Spectrophotometric Determination of

Sulphide and Fluoride

Ho Chak Ming

(何澤明）

A thesis submitted in partial fulfilment of

the requirement for the degree of

Master of Philosophy in

Division of Chemistry

Graduate School

The Chinese University of Hong Kong 1994

Thesis Committee: Dr. K. 丫. Hui, Chairman Dr. O. W. Lau Dr. R. W. M. Kwok

Prof. J. D. R. Thomas, External Examiner •

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/ 1 Acknowledgment

I wish to express my deepest gratitude to my supervisor, Dr. O. W. Lau, for her invaluable advice and encouragement during the course of the research and the preparation of this thesis.

Department of Chemistry

The Chinese University of Hong Kong

June, 1994.

Ho Chak Ming

i Table of Contents Page

Acknowledgment i

Table of contents ii List of figures iv List of tables v Abstract ix

Part 丨 Determination of sulphide General introduction 1

Spectrophotometric determination of sulphide 5

1. Introduction 5 A. Review of the reported methods for the determination of sulphide 5 B. General description of the proposed method 13

2. Experimental section 18 A. Preservation of samples 18 B. Sample pre-treatment 19 C. Separatio门/preconcentration of sulphide 19 D. Spectrophotometric determination of sulphide by proposed method 24 E. Spectrophotometric determination of sulphide by standard methylene blue method as the counter-check method 32 3. Results and discussions 34 A. Optimization of the proposed spectrophotometric method 34 B. Optimization of the separation/preconcentration methods 45 C. Construction of the calibration graph for the spectrophotometric determination of sulphide and determination of the molar absorptivity coefficient 53 D. Precision and detection limit of the proposed spectrophotometric method 54 E. Interferences studies of the proposed method 56 F. Spectrophotometric determination of sulphide in water 60 G. Spectrophotometric determination of sulphide in beers 63 H. Spectrophotometric determination of sulphide in orange juices 66 4. Conclusions 68 References 70

ii Part II Determination of fluoride General introduction 75 Spectrophotometric determination of fluoride 78 1. Introduction 78 A. Review of the reported methods for the determination of fluoride 78 B. General description of the proposed scheme for the spectrophotometric determination of fluoride in animal feeds 84 2. Experimental Section 87 A. Ashing of the animal feed samples 87 B. Reduction of interferences in the ashed sample solutions using anion exchange resin 89 C. Preparation of solutions for the calibration graph and the ashed sample solutions for the spectrophotometric measurement 90 D. Spectrophotometric determination of fluoride in the ashed samples 92 E. Potentiometric determination of fluoride in the ashed sample solutions by the FISE method as the counter-check method 93 3. Results and discussions 94 A. Ashing conditions of the animal feed samples 94 B. Removal of the interferences in the ashed sample solutions 99 C. Construction of the calibration graph for the spectrophotometric determination of fluoride 112

D. Precision of the spectrophotometric method 113 E. Spectrophotometric determination of fluoride in animal feeds 114 F. Potentiometric determination of fluoride in animal feeds by the FISE method as the counter-check method 117

4. Conclusions 120 References 122

iii List of Figures Figure no. Figure title Page Part I Determination of sulphide Summary of the sources of sulphide and its effect 4 1-1 Reaction of ortho-hyclroxy(mercuri)-benzoate with sulphide 7 1-2 Reaction of para-chIoro(mercuri)-benzoate with sulphide 9 1-3 Structures of dithizone and the complex with

organomercury compound 14 1-4 Equilibria in two-phase system (water-tetrachloromethane) involving dithizone, o-carboxyphenylmercury cation and the complexes formed over different pH ranges 14 1-5 Reaction of para-hydroxy(mercuri)-benzoate with sulphide 15 1-6 Illustration of the Beer's law 16 1-7 Experimental set-up for purging 21 1-8 Experimental set-up for distillation 23

1-9 UV spectrum of the reaction system at various concentrations of sulphide after the extraction 35 I-10 Calibration graph of sulphide with absorbance at 237 nm

plotted against the amount of sulphide (^g) 54

Part II Determination of fluoride II-1 Structure of SPADNS 86

11-2 XRF spectrum of a rat feed sample 95

11-3 Calibration graph of fluoride with absorbance at 570 nm plotted against the concentration of fluoride (ppm) 112

iv List of Tables Table no. Table title Page Part I Determination of sulphide Summary of the reported methods for the determination of sulphide 5 1-1 Tables showing the preparation of the buffers used 27 1-2 Effect of pH on the spectrophotometric method 37

1-3 Effect of the addition of saturated sodium chloride solution to the reaction system on the absorbance measured and the time of separation of phases required 39 1-4 Effect of the extracting medium to the spectrophotometric method 40 1-5 Effect of time on the spectrophotometric measurement after

the extraction 41

1-6 Effect of the amount of dithizone used on the

spectrophotometric method 42

1-7 Effect of the amount of the mercury reagent used on the

spectrophotometric method 43

1-8 Effect of the order of addition of the reagents on the spectrophotometric method 44 1-9 Effect of purging time at a purging rate of 7 ml per minute on the recovery of 5.00 p-g of sulphide in 80 ml of sample solution using the set-up of Lindell,包 0 46 1-10 Effect of purging rate with a purging time of 25 minutes on the recovery of 5.00 jig of sulphide in 80 ml of sample using the set-up of Lindell,包 0 46 1-11 Effect of purging rate for 25 minutes of purging on the recovery of 5.00 ^g of sulphide in 80 ml of sample using the modified experimental set-up 48 1-12 Recoveries of 5.00 ^g of sulphide in 80 ml of solution

using the modified experimental set-up at different purging

time, the flow rate being 250 ml/min 48

1-13 Effect of the volume of 0.10 M sodium hydroxide solution

used for the absorption of sulphide generated from 80 ml

of solution containing 5.00 |ig of sulphide 49

vii 卜14 Effect of the concentration of 1.00 ml of sodium hydroxide solution used for the absorption of sulphide generated from 80 ml of solution containing 5.00 jig of sulphide 50

