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ml levels of sulphide by a chemiluminescent reaction
T~,~w~ro. Vol 27.pp 309,o314 0 PergamonPresLld 19X0PrmtedI"GreatBritam
DETERMINATION OF ng/ml LEVELS OF SULPHIDE BY A CHEMILUMINESCENT REACTION J. L. BWRGUERA
and ALAN TOWNSHEND@
Chemistry Department, Birmingham University, P.O. Box 363, Birmingham, B15 2TT, U.K. (Receioed
10 May 1979. Accepted
19 October
1979)
Summary-Sulphide
(20.1 ng/ml) is determined by measuring the chemiluminescence given by its oxidation in aqueous solution by hydrogen peroxide, catalysed by peroxidase. The detection limit is 0.05 ng/ml. Several other, rather less sensitive, systems are also described. Sulphite and thiocyanate, which depress the emission, are masked with formaldehyde.
sulphide solutions is well known. In the present investigation, flasks containing sulphide solutions were tightly stoppered, and protected from light. Under these conditions, l-10 ng/ml sulphide solutions remained stable for 2&30 min. For 0.01-10 ng/ml solutions, no change was detected during the first 10 min after preparation; after 25 min the chemiluminescence response had decreased by 50%. For this reason, the calibration solutions used for < 10 ng/ml sulphide were prepared serially from the solution of next highest concentration and analysed immediately. Sodium hypochlorite (l-10 x 10-3M) and hydrogen peroxide (0.8-4.OM) solutions were prepared from a commercial sodium hypochlorite solution, 12% w/v available chlorine, and IOO-volume hydrogen peroxide, respectively. Osmium tetroxide solution (0.01 M) was prepared by dissolving 0.253 g of 99.9% pure osmium tetroxide in 100 ml of 0.05M sulphuric acid. Sensitizer solutions (2 x 10e3M) were prepared by dissolving 0.0884 g of Rhodamine B or 0.0644 g of fluorescein in 100 ml of sulphide working solution. Catalase stock solution (34 x 10’ Sigma units/ml) was made by diluting 4.0 ml of catalase solution (beef liver, twice-crystallized, 25 mg/ml aqueous suspension with O.Io/, thymol; activity 3 x 104-4 x IO4 Sigma units/mg; Sigma Laboratories) to 100 ml with water. and stored at 4”. Peroxidase stock solution (28.5 purpurogallin units/ml) was made by dissolving 0.0285 g of horseradish peroxidase (salt-free powder, activity 370-600 purpurogallin units/mg; Sigma Laboratories) in IO ml of water, and stored at 2”. Standard solutions of enzymes were prepared daily from the stock solutions by appropriate dilution with water, and were kept in an ice-bath.
Chemiluminescent and bioluminescent systems exhibit considerable diversity in the determination of many inorganic and organic compounds,‘-5 and newer systems are continually expanding their scope. 6.7 Few methods for the determination of sulphide by chemiluminescence measurements are available. Lukovskaya et al.8,9determined 5-20 ng of sulphide by means of its quenching of the chemiluminescent reaction of luminol with iodine, or its reduction of vanadium(V) to vanadium(W), the vanadium(W) then catalysing the chemiluminescent reaction of luminol with hydrogen peroxide. Klockow and Teckentrup’” studied the determination of sulphide by means of its chemiluminescent oxidation by hypohalites in the presence of Methylene Blue. Linear calibration graphs were obtained for 1.x lo-‘p 5 x 10m6M sulphide. The sensitivity of the Klockow and Teckentrup procedure is already high. However, we decided that the reaction should be studied in some detail in order to improve its sensitivity, to evaluate and eliminate interferences, and to obtain some insight into the mechanism. To improve the sensitivity of emission the use of other sensitizers, such as fluorescein and Rhodamine B, has been investigated. In addition, hydrogen peroxide (accompanied by known catalysts for peroxide reactions, such as catalase,“*‘* peroxidase13.14 and osmium tetroxide15,‘6 has been investigated in detail as an alternative oxidant.
Basic procedure for cherniluntinc,scence measurements A 0.5-ml portion of the oxidant solution was transferred to the cell and 0.5 ml of the catalyst solution added. The syringe was filled with 1.0 ml of the buffered sulphide solution of concentration 2 x lo-‘-2 x lo3 pg/ml. The reaction was started by injecting the sulphide solution very rapidly (in ~0.4 set). The emission intensity was recorded at 440 nm as a function of time during 20 sec. For calibration purposes the experiment was repeated with each standard solution of sulphide. The calibration graph was a plot of maximum emission intensity us. sulphide concentration. The same procedure was used to determine “unknown” samples, with a I.O-ml sample.
EXPERIMENTAL Appuratus
The instrumentation and experimental were as described previously.’ ’
technique
RefXJent.5 All reagents were laboratory reagent grade, unless otherwise stated. The water used was doubly distilled. Stock sulphide solutions (2000 pg/ml) were prepared by dissolving 1.50 g of sodium sulphide enneahydrate (Na,S .9H,O) in 100 ml of 0.1 M sodium carbonate or borate buffer (pH 9-12). Working solutions were prepared daily from this stock solution by appropriate dilution with the buffer solution chosen. The instability of very dilute
RESULTS
Injection hypochlorite 309
of a sulphide solution into a sodium solution or hydrogen peroxide-catalyst
J. L. BUKGUERA
310
and ALAN
T~WNSHENU
IL
0.
