Vol. 5(4), pp.

66-72, April, 2013

DOI 10.5897/JTEHS13.0263 Journal of Toxicology and Environmental
ISSN 2006-9820© 2013 Academic Journals
http://www.academicjournals.org/JTEHS
Health Sciences

Full Length Research Paper

A spectrophotometric method for quantification of

sulphite ions in environmental samples
Peter Musagala1, Henry Ssekaalo2, Jolocam Mbabazi2 and Muhammad Ntale2*
1
Department of Chemistry, Busitema University, P. O. Box 236, Tororo, Uganda.
2
Department of Chemistry, Makerere University, P. O. Box 7062, Kampala, Uganda.
Accepted 12 April 2013

This study described an alternative method developed for the quantification of sulphite ions in
environmental samples. The method was based on results of an investigation of the reaction of excess
pentacyanidonitrosylferrate(II) popularly known as nitroprusside (NP) and the sulphite anion. NP-SO32-
reaction product by use of zinc-ethylenediamine complex cation(s) was stabilized. The NP-SO32-
reaction product was stabilized for 30 minutes by use of zinc ethylenediamine complex cation(s) and
the absorbance was enhanced, making determination of sulphite possible. The method has a limit of
quantification of 2.321 μg SO32- mL-1. Good accuracy was achieved for samples spiked with SO32- in the
range from 1 to 10 µg SO32- mL-1, which demonstrated the validity of the proposed procedure. The
repeatability (CV) was not more than 2.37% and the limit of detection was estimated at 0.99 μg SO32- mL-
1
. The method was applied to determine the concentration of sulphite ions in sugar and wine brands
sold in local market. Comparable results were obtained between this method and an iodometric
procedure for determination of sulphite in environmental samples.

Key words: Determination of sulphite ions, wine, sugar, environmental samples.

INTRODUCTION
2-
Sulphite anion (SO3 ) is a major species of sulphur in acid or acidified foods.
which oxidation state IV is expressed. The detection of However, excess sulphite in boiler waters is
the anion has long held the interest of the analytical deleterious, because it lowers the pH and promotes
community, because of the large number of roles that it corrosion. Control of SO32- in wastewater treatment and
can play within environmental and physiological systems discharge may be important environmentally, principally
(Isaac et al., 2006). It may occur in boilers and boiler feed because of its toxicity to fish and other aquatic life and its
waters treated with it for dissolved oxygen control, in rapid oxygen demand (American Public Health
natural waters or wastewaters as a result of industrial Association, 1998). Sulphites as additives can cause an
pollution, and in treatment plant effluents dechlorinated asthmatic reaction; presence of excessive amounts of
with sulphur dioxide. Sulphites or sulphiting agents are sulphites is respon-sible for off flavour in food products
the most common preservatives used in winemaking (McFeeters and Barish, 2003; Machado et al., 2008).
(Koch et al., 2010), and are also important additives in Some of it added to foods often disappears as a result of
many food products, because they inhibit development of reversible and irreversible chemical reactions. Thus, it is
both enzymatic and non-enzymatic browning in a variety often important to measure both free and bound forms of
of processing and storage situations (American Public sulphite that are present in foods.
Health Association, 1998; Claudia and Francisco, 2009). Several techniques have been developed to quantify
The sulphite ion is a very effective microbial inhibitor in the sulphite anion alone or in combination with other

*Corresponding author. E-mail: [email protected]. Tel: +256-392-967 223.