1-15 Recoveries of sulphide using purging as the separation method 51 1-16 Recovery test for the distillate collected at different period of time for 100 ml of sample solution containing 4 ^g of sulphide (0.04 ppm sulphide solution) 52 1-17 Recovery of the distillation procedure in couple with the proposed spectrophotometric method utilizing 100 ml of sample of various concentrations of sulphide for the collection of about 15 ml distillate 52 1-18 Data for the calibration graph for the determination of sulphide using para-hydroxy(nnercuri)-benzoate as a reagent 53 1-19 Precision test for the determination of sulphide 55 1-20 Effect of some common ions in water samples on the determination of 4 ^g of sulphide (20 ml of 0.2 ppm sulphide solution) using the proposed spectrophotometric method but without purging or distillation 57 1-21 Effect of some ions on the determination of 4 fig of sulphide (40 ml of 0.1 ppm sulphide solution) using the proposed spectrophotometric method with purging 58 1-22 Effect of some ions on the determination of 4.00 |j.g of sulphide (100 ml of 0.04 ppm sulphide solution) using the proposed spectrophotometric method with distillation 58 1-23 Comparison of the tolerance limits of various ions

obtained using respective purging and distillation before spectrophotometric measurements 60 1-24 Determination of sulphide in water samples using the proposed spectrophotometric method with purging and distillation procedure and the methylene blue method 61 1-25 Recovery test for the determination of sulphide ion in the water samples using the proposed spectrophotometric method with purging 62

1-26 Recovery test for the determination of sulphide ion in the

water samples using the proposed spectrophotometric

vi method with distillation 63 1-27 Determination of sulphide in beer samples 65 1-28 Recovery test for the determination of sulphide ion in beer samples using the proposed spectrophotometric method with purging 66 1-29 Determination of sulphide in orange juice samples with purging as the separation/preconcentration for both the proposed spectrophotometric and the methylene blue method 67 1-30 Recovery test for the determination of sulphide ion in orange juice samples using the proposed spectrophotometric method with purging 67

I-31 Comparison of the methylene blue method and the proposed spectrophotometric method 69

Part II Determination of fluoride Suggested tolerance of fluoride ion to various kinds of animals 77 II-1 Time required for ashing 1 g of sample with 2.5 g of sodium hydroxide at different ashing temperatures 97 11-2 Effect of the diameters of the column on the adsorption and elution of fluoride from the ashed sample solutions with fluoride determined using the FISE method 105 11-3 Effect of the amount (length) of the resin used in the separation of the interfering substances in the ashed sample solutions with 0.15 M sodium hydroxide as the eluent with fluoride determined using the FISE method 107 11-4 Effect of the amount (length) of the resin used in the separation of the interfering substances in the ashed sample solutions with 0.15 M sodium hydroxide as the eluent with fluoride determined using the spectrophotometric method 108 11-5 Effect of the concentrations of sodium hydroxide on the elution of fluoride from a column with a resin length of 50 cm with fluoride determined using the FISE method 109

11-6 Suitable lengths of resin required for the separation of fluoride with the use of different concentrations of

vii sodium hydroxide solution 110 11-7 Data for the calibration graph for the spectrophotometric determination of fluoride 113 11-8 Animal feed types and their sources 114 11-9 Recoveries of fluoride in the ashed sample solutions determined by the spectrophotometric method after the anion exchange clean-up 115 11-10 Analysis of animal feed samples by the proposed method and results for the recoveries of added fluoride 116 11-11 Determination of fluoride content in animal feed samples using the proposed method 116 11-12 Composition and preparation of buffers for the FISE method 118 11-13 Amount of fluoride in animal feed samples found using the FISE method with the inclusion of the results obtained from the proposed spectrophotometric method 120

viii Abstract

A spectrophotometric method for the determination of sulphide has been developed. The method is based on the reaction between sulphide and para- hydroxy(mercuri)-benzoate where the excess reagent is removed by extraction with dithizone dissolved in chloroform. The working range of the method was 0.25 to 10.0 g of sulphide for 30 ml of sample solution and the precision was within 1 %. Owing to the low concentration of sulphide in water, two separation/preconcentration techniques

had been incorporated into the spectrophotometric method. The techniques involve the generation of hydrogen sulphide from the sample under an acidic, inert

atmosphere. Hydrogen sulphide is then separated by distillation or purging with

nitrogen and is subsequently absorbed in sodium hydroxide solution. Waste water

samples obtained from several sewage treatment works were tested and satisfactory

results were obtained and compared with those obtained using the reference

methylene blue method. The average recoveries of the proposed spectrophotometric

method following purging or distillation were 98 % and 101 %, respectively.

An ashing procedure for the determination of fluoride in animal feeds has been

developed, which includes ashing the animal feed samples with sodium hydroxide and

silica in a nickel crucible at 550°C for three hours, dissolving the cake formed after the

ashing with hot water, and adjusting the pH of the solution to 8 with hydrochloric acid.

Interferences were observed for the spectrophotometric determination of fluoride

usinq Zr4+ -SPADNS (sodium 2-(parasulfophenylazo)-1,8-clihydroxy-3,6-naphthaIene

disulfonate) as a reagent, and were removed by anion exchange with Amberlite IRA-

400 (0H") using 0.15 M sodium hydroxide solution as the eluent. The amount of

fluoride in the eluate, which was adjusted to pH 8 with hydrochloric acid, was then

determined. Using the proposed procedure, the recovery of fluoride was 93 % and the

ix precision was 3 %. Potentiometric determination using a FISE (fluoride ion selective electrode) was used as a counter-check method for the proposed procedure.

X Part 丨 Determination of sulphide General introduction

Sulphide and its acidic form, hydrogen sulphide, are toxic JThey react with metals and metal compounds and affect metallic structures and functions J In addition, hydrogen sulphide has an unpleasant odourJ"^'

Exposure of human under the environment containing high levels of hydrogen sulphide can cause disturbances to the respiratory and centra卜nervous systems and even death.18 Even at low concentrations of hydrogen sulphide, prolonged exposure will affect the reflex response^, heart function^ ‘ ^ and the eyes^ ‘ 3-4, 6

Other symptoms such as headache^'®, nausea and vomiting^ have also been reported. The toxicity of sulphide (or hydrogen sulphide) originates from its great tendency to combine with metal ions essential to the metabolism of the living organisms^, (under physiological pH value, one of the dissociation products of hydrogen sulphide is sulphide^®) and results in metabolic disturbances. Under high concentrations of sulphide, inhibition of oxygen transfer and damage of nerve tissues will occur.6 In addition, sulphide is harmful to other living organisms such as birdsJ

Sulphide can cause direct^ ‘ 6' 12 and indirect damages13-14 to metallic and concrete structures because hydrogen sulphide is corrosive and its oxidation product, sulphuric acid, formed through bacterial action can attack concrete sewer J 2-14

Owing to the high affinity of sulphide for metals, it is a poison to the metal catalysts. The metallic sulphides formed in the electric metallic circuitries affect the electric function.‘‘ Sulphide changes the color of paint^' 6' 12 and plastics】 as metals (such 3S lead compounds) have been used as the color pigments or internal fungicides and internal stabilizer or lubricant, respectively.