6
2. 0.
.
. .
_ 0
10’
0 10 ”
!_
0 10 n 0 10 'I 0 10
.
0 10
*
0 10 ”
0 10 u 0 10 ’
0 10
Time (secl
Fig. 1. Intensity OS.time signals from (1) 100 ppm and (2) 10 ppm sulphide, O.lM and (4) 1 ppm sulphide, 10m3M fluorescein, O.lM NaOCl; (5) 0.1 ppm and (6) 0.01 H202. 0.25 ppm 0~0~; (7) 0.01 ppm and (8) 0.005 ppm sulphide, 0.4M H202, catalase; (9) 0.01 ppm and (10) 0.001 ppm sulphide, 0.4M H202, 1.5 purpurogallin (l))(4):
pH 11.5; (5)(lO):
pH 10.0, carbonate
buffer. (1). (2) (S)(lO)
NaOCl; (3) 10 ppm ppm sulphide, l.OM 750 Sigma units/ml
units/ml peroxidase. at 440 nm; (3) (4) at 520 nm.
mixture produced a chemiluminescence emission which reached maximum intensity after 5 set, and then decayed rapidly. Typical emission us. time responses are shown in Fig. I. The coefficient of variation for the determination of 200 ng of sulphide (equivalent to 100 ng/ml in the reaction mixture) based on such a response was ca. 5% in all cases. The spectra of the chemiluminescence emission given on oxidation of sulphide by sodium hypochlorite or hydrogen peroxide have not been reported Wavelength
Fig. 3. by the chlorite 0 with
Lm
L60 520 560 Wavelength Inm)
Inml
Spectra of the chemituminescence emissions given oxidation of 100 ppm of sulphide by O.lM hypoat pH 11.5 (carbonate buffer). l without sensitizer, IOM3M fluorescein, q with IO-‘M Rhodamine B.
600
Fig. 2. Spectra of the chemiluminescence emissions given by the oxidation of sulphide under different conditions. (I) 100 ppm sulphide, 0.1M NaOCI, pH 11.5; (2) 100 ppm sulphide, l.OM HZOz, 0.25 ppm 0~0,. pH 10.0; (3) I ppm sulphide, 0.4M HzO1. 1.5 purpurogallin units/ml peroxidase. pH 10.0. (I) I 12 mV; (2). (3) 0 80 mV.
Log sensitizer
concentration
IMI
Fig. 4. Effect of sensitizer concentration on the emission intensity given by the oxidation of 100 ppm sulphide by 0. I M hypochlorite at pH I 1.5 (carbonate buffer). (a) Fluores&n. 520 nm: tb) Rhodamine B. 610 nm.
Determination
311
of ng/ml levels of sulphide
5 g 0.6 x ‘tl g E 0.4 .-S I ‘E 0.2
s” 0
Fig. 5. Effect of pH on the chemiluminescence intensity for the uncatalysed oxidation of 100 ppm sulphide by O.lM hypochlorite. (a) Carbonate buffer; (b) borate buffer. and are shown in Fig. 2. They were obtained by measuring the maximum emission intensity produced at various wavelengths by a constant amount of sulphide. The maximum intensity is at 440 nm in all instances, so this wavelength was used for all subsequent experiments. The spectra all have an identical single peak and differ only in their amplitude, indicating the formation of a common emitter, which has not been identified. The effect of the sensitizers fluorescein and Rhodamine B on the emission spectrum given on oxidation of sulphide by hypochlorite is shown in Fig. 3. The spectra obtained were similar to the fluorescence emission spectrum of the sensitizer added, and confirm that the sensitizers are activated by an energytransfer process. The effects of sensitizer concentration on the emission intensity are shown in Fig 4. Maximum intensity was observed with a final concentration of 10d3M before
-/ -3
-2 Log
0 concentration
(MI
Fig. 7. Variation of chemiluminescence intensity with sodium hypochlorite concentration: 10 ppm sulphide, pH 11.5 (carbonate buffer).
fluorescein or Rhodamine B. An advantage of using fluorescein as sensitizer was an increase in sensitivity, as described later. In investigation of the optimum reagent parameters, only the quantities indicated by points on the graphs were tested, and the true optimum may not lie exactly at the value stated in the text though it will be near it. The effect of pH (9.0-12.0) on the emission intensity was investigated for all the systems mentioned. Chemiluminescence was observed only in an alkaline medium. The uncatalysed oxidation of sulphide by sodium hypochlorite gave maximum intensity at around pH 11.5 in both buffers (Fig. 5), the emission intensity being higher for the carbonate buffer. The emission intensity during catalysed oxidation by hydrogen peroxide in a carbonate buffer was maximal at about pH 10.0 (Fig. 6). The effect of oxidant concentration on the emission intensity was strongly dependent on the oxidant concentration, and was maximal with z O.lM hypochlorite (Fig. 7), and -0.4M hydrogen peroxide in the presence of either enzyme as catalyst, or _ l.OM hydrogen peroxide with osmium tetroxide as catalyst (Fig. 8); the concentrations refer to the reaction mixture.
160
t
.
/
z
i
h.0,
.N
5
.-*
r5
/
.5 40,
E
IfI
b1..