 Musagala et al. 67

sulphur species like sulphate, thiosulphate and an alternative spectrophotometric procedure for the
dithionate. These include: titrimetric methods with quantification of the sulphite ion in solution. Our method
potassium iodide-potassium iodate (American Public has been compared with an iodometric titration method
Health Association, 1998), copper sulphate (Shahine and for determining the concentration of sulphite ions in wine
Ismael, 1979), cerium(IV), mercury(II) (Crompton, 1996), and other beverages such as sugar that are commonly
spectro-photometric methods with 1,10-phenanthroline sold in local markets.
(American Public Health Association, 1998), Fuchsin N
solution (Badri, 1988), mercuric chloranilate (Humphrey
3+ MATERIALS AND METHODS
and Hinze, 1971), mercuric thiocyanate and Fe , electro-
chemical methods with mercury(I) chloride-mercury(II) Apparatus
sulphite electrodes (Marshall and Midgley, 1983) and the
sulphite oxidase enzyme electrode (Smith, 1987). Other Weighing was done on an AAA 160DL dual range balance (Adam
methods include molecular emission spectrometry Equipment Co. Ltd UK). The absorption spectra were recorded on a
UV-VIS Shimadzu UV-1700 CE double beam spectrophotometer
(Schubert et al., 1979), high performance liquid (Shimadzu Corporation, Japan) and absorbance measurements at
chromatography (HPLC) with ultraviolet (UV) detection a fixed wavelength were made with the same instrument in the
(McFeeters and Barish, 2003), ion-exchange chroma- photometric mode. pH measurements were done with a Corning
tography (Edmond et al., 2003), chemiluminescence Pinnacle 555 pH/ion meter (Corning Incorporated Life Sciences
methods (Al-Tamrah, 1987; Koukli et al., 1988) and flow Corning, New York, 14831 USA). The addition of aqueous
injection analysis techniques (Thanh et al., 1994; Araujo nitroprusside (NP) to aqueous SO32- was carried out using
Transferpette micro pipettes (BRAND GMBH + CO KG Postfach, 11
et al., 1998; Xiaoli and Wei, 1998; Atanassov et al., 2000; 55 97877 Germany).
Hasson and Spohn, 2001; Claudia and Francisco, 2009). All chemicals used were of analytical grade. The solutions used
The phenanthroline method is currently adopted as the were prepared as subsequently described.
standard one for determination of sulphite in water and
wastewaters, while the sulphite in foods and beverages is Starch indicator solution
determined by the traditional AOAC Official Method
990.28 (AOAC, 1995; Cunniff, 1995). The phenanthroline The starch indicator solution was prepared by dissolving analytical
method requires elaborate technical specification and grade soluble starch (2 g) and salicylic acid (0.2 g) in hot deionized
user expertise and as such, can incur substantial running water (100 mL).
costs. It involves purging of an acidified sample with
nitrogen gas and trapping the liberated sulphur dioxide Standard potassium iodate solution
gas (SO2) in an absorbing solution containing Fe3+ ion
and 1,10-phenanthroline. The Fe3+ ion is reduced to Fe2+ Potassium iodate (0.0021 mol/L) solution was prepared by
dissolving the solid (812.4 mg) in a minimum amount of deionized
by SO2, producing the orange tris(1,10-
water and diluted to 1000 mL.
phenanthroline)iron(II) complex as illustrated in the
following reaction schemes:
Standard sodium thiosulphate
2Fe3+(aq) + SO2(g) + 2H2O(l) → 2Fe2+(aq) + SO42-(aq) +
Sodium thiosulphate pentahydrate (Na2S2 O3.5H2 O, 6.204 g) was
4H+(aq) placed in a volumetric flask (1000 mL) and dissolved in deionized
water (50 mL). Sodium hydroxide (0.4 g) was added and the
Fe2+(aq) + 3phen(aq) → [Fe(phen)3]2+(aq), [phen = solution made to the mark with deionized water. The solution was
C12H8N2] then standardized using a standard potassium hydrogen iodate
solution as follows. Potassium iodide (2 g) was dissolved in an
In solution, the sulphite anion reacts with Erlenmeyer flask with deionized water (150 mL). Concentrated
sulphuric acid (3 drops) and starch solution (20 mL, 0.0021 mol/L)
pentacyanidonitrosylferrate(II) ion to form an unstable, were added, respectively. The solution was diluted to 200 mL and
red sulphite-nitroprusside ion ([Fe(CN)5(NOSO3)]4-) (Fogg the liberated iodine was titrated against the sodium thiosulphate
et al., 1966; Andrade and Swinehart, 1972; titrant with starch as the indicator. The concentration of sodium
Leeuwenkamp et al., 1984; Araujo et al., 2005). In our thiosulphate was found to be 0.025 mol/L.
laboratory, we succeeded in stabilizing both the sulphate
(Mbabazi et al., 2011) and the sulphite anions through a Standard iodine solution
series of spectrophotometric tests. The red sulphite-
nitroprusside reaction product was stabilized by Potassium iodide (25 g) was dissolved in a minimum amount of
water and iodine (3.2 g) was added. After dissolution of the iodine,
ethylenediamine complexes of zinc; the general reaction
the solution was diluted to 1000 mL and standardized against
scheme for this behavior being; Na2 S2O3.5H2O (0.025 mol/L) using starch solution as indicator.