The rotten-egg smell of hydrogen sulphide is very offensive''''^' 6 and causes irritation to the olfactory function of human^' 7. its odor threshold of human is very

1 low, and reported to be about 0.0005 ppm by volume^' ”」^ [other different threshold levels (0. O25I, 0.0353 or 0.10l' 3 ppm) had also been reported].

Basically, there are two sources of sulphide: operations involving biological activities and those involving industrial processes. Processes such as the paper and pulp making Kraft process^‘ those in the petroleum refining^' 12, 14, coal gasification 1' ^^ and refuse disposal^, tar distillation^, grease refining^, natural gas processing^ ‘ 14, viscose processing^ ‘ 12, chemicals production^ ‘ 4' 12, 14, dye-making and dyeing (textile factories using sulphur dyesp' 14, meat processing 1 and sewage treatment or purification^ ‘ 14, artificial silk production^ are the ways where sulphide can be found and produced. Industrially, hydrogen sulphide is formed from the reductive conversion of the elemental sulphur or its-containing compounds present in the industrial materials」Biologically, it is generated by the anaerobic action of the micro-organisms in nature on the organic matters containing sulphur such as thio-proteins, thio-amino acids''' 4' 6-7, 14, and mineral compounds containing sulphur^ 16. Apart from the conversion of sulphur- containing compounds, sulphide can be found in processes where it is used as a reagent. Sodium sulphide is used in the Kraft process. The concentration of sulphide solution used in the process needs to be controlled J ^

Three kinds of sulphides can be determined in the waters. They are the total sulphide, the dissolved sulphide, and the un-ionized (free) hydrogen sulphide.12, 16,

18 Total sulphide in the water sample consists of the dissolved sulphide (that is described below), the hydrogen sulphide anion (HS"), and the acid-soluble metallic sulphides that are present in the suspended solids of the samples. The amount of the dissolved sulphide is found from the amount of sulphide present in the water sample after excluding the suspended solids or particles. The concentration of the un-ionized hydrogen sulphide in the water is affected by a number of factors: the pH of the

2 samples, the concentration of the dissolved sulphide and the practical ionization constant of hydrogen sulphide.

The amounts of sulphide (or hydrogen sulphide) present in foods affect their aroma and flavour and the levels of sulphide in natural foods are taken as guidelines for the levels of sulphide in artificial or processed foods J Hydrogen sulphide in food is coming from the oxides of sulphur and the sulphur-containing amino acids such as cysteine, methionine, and proteins where they are broken down by enzymatic processes or Strecker degradation during heating process. Hydrogen sulphide is found in the headspace gases above fresh juices from all the major citus cultivars.22-26 Foods such as milk22-23, 27 gnd meat22-23, 28 have generated hydrogen sulphide (from p-lactoglobulin in milk and glutathione and cystine in meat) during cooking, which is one of the sources of aroma of the food. Other foods, including natural cheese^^' vegetables such as onion, tomato, cabbage and pea, etc., marine products such as canned salmon, and nonalcoholic beverages such as coffee (when overheated or kept too long} and tea^^, have hydrogen sulphide found in the flavor. Also, it has been reported that the odour produced by a mixture of compounds is distinct and cannot be found in the corresponding individual component.

In addition, the strong odor of hydrogen sulphide is the reason for the necessity to lower its formation during fermentation in alcoholic beverages, including beers27 and wines22-23, 27 Another reason for its removal is owing to its reducing character that affects the redox balance in beer production.27

The above facts and reasons lead to the need for the development of sensitive analytical methods for the monitoring of the level of sulphide in the materials in the industrial processes and the environment around us. A typical example is that in Hong Kong, the Environment Protection Department has set up the

3 upper limit for the amount of sulphide present in the effluents that discharges into various waters.扣

A summary of the sources and effect of sulphide is shown below.

Other forms of sulphide

J- ,.., ^ sulphide from sulphide present as . ,..., • . , . . . . organic compounds sulphide forms inorganic sulphide m . . ^ . , .‘ • • ° , , . . , such as thio-proteins from its inorganic the form of sulphide, , “ .. ,... , from meat species such as hydrosulphide and , , . , ,, ,.... processing and sulphate and hydrogen sulphide in 尸 ^ " . , . ^ .."., + sewage treatment thiosulphate industrial wastes , ^ ^ 卜 plant,etc.

X formation of sulphide by bacterial action 4 under anaerobic conditions ^

X

total sulphide

Toxic to living . organisms Odor Co?sive by (by nusiance atta，ng Affects the combining to human metals and aroma and with metal (rotten egg J^onci^ete flavour of ions present smell) directly or foods in the indirectly metabolism)

Summary of the sources of sulphide and its effect

4 Spectrophotometric determination of sulphide 1. Introduction (A). Review of the reported methods for the determination of sulphide

In the determination of sulphide, a number of difficulties were encountered.

Its determination was subjected to errors because of oxidation, probably by dissolved oxygen, volatilization of hydrogen sulphide in an acidic samples and precipitation with metal ions usually under alkaline medium.

From the literature, a variety of methods had been used for the determination of sulphide. They involve titrimetry, polarography, potentiometric method using ion selective electrode, spectroscopy including spectrophotometry and spectrofluorimetry and chromatography including ion chromatography and high performance liquid chromatography, etc., and are summarized below.

Summary of the reported methods for the determination of sulphide

Methods employed I the main reagent(s) used References

1. Titrimetry

(a), lodometry 14, 15, 16, 18, 31

(b). Use of ortho-hydroxy(mercuri)-benzoate 32, 33, 34

2. Spectrophotometry (a). Methylene blue method 1, 12, 14, 15, 16, 18, 35, 36 (b). Nitroprusside method 37, 38 (c)_ Formation of molybdenum blue 39, 40 (d). Use of iron (III) and 1,10-phenanthroline 41 (e). Use of iron (III) and nitrilotriacetic acid 42 (f). Use of iodate and 2',7'-dichlorofluorescein

5 3. Spectrofluorimetry (a).(i). Use of Hg"-2,2'-pyridylbenzimidazole 44 (ii). Use of Cu"-2-(o-hydroxyphenyl)benzoxazole 45 (b). Reduction of 1,2-naphthoquinone-4-sulphate 9, 46 4. Photometric titration

Use of para-chloro(mercuri)-benzoate £7

5. Polarography (a). Anodic polarography (i). Rapid direct current polarography 48 (ii). Differential pulse polarography 49 (iii). Normal pulse polarography 50 (b). Cathodic stripping polarography

6. Chromatography (a). Ion chromatography 52, 53

(b). High performance liquid chromatography 54, 55

(c). Gas chromatography 56, 57

7. Electrochemical methods

The use of ion selective electrode 14, 58, 59

8. Miscellaneous

(a). Molecular emission cavity analysis 60, 61, 62 (b). Atomic absorption spectroscopy 63

(c). Gravimetry 64 (d). Catalytic and kinetic (iodine and azide) reaction 65 (e). Enzymatic (oxidases and peroxidase) reaction ^

The determination of sulphide using iodine solution as a reagent through back-titration with thiosulphate solution''^-l6, 18, 31 are suitable for samples with high level of sulphide ion, however, the method is subjected to interferences such as thiosulphate present in the samples.