.-
_v--•’
:-:
o7
0.2
Fig. 6. Effect of pH on the chemiluminescence intensity from oxidation of 100 ppm of sulphide by hydrogen peroxide in the systems: (a) 1.5 purpurogallin units/ml peroxidase, 0.4M H,O,; (b) 0.25 ppm 0~0~. l.OM H,O,; (c) 750 Sigma units/ml catalase, 0.4M H202.
-1
hypochlorite
04
0.6
Hydrogen
---=.. 0.8 peroxide
1.0 concentration
1.2
14 (Ml
Fig. 8. Effect of hydrogen peroxide concentration on chemiluminescence intensity. (a) 1.5 Purpurogallin units/ml peroxidase; (b) 0.25 ppm 0~0,; (c) 750 Sigma units/ml catalase; 0.1 ppm sulphide, pH 10.0 (carbonate buffer).
J. L. BURGUERA and ALAN TOWNSHEND
312
01
koxidasel,
c$ 29
ipurpurcgallin
$5 2,o 2.5 750 1000 1250 [Cotalasel, (slgma units/ml1
$0 SW
The effect of catalyst concentration on the chemiluminescence intensity in the peroxide oxidation was also investigated (Fig. 9). The emission intensity is dependent on enzyme concentration up to - 1.5 purpurogallin units/ml final concentration of peroxidase and - 750 Sigma units/ml final concentration of catalase. The final osmium tetroxide concentration chosen was 0.25 pg/ml (Fig. 10). calibration graphs were obtained for each of the systems described above, and are shown in Fig. Il. They demonstrate that sulphide can readily be determined over a wide concentration range, down to < 10m4 ppm by chemiluminescence measurements. Because the curves pass through a maximum, it is suggested that as a safety precaution in interpreting 1. Analytical
the results, a second sample should be tested, a lower concentration being used. The suggested range of application, together with detection limit, is given in Table 1. Figure 12 shows the blank and sulphide responses near the detection limit. Interferences
The effect of some other sulphur anions (sulphate, thiocyanate and sulphite) commonly found with sulphide was studied. It was found that sulphate had no significant effect on the determination of 1 ppm of sulphide, even in lOOO-fold w/w ratio, in all the systems studied. Thiocyanate (IO-fold w/w ratio) suppressed the emission only in the hydrogen peroxide systems, whereas sulphite affected all the systems (Table 2). It was found that these interference effects were eliminated by addition of formaldehyde to give a final concentration of 4.0 mg/ml (Table 2). Low concen-
parameters for the determination in some different systems Range of application, PPfi'
System NaOCl NaOClMuorescein HzOZ-0~0, H,O,-catalase H,O,-peroxidase Conditions
045 l&d1
Fig. 10. Effect of osmium tetroxide concentration on the chemiluminescence intensity from 100 ppm sulphide: l.OM H,02, pH 10.0 (carbonate buffer).
Fig. 9. Effect of enzyme concentration on the chemiluminescence emission from 100 ppm of sulphide. (a) Peroxidase, 0.8M HzOz (C&100 mV scale); (b) catalase, 0.4M H202 (O-30 mV scale): pH 10.0 (carbonate buffer).
Table
0.25 0.35 Osmiumtetroxlde concentration
015
units/ml)
IO_‘-10’ lo-*-lo2 lo-‘-lo2 lo-‘-lo-’ lo-4- 10
of sulphide
by oxidation
Detection
limit, t1g/nd
Y
20 2.0 0.2 2.0 0.1
10 1.0 0.1 1.o 0.05
as in Fig. 11.
Table 2. Effect of interfering
System*
SCN-
NaOClt NaOClMuorescein H,O,-0~0, H,O,-catabase H,O,-peroxidase
0 0 - 18 -34 -45
ions in the presence
* 1 ppm sulphide, 10 ppm interfering t 10 ppm sulphide.
and absence
of formaldehyde
Change in emission intensity, SCN- + HCHO so:0 0 -1 0 -2
- 10 - 12 -5 -4 - 12
ion, 3.9 mg/ml formaldehyde.
“/, SO;-
+ HCHO 0 -1 -2 0 -2
Determination
of ng/ml levels of sulphide
313
Wavelength
I nml
Fig. 13. Spectrum of the weak chemiluminescence emission given by the osmium tetroxide-H20, 0.25 ppm 0~0,. l.OM H202, pH 10.0 (carbonate Log sulphide
concentration
lppml
Fig. 11. Effect of sulphide concentration on maximum emission intensity. 0 O.lM NaOCl, pH 11.5; 0 O.lM NaOCl, 10m3A4 fluorescein. pH 11.5, 520 nm; q 750 Sigma units/ml catalase, 0.4M H202, pH 10.0, 0 1.5 purpurogallin units/ml peroxidase, 0.4M H,OZ, pH 10.0; n 0.25 ppm 0~0,. l.OM H,Oz. pH 10.0. All at 440 nm except where
stated otherwise.
10
I
z5 z. f0
sf
10
1
2
0
t
% 65
3
L
Lr-h.
20
0
20
0
2-a
0
20
5
EO-- I.
RLL 0
20
0
20
0
20
blank system: buffer).
0
20
Time kc) Fig. 12. Responses near the detection limit for (1). (3), (5). (7) no S’~ and (2) 20, (4) 0.20, (6) 0.20, (8) 2.0 ng of St- in the sodium hypochlorite and hydrogen peroxide-osmium tetroxide, -peroxidase and -catalase systems, respectively. Conditions as in Fig. Il.
trations of formaldehyde did not affect the chemiluminescence emission intensity, but 3 8.0 mg/ml increased it. Thus, the final formaldehyde concentration should not exceed 8.0 mg/ml.