[Fe(CN)5(NOSO3)]4-(aq) + [Zn(en)x]2+(aq) →
2- Sodium sulphite stock solution
[Fe(CN)5(NOSO3)][Zn(en)x] (aq)
The main objective of this study therefore was to utilize Fresh sodium sulphite stock solutions were prepared by weighing
the sulphite-nitroprusside stabilized product and describe sodium sulphite (0.5 g) and dissolving it in deionized water to make
68 J. Toxicol. Environ. Health Sci.

100 mL of aqueous solution. Determination of the sulphite ion mol/L) was pipetted into four different 50 mL volumetric flasks;
concentration was carried out iodometrically. ethylenediamine (30 µl, 13.66 mol/L) was added followed by
ethanol (10 mL) to stabilize SO32- in aqueous solution. A solution of
0.025 g of gelatin in 5 mL of deionized water was added followed by
Aqueous sodium nitroprusside (SNP) a sugar solution (200 µl). Three of the flasks were then spiked with
6, 8 and 10 µg SO32- mL-1. Zinc acetate solution (0.5 mL, 1 mol/l)
A fresh stock solution of SNP (0.02 mol/L) was used. This was was added; the mixture was diluted to the mark by addition of an
prepared by dissolving SNP crystals (6 g) in deionized water and appropriate amount of deionized water and was uniformly mixed for
the volume was made up to 1000 mL. 5 s. The resultant red solution was immediately transferred to
cuvettes which were inserted in the spectrophotometer to read the
absorbance. The concentration of the sulphite anion in the sample
Aqueous zinc acetate solution that was not spiked with SO32- was obtained from absorbance
versus concentration curve.
Zinc acetate (220 g) was weighed into a volumetric flask and
dissolved in deionized water. The solution was made up to 1000
mL. Iodometric determination of sulphite ion

Standard iodine (10 mL, 0.0125 mol/L) was measured from a

Aqueous ethylenediaminetetraacetic acid (EDTA) solution burette into a 250 mL conical flask. Sodium sulphite solution (2 mL)
was added, the excess iodine was back titrated against standard
The sodium salt of EDTA (0.0372 g) was dissolved in deionized Na2 S2O3.5H2O (0.025 mol/L) using starch as the indicator.
water and the solution was made up to 10 mL.

Iodometric determination of sulphite ion in sugar

Spectrophotometric procedure for determination of sulphite
ion Sugar (1.0 g) was dissolved in deionized water to make 10 mL of
solution. Starch (0.5 mL) was added and the resultant mixture was
This was done as per our validated stabilization procedure. SNP titrated against standard iodine solution.
(4.5 mL, 0.02 mol/L) was pipetted into in a 50 mL volumetric flask;
EDTA (100 µl, 0.01 mol/L) was added followed by ethylenediamine
(30 µl, 13.66 mol/L) and ethanol (10 mL). A solution of 0.025 g of Iodometric determination of sulphite ion in wine
gelatin in 5 mL of deionized water was added followed by perchloric
acid to lower the pH to 6.5. An aliquot of intermediate standard Wine (10 mL) was pipetted into a conical flask. Sodium hydroxide
aqueous SO32- followed by zinc acetate solution (0.5 mL, 1 mol/L) solution (1.6 mL, 4 mol/L) was added to release the bound SO 32-.
was added; the mixture was diluted to the mark by addition of an The mixture was left to stand for 5 min. Sulphuric acid (1.7 mL, 10%
appropriate amount of deionized water and uniformly mixed for 5 s. v/v) was added followed by starch solution (0.5 mL). The resultant
The resultant red solution was immediately transferred to cuvettes mixture was titrated against standard iodine solution.
which were inserted in the spectrophotometer for scanning its
spectrum. A stable absorbance at λ max 482 nm was read off and
then using a pre-prepared absorbance versus concentration Method validation
calibration curve, the concentration of the sulphite ion was
determined. A validation was carried out on the developed method and the
following characteristics were evaluated; working range and
linearity, accuracy, precision, selectivity and detection limits.
Spectrophotometric determination of total sulphite in wine