A more selective titrimetric method has employed ortho-hydroxy(mercuri)- benzoate32-34 (o-HMB) as a titrant which reacts with sulphide, using dithizone as an indicator of the end-point. Figure 1-1 shows the equation for the reaction. The color

6 change at the end-point is affected by the pH and the volume of the sample used with a comparatively less distinct end-point under a low pH and high sample volume.

These titrimetric methods are not accurate enough in comparison with other methods such as spectrophotometric method.

COO —

ortho-hydroxy(nnercuri)-ben2oate (o-HMB)

coo 一 "OOC \

Figure 1-1 Reaction of ortho-hvdroxv(mercuri)-benzoate with sulphide

Most of the handbooks have suggested the methylene blue method 1'

14-16, 18, 35-36 fQp the determination of sulphide in water samples. This method involves the measurement of absorbance of methylene blue that is formed from the reaction of sulphide with N,N-dimethyl-p-phenylenediamine in the presence of iron

(III) ion. The accuracy of this method is not good and is reported to be 土 10% if very careful use of the experimental conditions and technique are made J ® Moreover, the presence of some common ions in the water such as sulphite, thiosulphate and iodide, etc., interfere with the reaction by reducing the iron (III) ionJ® Side reactions, including dimerization of the dye and formation of other dyes such as methylene red'', are also found and affect the results. Also, it has been reported that the complete formation of the blue color of methylene blue requires as long as three hours. The lower the pH value of the solution, the higher the amount of sulphide exists as hydrogen sulphide form J ^ (under a temperature range of 20° to 30°C and a dissolved mineral content below 2000 mg per liter). As the reaction is carried out in an acidic medium and the reaction is not immediate, these may contribute to an

7 error to the determination due to the loss of sulphide as its acidic gaseous form.

Further, it is difficult to analyze turbid or colored waters using this method because

these substances affect the absorption of the system and hence the accuracy,

precision and sensitivity of this analytical method may be reduced.

Some spectrophotometric methods utilize the reducing character of sulphide,

including the reaction of sulphide with nitroprusside37-38, the formation of

molybdenum blue from molybdate in an acidic medium^^"^^ and iron-(1,10-

phenanthroline) (II) from iron (III) and 1,10-phenanthroline^^. Probably, the presence

of reducing substances influence these reactions that in turn affect the accuracy of

the results. The color product from the reaction of sulphide and nitroprusside is

found to be unstable and decomposed after 150 seconds of the mixing of the

reagents. Also, the method has low sensitivity.37-38 The reaction between sulphide

and molybdate is affected by the impurities in the reagents, the pH and the

temperature of the solution. Time (1 hour) is required to reach the stable absorbance

for the molybdenum blue development.39-40 丁he reaction of sulphide with 1,10-

phenanthroline is interfered by oxidizing and complexing agents, including

thiosulphate, sulphite, nitrite, citrate, tartrate and oxalate, (that may complex with

iron (II) and iron (III) ions), and zinc. Time (30 minutes to 2 hours) for this reaction is I needed.41

The determination using the reaction of sulphide with iron (III) and

nitrilotriacetic acid^^ is not sensitive enough and is suitable for samples containing

high concentration of sulphide (> 8 ppm). Also, the measurement should be carried

out within a certain period of time (2-12 minutes after mixing the solutions) owing to

the instability of the complex formed.

Indirect determination of sulphide has also been reported. The concentration

of sulphide in the air is found from the absorbance of the reaction between 2,,7'-

dichlorofluorescein and ICI, formed from the interaction of iodate and sulphide in an

8 acidic solution. The interferences studies reported are restricted to the effect of sulphur and nitrogen oxides present in air and sulphur dioxide is found to interfere at all levels

Some spectrofluorimetric methods reported are indirect methods and are based on the displacement of the cation from the chelate by sulphide. Some reported spectrofluorimetric methods have used metal complexes such as Hg"-PBI (2,2'- pyridylbenzimidazole) complex^^ and Cu"-HPB [2-(o-hydroxyphenyl)benzoxazole] complex45 utilizing the release of respective nonfluorescent PBI (quenching action) and fluorescent HPB. These methods suffer from interferences from compounds such as thiosulphate that react with the metals leading to the decomposition of the meta卜 organic chelates.

A relatively new spectrofluorimetric method makes use of the reduction of potassium 1,2-naphthoquinone-4-sulfonate (NS) by sulphide to form a fluorescent product.^' This method is subjected to interferences from some reducing substances such as sulphite and nitrite ions. Another disadvantage is that a reaction time of 30 to 40 minutes is needed for the constant fluorescence intensity to attain.

The determination of sulphide utilizing the change in the absorbance at 250 nm by mixing para-chloro{mercuri)-benzoate (p-CMB) with samples containing sulphide in a photometric titration^? has limited sensitivity as only small amount of sample is allowed to add to the cuvette. The reaction is shown in Figure 1-2.

2 "OOC(^)HgCI + S 2-

para-chloro{mercuri)-ben2oate

2CI > - OOC(^)Hg-S-Hg~(^)COO ~

Figure 1-2 Reaction of para-chloro(mercuri)-benzoate with sulphide

9 Anodic wave using the technique of rapid direct current^^ has improved the conventional anodic direct current polarography by reducing the multiple anodic waves originated from the oxidation of mercury and the multiple formation of insoluble mercury sulphide in the mercury drop. The lack of sensitivity of this method due to the double-layer charging current in the initial period of the mercury drop expansion is improved by the technique of differential pulse polarography.^^

However, this technique is limited to the point where the determination is feasible below the formation of the insoluble mercury sulphide layer.^^ The use of normal pulse^O technique has eliminated the double-layer charging current with a higher concentration range allowed but the sensitivity is not so good as the differential one.