DISCUSSION
The emission spectra suggest that the same excited species produces the luminescence, regardless of the oxidant or catalyst. The final oxidation product is sulphate in all instances, but the intermediate reaction steps are not well established.” Since ‘OH* radicals are assumed to be the source of energy for forming the excited species producing the luminescence, attempts were made to demonstrate their existence in the systems studied, by measuring the spectrum of the very weak chemiluminescence produced by reaction of the hydrogen peroxide-catalyst mixture with a buffered alkaline solution without sulphide. These attempts failed for the catalase and peroxidase systems, probably because the emission intensity was too
weak. However, a spectrum for the osmium tetroxide system was obtained (Fig. 13) and the maxima at 450, 480 and 580 nm correspond to those attributed to the emission from the excited double oxygen molecule (0202)*.19 This excited molecule is produced by collision of ‘OH2 radicals from the hydrogen peroxide decomposition.20~22 The nature of the emitting species formed by oxidation of the sulphide is not known. It is not the excited oxygen molecule described above, but it could be an excited S2 molecule similar to that involved in flame chemiluminescence emission,23,24 formed as an intermediate in the ‘OH2 oxidation of sulphide ions. However, the chemiluminescence measurements give little further evidence about the oxidation reaction mechanism. Other studies, e.g., ESR measurements, will be needed even to begin understanding the system. It has been established that the chemiluminescence measurements provide an extremely sensitive, precise, simple and rapid (analysis time 2 min per sample) method for the determination of sulphide. The methods described should be applicable to the determination of sulphide in natural waters, such as river, rain and drinking water, with a precision of 676 at the 10-3-102 ppm level. Sulphate does not interfere at concentrations likely to be found in natural water systems, and ions such as sulphite and thiocyanate can readily be eliminated by masking them with formaldehyde. Acknowlcdllrn~mr-J. L. Burguera zuela. for a scholarship.
thanks
Foninves.
Vene-
REFERENCES 1. D. B. Paul, Talanta, 1978. 25, 377. 2. S. S. Stieg and T. A. Nieman, Anal. Chen7.. 1977, 49. 1322. 3. W. R. Seitz and M. P. Neary. in Contmporary Topics in Analytical and Clikul C/tmistry. D. M. Hercules. G. M. Hieftje, L. R. Snyder and M. A. Evenson (eds), Vol. I, pp. 49-125. Plenum Press. New York, 1977. 4. S. N. Lowery. P. W. Carr and W. R. Seitz. Anal. Lett.. 1977, 10, 931. 5. W. R. Seitz and D. M. Hercules, in Chr,,,i/ur,lirlrscc~~~~,
314
6.
7. 8. 9. 10.
I I. 12. 13.
J. L. BURCUERA and ALAN TOWNSHEND or~d Bio/u,tli,le.scc~nce,M. J. Cormier, D. M. Hercules and J. Lee, eds., Plenum Press. New York, 1973. Internurionul Symposium on Anulytical Applications of Biol~mlirlescr,lcr end Cltrmi/urni,lescrilce, Brussels, 6-8 September, 1978. G. Wettermark, S. E. Brolin and S. Hjertin, Cell. Molec. Biology, 1977, 22, 329. N. M. Lukovskaya and L. V. Markova. J. Anal. Chem. USSR, 1969, 24, 1868. N. M. Lukovskaya and N. I. Anatienko. Ory. Reayenty Analit. Khim. Tezisy Dokl., VW. Konj:, 1976, 2. 80. D. Klockow and J. Teckentrup, Talanta, 1976,23,889. K. Weber, A. Reiek and V. Vouk, Ber.. 1942, 75B, 1141. H. A. Neufeld, C. J. Conklin and R. D. Towner, Anal. Biochenl.. 1965. 12, 303. D. Slawinska, J. Slawinski, W. Pukacki and K. Polewski, in lnrrrnational Symposium on Analytical Applications ofBioluminescence and Chemiluminescence, Brussels. 6-8 September, 1978, Abstr. 43, p. 44.
14. G. AhnstrGm, G. Ehrenstein and R. Nilsson, Actor Chem. Stand., 1961, 15, 1417. 15. K. Gleu and K. Pfannstiel. .I. Pratt. Chem.. 1936, 146. 137. 16. L. Erdey, lnd. Chemist, 1957, 459, 523. 575. 17. J. L. Burguera and A. Townshend, Talanta, 1979, 26, 795. 18. C. H. Bamford and C. F. H. Tipper, Chemical Kinetics, Vol. 6. Elsevier, Amsterdam, 1972. 19. J. W. Haas. J. Chem. E&c., 1967, 44, 396. 20. J. Staufl, Z. Physik. Chem. (Frunkjitrt). 1964. 40, 64; 1966. 49, 58; 1967. 55, 39. 21. J. StauiT and H. Schmidkunz, ibid., 1962, 35. 295. 22. A. V. Khan and M. Kasha, J. Am. Chem. Sot.. 1966. 88. 1574. K. C. Thompson and T. S. West, 23. R. M. Dagnall, Analyst, 1967, 92, 506. D. J. Knowles and A. 24. R. Belcher. S. L. Bogdanski, Townshend. Anal. Chim. Acta. 1973. 67, I and references therein.