Wine was treated to release matrix bound SO32- as follows: sodium

Working range and linearity
hydroxide solution (1.6 mL, 4.0 mol/L) was added to 10 mL of the
sample to release the bound SO32-. The mixture was left to stand for
The linearity of the method was evaluated by using calibrators in
5 min and the pH of the solution was adjusted to 8.5 using sulphuric
the entire working range of 1 to 10 µg mL-1 for the analyte. The
acid. SNP solution (4.5 mL, 0.02 mol/L) containing EDTA (4.5 µl,
curve was a plot of the absorbance of the sulphite-nitroprusside
0.01 mol/L) was pipetted into four different 50 mL volumetric flasks.
reaction product against concentration. The regression equation
Ethylenediamine (30 µl, 13.66 mol/L) was added to each flask
with the slope, intercept and correlation coefficient (r 2) was
followed by ethanol (10 mL) to stabilize SO32- in aqueous solution. A
generated using Microsoft Excel. Limit of quantification formed the
solution of 0.025 g of gelatin in 5 mL of deionized water was added
lower end of the working range. The linearity was established using
(to avoid bubbling in the ethanol stabilized solutions) followed by
the square of correlation coefficient value (r2) of the line of best fit.
treated wine (200 µl). Three of the flasks were then spiked with 6, 8
and 10 µg SO32- mL-1. Zinc acetate solution (0.5 mL, 1 mol/L) was
added; the mixture was diluted to the mark with deionized water
Accuracy and precision
and was uniformly mixed for 5 s. The resultant red solution was
immediately transferred to a cuvette which was inserted in the
Accuracy was determined by calculating the mean recovery of the
spectrophotometer to read the absorbance. The concentration of
seven portions spiked with standard SO32- solutions at three
the sulphite anion in the sample that was not spiked was obtained
concentration levels, all within the working range. The accuracy was
from absorbance versus concentration curve.
then expressed as;

Spectrophotometric determination of sulphite ion in sugar y

Accuracy   100%
SNP solution (4.5 mL, 0.02 mol/L) containing EDTA (4.5 µl, 0.01 z
 Musagala et al. 69

Table 1. The accuracy and precision of the developed nitroprusside method at three concentration levels.

2- -1 2- -1
Nominal concentration (μg SO3 mL ) Concentration measured (μg SO3 mL )*( mean ± SD) CV (%; n = 7)
1.521 1.466 ± 0.035 2.368
2.500 2.457 ± 0.046 1.888
5.031 4.846 ± 0.098 2.028
*Mean ± SD, at 95% confidence interval.