Cathodic stripping polarography is a relatively simpler way.51 However, polarographic methods suffer from the interferences from heavy metals and organic matters in the samples.

Adsorption of sulphide ion on the analytical column^^ jg one of the major problems in ion chromatography for its determination. Although saturation of the active site of the column with sulphide would reduce the adsorption, sulphide would slowly release from the active site at low sulphide concentration and this would affect the results.52 Moreover, only synthetic samples have been used in the experiment. The applicability of the method to the real samples is not clear and the analysis may be complicated by the matrix of the samples. The determination of

sulphide involving the use of conductivity^^ gnd UV absorption^S by employing the change in composition of the substance in the eluent is indirect. As sulphide is very easily oxidized, the accuracy of the method may be affected due to the time-span of the sample in the column during the separation process.

A high performance liquid chromatographic method determined methylene blue formed from sulphide and p-phenylenediamine54-55 jg time-consuming because

10 time is needed for the completion of the reaction (10 minutes) and the chromatographic resolution of the methylene blue MO minutes for one injection).

Regular injection of sulphide to the column in headspace gas chromatography^^ is necessary to saturate it (due to the adsorption effect of sulphide to the column) in order to obtain results with good accuracy. Moreover, deposition of sulphide in the metal syringe used for the injection may cause errors in the analysis. Interfering substances, including surfactants, sulphur dioxide, and thiosulphate, affect the results. Another gas chromatographic method uses three methylating agents to derivatize sulphide. However, the derivatization yields are 40 to 60 %. The derivatization reaction time of 30 minutes to 1 hour and temperature control are needed.^^

The precision of the potentiometric method using silver sulphide solid-state ion-selective electrode''58 jg poor owing to the high level of noise, drift in the signal and interferences from chemicals in the sample matrix^^. Accurate results can be achieved in solutions of constant and known ionic strength. Moreover, the electrode is subjected to memory effect when a low concentration of sulphide is to be measured after a relatively high concentration of sulphide is measured. The time for the equilibrium when measuring low sulphide concentration would be long.

The molecular emission cavity analysis (MECA) involving the measurement of the intensity of the $2 emission at 384 nm after the introduction of the sample in the cavity into the nitrogen-diluted hydrogen flames suffers from the two basic interferences.60-62 Sulphur compounds, such as thiosulphate, that decomposed on heating give the same peak as sulphide.-62 jhe appearance of the sulphide peak is

delayed in the presence of cation in the samples that form stable compound^^ with sulphide.

The determination of sulphide via the precipitation processes, by measuring the amount of the precipitated zinc in zinc sulphide after filtering and redissolving

11 using atomic absorption spectroscopy®^ or barium sulphate precipitation after oxidation of sulphide to sulphate and adding an excess barium to the solution®^, are subjected to interferences by the process of occlusion, where the ions (zinc ion and barium ion) are trapped inside the precipitate during the course of precipitation.

The measurement using the catalytic action of sulphide on the iodine-azide reaction is interfered by other sulphur anions such as thiosulphate that can also catalyze the reaction.®^

Inhibitory action of sulphide on various oxidative enzymes, including oxidases and peroxidase, on the reaction of nonfluorescent homovanillic acid with hydrogen peroxide to a fluorescent compound has been used to determine sulphide concentration.®® However, the reaction is not specific to sulphide, where ions including cyanide, iron (II) and (III), and cobalt (II), have also inhibitory action on the enzymes. The experimental conditions involving enzymes need to be carefully controlled.

Recently, there is a greater tendency for the separation/preconcentration methods to be coupled with the established analytical methods. Obviously, the reasons are to remove the interferences due to the suspended solid and interfering ions in the samples and to concentrate sulphide. There are several kinds of separation methods of sulphide. Commonly, hydrogen sulphide is generated from the acidified sample and is either distilled from a heated inert atmosphere^' 46 or purged using an inert gaslO, 15, 51, 67, which may then be collected in an absorbing agent. An inert environment is needed because sulphide is susceptible to oxidation.

However, separation methods are not wsll-optimized. For example, the previous described spectrofluorimetric method using the reduction of NS by sulphide has employed distillation to remove interferences, but the distillate collected is large (70 ml of distillate from 200 ml of sample). This lowers the concentration effect.^' 46

With purging as a separation 10, the recovery of sulphide is not determined after

12 purging for a fixed interval of time (25 minutes) which indicates an unreliable accuracy of this determination. Other separation methods involve the use of ion exchange resin^^ including zeolite^^, precipitation^^' 63, microdiffusion^^ and filtration®^. After the separation, the procedure of the analytical methods is followed.

(B). General description of the proposed method

(i). The objective and principle of the proposed method for the determination of sulphide

As described above, different mercury compounds had been employed for the determination of sulphide. In the photometric titration, p-CMB was used and the amount of sulphide was found from the change in the absorbance at 250 nm. In the titration with o-HMB {ortho-hydroxy{mercuri)-benzoate), the reagent was used as a complexing agent for sulphide with dithizone as an indicator for the end-point.

The objective of this project was to develop a spectrophotometric method to determine sulphide using either o-HMB or p-HMB《para-hydroxy{mercuri)-benzoate) as

reagent. In the preliminary study, the reagent as well as the organomercury-sulphur

complex absorbed UV light in the same region. Therefore, the reagent needed to be

removed after reaction.

From the titrations described above, it could be seen that the organomercury

complexing agent was preferentially combined with sulphide rather than the

indicator, dithizone. Dithizone (diphenylthiocarbazone) is a chelating reagent for many

metals and it is soluble in chloroform and tetrachloromethane but insoluble in water

(and slightly soluble in alcohol).69 p-HMB and o-HMB had been found to be

extractable from aqueous medium to tetrachloromethane containing dithizone. This

was due to the formation of 1:1 complex via the bonding between sulphur in

dithizone and mercury in the organomercury compound as shown in Figure 1-3.

13 Hence, this could be employed for the removal of the excess complexing agent in water after the reaction of p-HMB or o-HMB with sulphide.