DETERMINATION OF ng/ml LEVELS OF SULPHIDE BY A CHEMILUMINESCENT REACTION J. L. BWRGUERA
and ALAN TOWNSHEND@
Chemistry Department, Birmingham University, P.O. Box 363, Birmingham, B15 2TT, U.K. (Receioed
10 May 1979. Accepted
19 October
1979)
Summary-Sulphide
(20.1 ng/ml) is determined by measuring the chemiluminescence given by its oxidation in aqueous solution by hydrogen peroxide, catalysed by peroxidase. The detection limit is 0.05 ng/ml. Several other, rather less sensitive, systems are also described. Sulphite and thiocyanate, which depress the emission, are masked with formaldehyde.
sulphide solutions is well known. In the present investigation, flasks containing sulphide solutions were tightly stoppered, and protected from light. Under these conditions, l-10 ng/ml sulphide solutions remained stable for 2&30 min. For 0.01-10 ng/ml solutions, no change was detected during the first 10 min after preparation; after 25 min the chemiluminescence response had decreased by 50%. For this reason, the calibration solutions used for < 10 ng/ml sulphide were prepared serially from the solution of next highest concentration and analysed immediately. Sodium hypochlorite (l-10 x 10-3M) and hydrogen peroxide (0.8-4.OM) solutions were prepared from a commercial sodium hypochlorite solution, 12% w/v available chlorine, and IOO-volume hydrogen peroxide, respectively. Osmium tetroxide solution (0.01 M) was prepared by dissolving 0.253 g of 99.9% pure osmium tetroxide in 100 ml of 0.05M sulphuric acid. Sensitizer solutions (2 x 10e3M) were prepared by dissolving 0.0884 g of Rhodamine B or 0.0644 g of fluorescein in 100 ml of sulphide working solution. Catalase stock solution (34 x 10’ Sigma units/ml) was made by diluting 4.0 ml of catalase solution (beef liver, twice-crystallized, 25 mg/ml aqueous suspension with O.Io/, thymol; activity 3 x 104-4 x IO4 Sigma units/mg; Sigma Laboratories) to 100 ml with water. and stored at 4”. Peroxidase stock solution (28.5 purpurogallin units/ml) was made by dissolving 0.0285 g of horseradish peroxidase (salt-free powder, activity 370-600 purpurogallin units/mg; Sigma Laboratories) in IO ml of water, and stored at 2”. Standard solutions of enzymes were prepared daily from the stock solutions by appropriate dilution with water, and were kept in an ice-bath.
Chemiluminescent and bioluminescent systems exhibit considerable diversity in the determination of many inorganic and organic compounds,‘-5 and newer systems are continually expanding their scope. 6.7 Few methods for the determination of sulphide by chemiluminescence measurements are available. Lukovskaya et al.8,9determined 5-20 ng of sulphide by means of its quenching of the chemiluminescent reaction of luminol with iodine, or its reduction of vanadium(V) to vanadium(W), the vanadium(W) then catalysing the chemiluminescent reaction of luminol with hydrogen peroxide. Klockow and Teckentrup’” studied the determination of sulphide by means of its chemiluminescent oxidation by hypohalites in the presence of Methylene Blue. Linear calibration graphs were obtained for 1.x lo-‘p 5 x 10m6M sulphide. The sensitivity of the Klockow and Teckentrup procedure is already high. However, we decided that the reaction should be studied in some detail in order to improve its sensitivity, to evaluate and eliminate interferences, and to obtain some insight into the mechanism. To improve the sensitivity of emission the use of other sensitizers, such as fluorescein and Rhodamine B, has been investigated. In addition, hydrogen peroxide (accompanied by known catalysts for peroxide reactions, such as catalase,“*‘* peroxidase13.14 and osmium tetroxide15,‘6 has been investigated in detail as an alternative oxidant.
Basic procedure for cherniluntinc,scence measurements A 0.5-ml portion of the oxidant solution was transferred to the cell and 0.5 ml of the catalyst solution added. The syringe was filled with 1.0 ml of the buffered sulphide solution of concentration 2 x lo-‘-2 x lo3 pg/ml. The reaction was started by injecting the sulphide solution very rapidly (in ~0.4 set). The emission intensity was recorded at 440 nm as a function of time during 20 sec. For calibration purposes the experiment was repeated with each standard solution of sulphide. The calibration graph was a plot of maximum emission intensity us. sulphide concentration. The same procedure was used to determine “unknown” samples, with a I.O-ml sample.
EXPERIMENTAL Appuratus
The instrumentation and experimental were as described previously.’ ’
technique
RefXJent.5 All reagents were laboratory reagent grade, unless otherwise stated. The water used was doubly distilled. Stock sulphide solutions (2000 pg/ml) were prepared by dissolving 1.50 g of sodium sulphide enneahydrate (Na,S .9H,O) in 100 ml of 0.1 M sodium carbonate or borate buffer (pH 9-12). Working solutions were prepared daily from this stock solution by appropriate dilution with the buffer solution chosen. The instability of very dilute
RESULTS
Injection hypochlorite 309
of a sulphide solution into a sodium solution or hydrogen peroxide-catalyst
J. L. BUKGUERA
310
and ALAN
T~WNSHENU
IL
0.
6
2. 0.
.