where y was the mean value of the concentration of the seven period of 30 min. The range of ethylenediamine concen-
replicates and x was the spiked (nominal) concentration. The intra- tration used was 5.5 to 22 mmol/L, above this concen-
batch variability in the measurement of the sulphite ion concen-
tration (precision) was calculated from seven repeat determinations
tration the absorbance was further enhanced but the
of spiked samples and was expressed as a coefficient of variation product was found to be very unstable. The spectro-
(CV). photometric determination of the SO32- from its reaction
with NP was carried out in aqueous slightly acidic media
Selectivity by using ethylenediamine and zinc concentrations of 11
and 10 mmol/L, respectively. This was done without alkali
Several cationic and anionic species such as sulphide, metal cations. Absorbance readings were made after a
thiosulphate, hydrogen phosphate, chloride, copper (II), iron (II) period of 5 to 10 min.
potassium and sodium ions were tested for interference. Different
amounts of the ionic species were added to a solution containing
Mbabazi et al. (2011) noted that an excess of NP not
sulphite ions of known concentration and the extent of interference greater than ten-fold is sufficient for analysis since the
caused by each species was investigated. absorbance increases with NP concentration. A similar
observation was also noted in this study. Again, it was
Detection and quantification limits noted that temperature, pH and the ratio of excess NP to
SO32- exerted a significant effect on stability of the pro-
The limit of detection (LoD) was defined as the lowest concentration 2-
duct of the NP-SO3 reaction. An optimum pH of 6.5 was
of sulphite ion in a sample that could be detected but not
found to be satisfactory for our purpose.
necessarily quantified under stated conditions of the developed
method. In this work, LoD was determined by analyzing 10 in-
dependent sample blanks for sulphite ion amount and the standard
deviation (SD) among the values determined was calculated. LoD Working range and linearity
was then expressed as the sulphite ion concentration
corresponding to the mean sample blank value + 3SD The working range was found to lie between 1 and 10 µg
The limit of quantification (LoQ) was defined as the lowest SO32- mL-1; above the upper limit, the decomposition of
concentration of sulphite ion that could be determined with an
the product was relatively fast and often characterized by
acceptable level of repeatability, precision and trueness. In this
work, LoQ was determined by analyzing 10 independent sample precipitation with time. The linearity of the method as
blanks for sulphite ion amount and SD was calculated. LoQ was measured by the correlation coefficient of inter-assay
then expressed as the sulphite ion concentration corresponding to linear regression curves (r2) was better than 0.99 in all
2- -1
the mean sample blank value + 10SD. cases in the measured range of 1-10 µg SO3 mL which
was indicative of a good linear relationship between SO32-
RESULTS AND DISCUSSION concentration and absorbance.

A comparison of sulphite content in wine and sugar using

both NP and an iodometric titration method was made Accuracy and precision
and the results are shown in Tables 3 and 4. A comparison of the nominal concentration of the spiked
A lot of attention has been drawn to the concentration solutions for seven replicates at three concentration
of sulphite ions in environmental systems because of levels with the concentration measured at each of the
their associated health effects. A method that is simple levels showed very good accuracy at all the three
but accurate is therefore essential for monitoring the concentration levels (Table 1), an indication that the
levels of the sulphite ions in such systems.
developed method was fit for the intended purpose. The
precision of the method was also very good (CV not more
Method development and validation than 2.368) in all cases, again suggesting that the me-
thod was precise in the concentration levels considered.
The optimum conditions chosen for this work were those
that had been previously validated in our laboratory
(Musagala, Busitema University, unpublished); in which Selectivity
ethylenediamine in the presence of zinc was found to
stabilize the sulphite-nitroprusside reaction product for a The effect of some cations and anions on the method
70 J. Toxicol. Environ. Health Sci.

Table 2. The effect of a number of anions and cations on the nitroprusside method for SO 32- in
the presence of ethylenediamine, using 3.10 μg SO 32- mL-1 for testing.

Foreign ion added Interference concentration (μg/mL) SO32- recovered (μg/mL)*

2-
S 1000 ND
2-
S2O3 1000 ND
HPO42- 1000 ND
-
Cl 1000 3.05 ±0.02
Cu2+ 100 ND
Fe2+ 100 ND
+
K 1000 3.05 ±0.02
+
Na 1000 3.05 ±0.02
*Mean ± SD, at 95% confidence interval; ND = Not detected.

Table 3. Comparison of the developed nitroprusside method to iodometric titration method for
the determination of sulphite in wine.

Concentration of SO32-µg-1 mL-1

Type of wine
Nitroprusside method Iodometric titration
65.0 ± 6.6 70.0 ± 8.4
68.0 ± 6.6 70.0 ± 8.4
Baron de Vignon semi-dry white wine 58.0 ± 6.6 60.0 ± 8.4
60.0 ± 6.6 60.0 ± 8.4
50.0 ± 6.6 50.0 ± 8.4

47.0 ± 8.4 50.0 ± 8.4

68.0 ± 8.4 70.0 ± 8.4
Bellingham dry white wine 58.0 ± 8.4 60.0 ± 8.4
60.0 ± 8.4 60.0 ± 8.4
50.0 ± 8.4 50.0 ± 8.4

69.0 ± 6.9 70.0 ± 7.4

50.0 ± 6.9 50.0 ± 7.4
King fisher strawberry fruit red wine 63.0 ± 6.9 65.0 ± 7.4
60.0 ± 6.9 60.0 ± 7.4
59.0 ± 6.9 60.0 ± 7.4
*Mean ± SD, at 95% confidence interval.