/N=N- /N=N-<0> / +R-Hg+ Z S=c R-Hg -S-C ^NH-NH-<0> \N-NH—<•>

Dithizone (H2DZ)

Figure 1-3 Structures of dithizone and the complex with organomercurv compound^^

Figure 1-4 below shows the extractability of dithizone and its complex with o-HMB between aqueous and organic phases under different pHs. The behaviour of p-HMB was found to be qualitatively similar to o-HMB. The extraction constant expressed in logarithm for extracting ortho-carboxyphenylmercury cation in water by dithizone in tetrachloromethane was reported to be within 4.52 to 5.41 "O

(magenta) -OOC.CgH4.HgDz- ‘ -OOC.CaH,.Hg{HDz) (yellovA/) -H + (yelknM HDz. (pH 11.5) / -H+ (PH9.0--10.5) 刷/-H+ J

HjDz +HOOC.C八.Hg、‘ HOOC.CgH^HglHDz) (yelloW 4H+

(pH 0-7) ‘ aqueous phase

HjDz rdithizone J ， organic phase

{C^Hi2N4S) HjDz (green) HOOC.CgH^.HglHDz) (vellovM

Figure 1-4 Equilibria in two-phase system (water-tetrachloromethane) involving dithizone. o-carboxvphenvlmercurv cation and the complexes formed over different pH ranges川

14 When the sample contained substances which absorbed in the UV region, it

was necessary to separate sulphide from the sample matrix. In this project, two

separation methods were attempted, including distillation and purging from an

acidified samples. It was expected that a concentration effect was introduced and

interferences from the sample matrix were reduced. Sulphide ion after separation

was allowed to react with either para-hydroxy{mercuri)-benzoate (p-HMB) or ortho-

hydroxy(mercuri)-benzoate (o-HMB). The reactions between the reagents with

sulphide are illustrated in Figures 1-1 above and 1-5 below.

, 2 - OOC"""" HgOH + S

para-hydroxy(mercuri)-ben2oate (p-HMB)

_00C ~(^)Hg-S-Hg^^00“

Figure 卜5 Reaction of para-hvdroxv(mercuri)-benzoate with sulphide

The reaction time was probably very short because the reaction had been used in a

titrimetric method before.32-34 J^Q excess complexing agent would then be

removed by solvent extraction with dithizone utilizing the method described above.

(ii). Brief review of the principle of the UV-VIS (ultra-violet and visible)

spectrophotometric method

The spectrophotometric method is based on the absorption of the

electromagnetic radiation by the substancels) in the sample solution. Electromagnetic

radiation possesses a certain amount of energy. A unit of the radiation is called

photon and the energy of a photon is proportional to a certain wavelength or

frequency corresponding to that radiation. The absorption of electromagnetic

radiation in the UV-VIS region corresponds to the electronic transitions by the

15 specific type of groups, bonds or functional groups in the molecule. In the process of radiation absorption, the electron(s) in the molecule or atom is (are) raised from the ground state (the lowest energy level) to a higher excitation energy state. The energy absorbed by the molecule is equal to the energy difference of the energy states. As the energy levels exist at discrete levels, the energies of the transitions (or the energy levels) are quantized. Usually, the rotational and vibrational transitions takes place with the electronic transition. Due to the fact that the rotational and vibrational energy levels are too numerous and close together with respect to those of the electronic energy levels, under excitation of UV-VIS radiation, the spectrum of an absorbing molecule is a broad band covering a region of wavelengths. Basically, the extent of electromagnetic radiation absorbed by the absorbing substance is related to the amount of that substance in the sample solution.^^

By the Beer-Bouguer-Lambert law, commonly called the Beer's law, the fraction of the monochromatic electromagnetic radiation absorbed by the absorbing analyte in the solution can be quantitatively related to its amount. Incident radiation with radiant power, P。， falls on a solution having an absorbing analyte with concentration, c, in a transparent《to the electromagnetic radiation) cell with path length, b, merging with transmitted radiation of radiant power, P.71-72 Figure 1-6 illustrates what is just described.

Po, power of incident radiation Po => c => P P, power of transmitted radiation c, concentration of the analyte b -> b, path length in cm

Figure 1-6 Illustration of the Beer's law

16 The fraction of the radiant energy transmitted is found to decay exponentially with the path length, b, of the cell and the concentration, c, of the analyte. Therefore, the transmittance, T , can be related by the following 71-72:

T = log^ = lO-ebc P where e = molar absorptivity, which is dependent on the wavelength and the nature of the absorbing material and is a constant for a specific molecule at a fixed wavelength. The above relation can be rearranged and expressed in term of ‘

I I absorbance, A:

A = -log T = ebc

By measurement of the absorbance (or transmittance) of the absorbing analyte at different concentrations at a known wavelength in a cell of constant path length, quantitative measurement can be carried out for the sample from the absorbances of the standards and the samples.

The nature of the absorption of radiation of a molecule is affected by a number of factors. The number of electrons in a molecule, the structure, the geometry and the symmetry of the molecule affect the electronic state of the molecule. Therefore, the absorption maximum will be different for the molecules having different orientations. For example, 3,8-phenanthroline and 1,10- phenanthroline have different UV-VIS spectra. Other factor such as the nature of solvent used has the effect of shifting the spectrum (in comparison with the use of other solvents) due to the interaction of the electronic states of the solvent and the solute. The solution medium used in the determination must therefore be compatible through the experiment.72-73

17 2. Experimental Section (A). Preservation of samples

Reagents

All reagents used were of analytical reagent grade and were used without further purification. The term "distilled water" used was distilled and deionized water passing through Millipore Milli-Q^® ultrapure water system and was used in all the experiments.

1 M sodium hydroxide solution -- 20 g of sodium hydroxide pellets (BDH

Chemical Ltd.) were dissolved and diluted to 500 ml with distilled water.

1 M zinc acetate solution - 22 g of zinc acetate dihydrate (E. Merck) were dissolved in 87 ml of distilled water and diluted to 100 ml with distilled water.

Procedure

Because sulphide could be easily and rapidly oxidized by air, the samples should be preserved after collection if the analysis was not started immediately.

According to Greenberg et the water sample collected for the analysis of sulphide should be preserved by adding 4 drops of 1 M zinc acetate per 100 ml of sample using either a glass or polythene bottle for storage. In addition, the water sample was collected with a minimum aeration that allowed to displace the

atmosphere by a nonsplashing rise in the bottle. Basically, the whole container

should be completely filled with the sample to reduce the chance of air-oxidation of

sulphide. The preserved samples were then shaken thoroughly to ensure good

mixing, and were refrigerated under 0°C. The sample could be stored for a maximum

of twenty-eight days. All the samples were preserved as described and each

determination was done in triplicate.