. .
_ 0
10’
0 10 ”
!_
0 10 n 0 10 'I 0 10
.
0 10
*
0 10 ”
0 10 u 0 10 ’
0 10
Time (secl
Fig. 1. Intensity OS.time signals from (1) 100 ppm and (2) 10 ppm sulphide, O.lM and (4) 1 ppm sulphide, 10m3M fluorescein, O.lM NaOCl; (5) 0.1 ppm and (6) 0.01 H202. 0.25 ppm 0~0~; (7) 0.01 ppm and (8) 0.005 ppm sulphide, 0.4M H202, catalase; (9) 0.01 ppm and (10) 0.001 ppm sulphide, 0.4M H202, 1.5 purpurogallin (l))(4):
pH 11.5; (5)(lO):
pH 10.0, carbonate
buffer. (1). (2) (S)(lO)
NaOCl; (3) 10 ppm ppm sulphide, l.OM 750 Sigma units/ml
units/ml peroxidase. at 440 nm; (3) (4) at 520 nm.
mixture produced a chemiluminescence emission which reached maximum intensity after 5 set, and then decayed rapidly. Typical emission us. time responses are shown in Fig. I. The coefficient of variation for the determination of 200 ng of sulphide (equivalent to 100 ng/ml in the reaction mixture) based on such a response was ca. 5% in all cases. The spectra of the chemiluminescence emission given on oxidation of sulphide by sodium hypochlorite or hydrogen peroxide have not been reported Wavelength
Fig. 3. by the chlorite 0 with
Lm
L60 520 560 Wavelength Inm)
Inml
Spectra of the chemituminescence emissions given oxidation of 100 ppm of sulphide by O.lM hypoat pH 11.5 (carbonate buffer). l without sensitizer, IOM3M fluorescein, q with IO-‘M Rhodamine B.
600
Fig. 2. Spectra of the chemiluminescence emissions given by the oxidation of sulphide under different conditions. (I) 100 ppm sulphide, 0.1M NaOCI, pH 11.5; (2) 100 ppm sulphide, l.OM HZOz, 0.25 ppm 0~0,. pH 10.0; (3) I ppm sulphide, 0.4M HzO1. 1.5 purpurogallin units/ml peroxidase. pH 10.0. (I) I 12 mV; (2). (3) 0 80 mV.
Log sensitizer
concentration
IMI
Fig. 4. Effect of sensitizer concentration on the emission intensity given by the oxidation of 100 ppm sulphide by 0. I M hypochlorite at pH I 1.5 (carbonate buffer). (a) Fluores&n. 520 nm: tb) Rhodamine B. 610 nm.
Determination
311
of ng/ml levels of sulphide
5 g 0.6 x ‘tl g E 0.4 .-S I ‘E 0.2
s” 0
Fig. 5. Effect of pH on the chemiluminescence intensity for the uncatalysed oxidation of 100 ppm sulphide by O.lM hypochlorite. (a) Carbonate buffer; (b) borate buffer. and are shown in Fig. 2. They were obtained by measuring the maximum emission intensity produced at various wavelengths by a constant amount of sulphide. The maximum intensity is at 440 nm in all instances, so this wavelength was used for all subsequent experiments. The spectra all have an identical single peak and differ only in their amplitude, indicating the formation of a common emitter, which has not been identified. The effect of the sensitizers fluorescein and Rhodamine B on the emission spectrum given on oxidation of sulphide by hypochlorite is shown in Fig. 3. The spectra obtained were similar to the fluorescence emission spectrum of the sensitizer added, and confirm that the sensitizers are activated by an energytransfer process. The effects of sensitizer concentration on the emission intensity are shown in Fig 4. Maximum intensity was observed with a final concentration of 10d3M before
-/ -3
-2 Log
0 concentration
(MI
Fig. 7. Variation of chemiluminescence intensity with sodium hypochlorite concentration: 10 ppm sulphide, pH 11.5 (carbonate buffer).
fluorescein or Rhodamine B. An advantage of using fluorescein as sensitizer was an increase in sensitivity, as described later. In investigation of the optimum reagent parameters, only the quantities indicated by points on the graphs were tested, and the true optimum may not lie exactly at the value stated in the text though it will be near it. The effect of pH (9.0-12.0) on the emission intensity was investigated for all the systems mentioned. Chemiluminescence was observed only in an alkaline medium. The uncatalysed oxidation of sulphide by sodium hypochlorite gave maximum intensity at around pH 11.5 in both buffers (Fig. 5), the emission intensity being higher for the carbonate buffer. The emission intensity during catalysed oxidation by hydrogen peroxide in a carbonate buffer was maximal at about pH 10.0 (Fig. 6). The effect of oxidant concentration on the emission intensity was strongly dependent on the oxidant concentration, and was maximal with z O.lM hypochlorite (Fig. 7), and -0.4M hydrogen peroxide in the presence of either enzyme as catalyst, or _ l.OM hydrogen peroxide with osmium tetroxide as catalyst (Fig. 8); the concentrations refer to the reaction mixture.
160
t
.
/
z
i
h.0,
.N
5
.-*
r5
/
.5 40,
E
IfI
b1..
.-
_v--•’
:-:
o7
0.2
Fig. 6. Effect of pH on the chemiluminescence intensity from oxidation of 100 ppm of sulphide by hydrogen peroxide in the systems: (a) 1.5 purpurogallin units/ml peroxidase, 0.4M H,O,; (b) 0.25 ppm 0~0~. l.OM H,O,; (c) 750 Sigma units/ml catalase, 0.4M H202.