was studied in detail by adding different amounts of ionic leading to low results.
species as shown in Table 2. The greatest anionic However, the effect of copper can be avoided by
interference to this method would be expected to come adding a complexing agent such as EDTA during sample
2-
from S that reacts with NP in a similar manner to collection as this would inhibit copper (II) catalysis and
produce a red product ([Fe(CN)5(NOS)]4-) with λmax 538 promote oxidation of iron (II) to iron (III) before analysis.
nm at pH above 10. Sulphide also forms a sparingly Sulphide can be removed by adding about 0.5 g of zinc
2+
soluble precipitate with Zn in the reaction mixture. Other acetate and thereafter analyzing the supernatant of the
ionic species were also found to interfere during the settled sample while thiosulphate can be determined
determination of sulphite as indicated in Table 2. The independently using a simple iodometric titration.
presence of oxidizable species such as sulphide, It is therefore recommend that the method be employed
thiosulphate, phosphate, and iron may lead to high levels mainly for the determination of sulphite in wine and
of sulphite. Copper is known to catalyse oxidation of sugar, but its application could be extended to relatively
sulphite to sulphate when the sample is exposed to air clean waters as is always the case with the iodometric
 Musagala et al. 71

Table 4. Comparison of the developed nitroprusside method with iodometric

titration method for the determination of sulphite in sugar.

2- -1 -1
Concentration of SO3 mg kg
Type of sugar
Nitroprusside method Iodometric titration
28.0 ± 5.4 30.0 ± 5.5
29.0± 5.4 30.0 ± 5.5
Kinyala white sugar 28.0 ± 5.4 30.0 ± 5.5
18.0 ± 5.4 20.0 ± 5.5
19.0 ± 5.4 20.0 ± 5.5

18.0 ± 5.9 20.0 ± 5.5

27.0 ± 5.9 30.0 ± 5.5
Kakira white sugar 29.0 ± 5.9 30.0 ± 5.5
30.0 ± 5.9 30.0 ± 5.5
18.0 ± 5.9 20.0 ± 5.5

30.0 ± 5.0 30.0 ± 5.5

Kenya Brown Sugar 21.0 ± 5.0 20.0 ± 5.5
19.0 ± 5.0 30.0 ± 5.5
28.0 ± 5.0 20.0 ± 5.5
29.0 ± 5.0 30.0 ± 5.5
*Mean ± SD, at 95% confidence interval.

method for the determination of sulphite in environmental inevitably shifted the equilibrium making the deter-
samples (American Public Health Association, 1998). mination of free sulphite difficult. In this work, relatively
Fogg et al. (1966) reported that several organic high values of sulphite were obtained for red wine and
compounds, including thiols, amines, and ketones were this was attributed to the colour of the wine.
also known to form coloured compounds with the NP ion,
but usually only in strongly alkaline solution. However, at
pH of 6.5 used for the nitroprusside-sulphite reaction, the Conclusion
aforementioned compounds did not interfere. Tin(II),
ferrocyanide and arsenates gave white precipitates with A manual spectrophotometric method has successfully
the reagents, but these also did not interfere with the been developed and validated for the quantification of
sulphite determination. micro quantities of sulphite ions based on the modified
reaction conditions of the nitroprusside-sulphite ion (NP-
SO32-) reaction.
Detection and quantification limits The method in the determination of sulphite ions has
also been applied in selected environmental systems
The LoD and LoQ for the NP-SO32- reaction method were such as wine and sugar. The results obtained by the de-
found to be 0.99 μgSO32- mL-1 (Blank + 3SD) and 2.321 veloped method compared well with those obtained using
2- -1
μgSO3 mL (Blank + 10SD), respectively. The obtained an iodometric titration method for SO32- determination.
detection limits showed that the method could be applied
in the detection and quantification of SO32- concentrations
as low as 1 μg mL-1. ACKNOWLEDGEMENT

The authors acknowledge with gratitude the financial

Applicability of the developed method for support of the Germany Academic Exchange Service
determination of sulphite ion in wine and sugar (DAAD).
The total sulphite content in wines is the sum of the free
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