18 (B). Sample Dre-treatmentl6, 18

It should be noticed that the precipitate of zinc sulphide formed on addition of the preservatives should not disturb and should allow it to settle. Sufficient 1 M

NaOH solution (about 1 ml per 100 ml of sample) was added to the sample to aid the coagulation of the precipitate. About 80 to 95% of the supernatant was then decanted from the bottles slowly and carefully so that no precipitate was lost in this process. All the remaining sample (including the zinc sulphide slurry) was poured into

I a volumetric flask (with volume, about 1/2 or 1/4 of the original sample volume) and was diluted to the mark with distilled water. The purpose of this was to concentrate

> the sample and to reduce soluble interfering substances such as the anions present in the samples.

(C). Separation/preconcentration of sulphide

In order to determine or widen the applicability of the analytical methods to samples containing suspended solids or colored matters or both, or to attain a greater sensitivity, the proposed spectrophotometric method were incorporated with separation/preconcentration techniques, where a suitable amount of concentrated mineral acid was added to the sample under an inert atmosphere to generate hydrogen sulphide, which was absorbed by an absorbing agent. Two methods had been used. One was by purging the sample with an inert gas and the other was by distilling hydrogen sulphide through heating. The inert gas used in the methods was nitrogen which prevented air oxidation of hydrogen sulphide generated. Nitrogen was first passed through an alkaline solution of pyrogallol before use for the removal of residual oxygen that might be present in nitrogen gas. The flow rate of nitrogen was monitored using a soap film flowmeter. Also, the sample in the apparatus was purged with nitrogen for some time before adding acid so as to remove the air inside the apparatus. An excess non-volatile acid was added to the samples to aid the

19 dissolution of sulphide bound to the suspended solids and the metal ions. Both separation methods were optimized such that better conditions were attained compared with that discussed in the review.

(i). by purging the acidified samples with an inert gas

The experimental set-up was a modification of that reported by Lindell, ^ al.lO I Apparatus 丨

125-ml wash bottle, Dreschel bottle head (with one more opening added),

Pasteur capillary pipettes, rubber tubings of various sizes, nitrogen gas supply. Dove

Brand 10-ml glass syringe, Hewlett-Packard 0101-0113 soap film flowmeter (1-10-

100-ml), 25-ml or 50-ml volumetric flasks.

Reagents

80% sulphuric acid - 80 ml of 95-98% sulphuric acid (Beijing Chemical

Works) were added to about 9 ml of distilled water.

0.1 M sodium hydroxide solution -- 4 g of sodium hydroxide pellets (BDH

Chemical Ltd.) were dissolved and diluted to 1 litre with distilled water.

Potassium Hydroxide solution -- 50 g of potassium hydroxide pellets (BDH

Chemical Ltd.) were dissolved and diluted to 100 ml with distilled water.

Alkaline pyrogallol solution'^ ^ -- 6 g of solid pyrogallol (BDH Chemical Ltd.) were dissolved and diluted to 20 ml with distilled water and mixed with 100 ml of the potassium hydroxide solution.

Procedure

The pre-treated sample was shaken thoroughly to ensure that the sample was mixed homogeneously. 10 ml to 80 ml of the sample were transferred into a

125-ml wash bottle, and a Dreschel bottle head was fitted into the wash bottle. The openings on the head were connected respectively to the nitrogen gas supply, the

20 syringe (with 10 ml of 80 % sulphuric acid) and a Pasteur capillary pipette through suitable size rubber tubings. The other end of the Pasteur capillary pipette was clipped into the bottom of a 50-ml volumetric flask containing 5.00 ml of 0.1 M sodium hydroxide solution, the absorbing agent. The experimental set-up was shown in Figure 1-7. All joints were ensure to be air-tighted. Nitrogen gas was passed through the apparatus at a steady rate of 250 ml/min for a fixed interval of time (say

10 minutes) to remove the air inside. Then, sulphuric acid in the syringe was slowly I injected into the apparatus. Nitrogen gas flow was continued for 1 5 minutes. Each . determination was done in triplicate. ；

[ I

i i

1 mm »nt«m«l dlam«t9f •“ tublno rubtm tvibtng 广斤：n connect to N^ gai cyHrKJtf 5 mm lnt»mtl dltrr>«t*f f

3 mm im^mtl dltmtw

[—一 」 — I n；— ~^~^ ^~Mo

SA , t25m1 W«h bottW MF 29/3/125 S«mpl« lohjtton Figure 1-7 Experimental set-UD for Duraina2 1 (ii). by heating and distilling the acidified samples under an inert atmosphere

The experimental set-up was basically the setting for simple distillation.

Apparatus

a Liebig condenser, an screwcap adapter, a receiver adapter, a still head.

Electrothermal EM 0500/C heating mantle, a three-neck 500-ml round bottom flask, a glass tubing, a stopper, a 100-ml dropping funnel, nitrogen gas supply, rubber tubings with various sizes, a Pasteur capillary pipette, 50-ml measuring cylinder, I Hewlett-Packard 0101-0113 soap film flowmeter {1-10-100-ml), and 25-ml or 50-ml j

I volumetric flasks. 丨

Reagents

85% phosphoric acid (Riedel-de HaSn)

1 M sodium hydroxide solution - 4 g of sodium hydroxide pellets (BDH

Chemical Ltd.) were dissolved and diluted to 100 ml with distilled water.

Procedure

A typical distillation set-up was used and shown in Figure 1-8. A three-neck

500-ml round bottom flask, with one neck connected to a dropping funnel, one to a still head and one to a stopper, was employed with a suitable heating mantle. The still head was connected to a water condenser and a screwcap adapter which fixed a glass tubing for nitrogen gas entrance. The glass tubing was positioned such that it nearly touched the bottom of the flask. The other end of the condenser was fitted to a receiver adapter, which was connected to a Pasteur capillary pipette through a suitable size rubber tubing. The Pasteur capillary pipette was dipped into 1.00 ml of

1.0 M sodium hydroxide solution contained in a 50-ml measuring cylinder. After shaking the pre-treated sample homogeneously, a suitable volume (100 ml) was added to the round bottom flask through the neck of the flask by removing the

stopper. The neck was fitted with the stopper. All the joints were air-tighted. A

nitrogen gas, with flow rate of 250 ml/min, was purged through the sample solution

22 for 15 minutes to remove the air inside the apparatus. A slow stream of nitrogen gas was maintained through the apparatus and the power of the mantle was turned on.