-1
hypochlorite
04
0.6
Hydrogen
---=.. 0.8 peroxide
1.0 concentration
1.2
14 (Ml
Fig. 8. Effect of hydrogen peroxide concentration on chemiluminescence intensity. (a) 1.5 Purpurogallin units/ml peroxidase; (b) 0.25 ppm 0~0,; (c) 750 Sigma units/ml catalase; 0.1 ppm sulphide, pH 10.0 (carbonate buffer).
J. L. BURGUERA and ALAN TOWNSHEND
312
01
koxidasel,
c$ 29
ipurpurcgallin
$5 2,o 2.5 750 1000 1250 [Cotalasel, (slgma units/ml1
$0 SW
The effect of catalyst concentration on the chemiluminescence intensity in the peroxide oxidation was also investigated (Fig. 9). The emission intensity is dependent on enzyme concentration up to - 1.5 purpurogallin units/ml final concentration of peroxidase and - 750 Sigma units/ml final concentration of catalase. The final osmium tetroxide concentration chosen was 0.25 pg/ml (Fig. 10). calibration graphs were obtained for each of the systems described above, and are shown in Fig. Il. They demonstrate that sulphide can readily be determined over a wide concentration range, down to < 10m4 ppm by chemiluminescence measurements. Because the curves pass through a maximum, it is suggested that as a safety precaution in interpreting 1. Analytical
the results, a second sample should be tested, a lower concentration being used. The suggested range of application, together with detection limit, is given in Table 1. Figure 12 shows the blank and sulphide responses near the detection limit. Interferences
The effect of some other sulphur anions (sulphate, thiocyanate and sulphite) commonly found with sulphide was studied. It was found that sulphate had no significant effect on the determination of 1 ppm of sulphide, even in lOOO-fold w/w ratio, in all the systems studied. Thiocyanate (IO-fold w/w ratio) suppressed the emission only in the hydrogen peroxide systems, whereas sulphite affected all the systems (Table 2). It was found that these interference effects were eliminated by addition of formaldehyde to give a final concentration of 4.0 mg/ml (Table 2). Low concen-
parameters for the determination in some different systems Range of application, PPfi'
System NaOCl NaOClMuorescein HzOZ-0~0, H,O,-catalase H,O,-peroxidase Conditions
045 l&d1
Fig. 10. Effect of osmium tetroxide concentration on the chemiluminescence intensity from 100 ppm sulphide: l.OM H,02, pH 10.0 (carbonate buffer).
Fig. 9. Effect of enzyme concentration on the chemiluminescence emission from 100 ppm of sulphide. (a) Peroxidase, 0.8M HzOz (C&100 mV scale); (b) catalase, 0.4M H202 (O-30 mV scale): pH 10.0 (carbonate buffer).
Table
0.25 0.35 Osmiumtetroxlde concentration
015
units/ml)
IO_‘-10’ lo-*-lo2 lo-‘-lo2 lo-‘-lo-’ lo-4- 10
of sulphide
by oxidation
Detection
limit, t1g/nd
Y
20 2.0 0.2 2.0 0.1
10 1.0 0.1 1.o 0.05
as in Fig. 11.
Table 2. Effect of interfering
System*
SCN-
NaOClt NaOClMuorescein H,O,-0~0, H,O,-catabase H,O,-peroxidase
0 0 - 18 -34 -45
ions in the presence
* 1 ppm sulphide, 10 ppm interfering t 10 ppm sulphide.
and absence
of formaldehyde
Change in emission intensity, SCN- + HCHO so:0 0 -1 0 -2
- 10 - 12 -5 -4 - 12
ion, 3.9 mg/ml formaldehyde.
“/, SO;-
+ HCHO 0 -1 -2 0 -2
Determination
of ng/ml levels of sulphide
313
Wavelength
I nml
Fig. 13. Spectrum of the weak chemiluminescence emission given by the osmium tetroxide-H20, 0.25 ppm 0~0,. l.OM H202, pH 10.0 (carbonate Log sulphide
concentration
lppml
Fig. 11. Effect of sulphide concentration on maximum emission intensity. 0 O.lM NaOCl, pH 11.5; 0 O.lM NaOCl, 10m3A4 fluorescein. pH 11.5, 520 nm; q 750 Sigma units/ml catalase, 0.4M H202, pH 10.0, 0 1.5 purpurogallin units/ml peroxidase, 0.4M H,OZ, pH 10.0; n 0.25 ppm 0~0,. l.OM H,Oz. pH 10.0. All at 440 nm except where
stated otherwise.
10
I
z5 z. f0
sf
10
1
2
0
t
% 65
3
L
Lr-h.
20
0
20
0
2-a
0
20
5
EO-- I.
RLL 0
20
0
20
0
20
blank system: buffer).
0
20
Time kc) Fig. 12. Responses near the detection limit for (1). (3), (5). (7) no S’~ and (2) 20, (4) 0.20, (6) 0.20, (8) 2.0 ng of St- in the sodium hypochlorite and hydrogen peroxide-osmium tetroxide, -peroxidase and -catalase systems, respectively. Conditions as in Fig. Il.
trations of formaldehyde did not affect the chemiluminescence emission intensity, but 3 8.0 mg/ml increased it. Thus, the final formaldehyde concentration should not exceed 8.0 mg/ml.
