC absorbance is carried out. 120,127 The selectivity of the methods for determining metal ions depends on the selectivity of the sulphur compound as a ligand. When this is not enough interferences may be avoided with masking agents132 or by previous separation by volati-l i ~ a t i o n ~ ~ ~ e x t r a c t i ~ n l ~ J ~ ~ or ion-exchange chroma-tography. 40,131 complexes. 126,134,136,138-141 Other Applications The determination of azide has been performed by titration of the unconsumed iodine with thiosulphate after the total consumption of azide in the presence of an excess of some stable catalysts.118 Thiocyanate has been recommended for this purpose. 119 The iodine - azide indicator reaction has been proposed for end-point detection in titrations with sulphide in acidic media. The first drop of excess titrant causes the evolution of H2S which is transferred by a nitrogen stream into a vessel containing the iodine - azide reagent.142 The reaction has also been employed for the semi-quantitative measurement of the properties of diverse materials. Thus the induction coefficients of cyclic tetra-sulphides of primary amines have been used to establish cross-linking activity in elastomer vulcanisation An increase of the molecular weight of the substituents related to cross-linking activity leads to higher induction coefficients.143 The photographic activity of some sulphur compounds, which are used as additives in photography is related to their catalytic activity.233144 However methods based on the measurement of the extent of the reaction after a relatively large time period are not suitable as the oxidation side reactions of the catalysts lead to wrong conclusions. Muller et al.23 proposed the use of ascorbic acid to avoid the influence of these side-reactions. 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