DISCUSSION
The emission spectra suggest that the same excited species produces the luminescence, regardless of the oxidant or catalyst. The final oxidation product is sulphate in all instances, but the intermediate reaction steps are not well established.” Since ‘OH* radicals are assumed to be the source of energy for forming the excited species producing the luminescence, attempts were made to demonstrate their existence in the systems studied, by measuring the spectrum of the very weak chemiluminescence produced by reaction of the hydrogen peroxide-catalyst mixture with a buffered alkaline solution without sulphide. These attempts failed for the catalase and peroxidase systems, probably because the emission intensity was too
weak. However, a spectrum for the osmium tetroxide system was obtained (Fig. 13) and the maxima at 450, 480 and 580 nm correspond to those attributed to the emission from the excited double oxygen molecule (0202)*.19 This excited molecule is produced by collision of ‘OH2 radicals from the hydrogen peroxide decomposition.20~22 The nature of the emitting species formed by oxidation of the sulphide is not known. It is not the excited oxygen molecule described above, but it could be an excited S2 molecule similar to that involved in flame chemiluminescence emission,23,24 formed as an intermediate in the ‘OH2 oxidation of sulphide ions. However, the chemiluminescence measurements give little further evidence about the oxidation reaction mechanism. Other studies, e.g., ESR measurements, will be needed even to begin understanding the system. It has been established that the chemiluminescence measurements provide an extremely sensitive, precise, simple and rapid (analysis time 2 min per sample) method for the determination of sulphide. The methods described should be applicable to the determination of sulphide in natural waters, such as river, rain and drinking water, with a precision of 676 at the 10-3-102 ppm level. Sulphate does not interfere at concentrations likely to be found in natural water systems, and ions such as sulphite and thiocyanate can readily be eliminated by masking them with formaldehyde. Acknowlcdllrn~mr-J. L. Burguera zuela. for a scholarship.
thanks
Foninves.
Vene-
REFERENCES 1. D. B. Paul, Talanta, 1978. 25, 377. 2. S. S. Stieg and T. A. Nieman, Anal. Chen7.. 1977, 49. 1322. 3. W. R. Seitz and M. P. Neary. in Contmporary Topics in Analytical and Clikul C/tmistry. D. M. Hercules. G. M. Hieftje, L. R. Snyder and M. A. Evenson (eds), Vol. I, pp. 49-125. Plenum Press. New York, 1977. 4. S. N. Lowery. P. W. Carr and W. R. Seitz. Anal. Lett.. 1977, 10, 931. 5. W. R. Seitz and D. M. Hercules, in Chr,,,i/ur,lirlrscc~~~~,
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6.
7. 8. 9. 10.
I I. 12. 13.
J. L. BURCUERA and ALAN TOWNSHEND or~d Bio/u,tli,le.scc~nce,M. J. Cormier, D. M. Hercules and J. Lee, eds., Plenum Press. New York, 1973. Internurionul Symposium on Anulytical Applications of Biol~mlirlescr,lcr end Cltrmi/urni,lescrilce, Brussels, 6-8 September, 1978. G. Wettermark, S. E. Brolin and S. Hjertin, Cell. Molec. Biology, 1977, 22, 329. N. M. Lukovskaya and L. V. Markova. J. Anal. Chem. USSR, 1969, 24, 1868. N. M. Lukovskaya and N. I. Anatienko. Ory. Reayenty Analit. Khim. Tezisy Dokl., VW. Konj:, 1976, 2. 80. D. Klockow and J. Teckentrup, Talanta, 1976,23,889. K. Weber, A. Reiek and V. Vouk, Ber.. 1942, 75B, 1141. H. A. Neufeld, C. J. Conklin and R. D. Towner, Anal. Biochenl.. 1965. 12, 303. D. Slawinska, J. Slawinski, W. Pukacki and K. Polewski, in lnrrrnational Symposium on Analytical Applications ofBioluminescence and Chemiluminescence, Brussels. 6-8 September, 1978, Abstr. 43, p. 44.
14. G. AhnstrGm, G. Ehrenstein and R. Nilsson, Actor Chem. Stand., 1961, 15, 1417. 15. K. Gleu and K. Pfannstiel. .I. Pratt. Chem.. 1936, 146. 137. 16. L. Erdey, lnd. Chemist, 1957, 459, 523. 575. 17. J. L. Burguera and A. Townshend, Talanta, 1979, 26, 795. 18. C. H. Bamford and C. F. H. Tipper, Chemical Kinetics, Vol. 6. Elsevier, Amsterdam, 1972. 19. J. W. Haas. J. Chem. E&c., 1967, 44, 396. 20. J. Staufl, Z. Physik. Chem. (Frunkjitrt). 1964. 40, 64; 1966. 49, 58; 1967. 55, 39. 21. J. StauiT and H. Schmidkunz, ibid., 1962, 35. 295. 22. A. V. Khan and M. Kasha, J. Am. Chem. Sot.. 1966. 88. 1574. K. C. Thompson and T. S. West, 23. R. M. Dagnall, Analyst, 1967, 92, 506. D. J. Knowles and A. 24. R. Belcher. S. L. Bogdanski, Townshend. Anal. Chim. Acta. 1973. 67, I and references therein.