The flow rate of nitrogen gas was adjusted during distillation such that a slow stream of gas bubbles were passed through the Pasteur capillary pipette. When the sample solution in the apparatus started to boil, 5 ml of 85% phosphoric acid were added to the apparatus through the dropping funnel. (The last drop of the acid was not added to the flask from the dropping funnel to prevent the escape of hydrogen sulphide through the dropping funnel.) The heating was continued until a suitable volume (15 ml) or a suitable time was attained for complete hydrogen sulphide collection. The absorbing agent was then transferred into a 50-ml volumetric flask with rinsing using distilled water. Each determination was done in triplicate.

connect to Nj gas cylinder

个

•rubber tubing

glas* tubing with 1 mm internal diameter

100ml dropping funn«i \\ 0 1/22 with H3PO4 ~f^ I Scrowcop odopter \ \ (^ST52/13

\ \ S Still h«»d water out VA U•狗 condenser

Round bottom Hask, 3 necks,-/^ \ U RA 1/12 500ml FR 500/2S/22A / ^ f • | V WBtBf In 广 Samp 丨,.olution || 7 扣柳

Pasteur captllary^ “ ” plp«tte

measuring cvllndar

Sodium hydroxicle - •、

Figure 1-8 Experimental set-up for distillation

23 (D). Spectrophotometric determination of sulphide by proposed method

As the sulphide standard was prepared from its hydrated sodium salt (see section 2D (i) below), which was hygroscopic, the exact concentration of the sulphide solution prepared needed to be standardized using iodometry.

(i). Standardization of sulphide solution

Reagents

Stock sodium sulphide solution - Sodium sulphide nonahydrate (Beiiing

Chemical Works) was used to prepare the sulphide solution. It was seen to be hygroscopic. The crystals were first washed with a small amount of water and 95% ethanol and then blotted dry using a filter paper. 0.751 g of sodium sulphide nonahydrate was dissolved and diluted to 100 ml in a volumetric flask with distilled water to obtain an approximately 1000 ppm sulphide solution.

Working standard sulphide solution, 20 ppm -- 20 ml of the stock sulphide solution were pipetted and made up to 1 litre with distilled water.

0.016668 M Potassium iodate solution - (Beiiing Chemical Works) The solids were heated at 110°C in an oven for an hour to remove the moisture on the solid surface and cooled in a desiccator before use. Then, 3.567 g of potassium iodate were weighed accurately, dissolved and diluted to 1 liter with distilled water.

Potassium iodide (Beiiing Chemical Works)

0.1 M Sodium thiosulphate solution -- 12.5 g of sodium thiosulphate pentahydrate (Riedel-de Haen) were dissolved and diluted to 500 ml in a volumetric flask with distilled water. (Three drops of chloroform were added to the solution for preservation.)

0.025 M sodium thiosulphate solution -- 25 ml of 0.1 M sodium thiosulphate

(prepared above) were pipetted and diluted to 100 ml in a volumetric flask with distilled water.

24 0.05 M Iodine solution ~ 10 g of iodate-free potassium iodide were dissolved with 15-20 ml of distilled water in a 500-ml volumetric flask. About 6.35 g of iodine

(Beijing Chemical Works) were weighed and transferred to the flask using a small funnel. The stopper was inserted to the flask which was shaken until all the iodine dissolved. The solution was allowed to cool and diluted to the mark with distilled water.

0.0125 M iodine solution -- 25 ml of 0.05 M iodine solution (prepared above) were pipetted and diluted to 100 ml in a volumetric flask with distilled water.

1 M Sulphuric acid -11 ml of 95-98% sulphuric acid (Beijing Chemical

Works) were diluted to 200 ml with distilled water.

Starch solution -- A paste of 1.0 g of soluble starch (J. T. Baker Chemical

Co.) was added with a little water and was poured into 100 ml of boiling water with constant stirring. The boiling was continued for one minute. Two to three grams of potassium iodide were added to the cooled solution and shaken to dissolve.

Procedure

(a). Standardization of 0.1 M sodium thiosulphate solution^^

25.00 ml of 0.016668 M potassium iodate solution were pipetted into a 250- ml conical flask and were added with 1 g of potassium iodide, followed by 3 ml of 1

M sulphuric acid and the liberated iodine was titrated with 0.1 M thiosulphate solution with constant shaking. When the color of the solution became pale yellow, the solution was diluted to 200 ml using distilled water, and 2 ml of starch solution

were added. The titration was continued until the color of the solution changed from

blue to colorless.

(b). Standardization of 0.05 M iodine solution^'^

25 ml of 0.05 M iodine solution were pipetted into a 250-ml conical flask and

diluted to 100 ml, and the solution was titrated with 0.1 M sodium thiosulphate

solution until the solution became pale yellow. Then, 2 ml of starch solution were

25 added and the addition of thiosulphate solution was continued slowly. The end point was reached when the solution just changed from blue to colorless,

(c). Standardization of the working standard sulphide solution^^

100.00 ml of -20 ppm sulphide solution were transferred to a 250-ml conical flask following 10.00 ml of 0.0125 M iodine solution and two drops of the concentrated hydrochloric acid. The excess iodine in the mixture was back-titrated with 0.025 M sodium thiosulphate solution. When the solution changed to pale yellow, 2 ml of starch solution were added to the solution and the titration was continued until the blue color of the solution changed to colorless.

(ii). Extraction procedure

Apparatus

100-ml separatory funnels, 50-ml volumetric flasks, Jenwav 3020 pH meter with a type no. PCP505, ser. no. 23820/9 combined pH electrode .

Reagents:

0.75m M para-hydroxy(mercuri)-benzoate (p-HMB) - It was prepared by dissolving 0.135 g of p-hydroxy(mercuri)-benzoic acid (Aldrich Chemical Co.) in 2 ml of 1 M sodium hydroxide to form p-hydroxy(mercuri)-benzoate. (Small amount of alkali was used which made the pH control easier.) Time (overnight) was allowed for the dissolution. The undissolved solids were filtered using Whatman no. 41 filter paper and the filtrate was diluted to 500 ml in a volumetric flask with distilled water.

Dithizone solution - 0.0144 g of dithizone (Beijing Chemical Works) was dissolved in 100 ml chloroform (E. Merck).

1 M hydrochloric acid

Saturated sodium chloride solution - Sodium chloride has a solubility of

39.12 g in 100 ml of hot water and 35.2 g in 100 ml of cold water.74 About 40 g of sodium chloride (Beijing Chemical Works) were added to 100 ml of boiling distilled

26 water. More sodium chloride solids were added until the solids added appeared to be undissolved. The solution was allowed to cool to room temperature. The solids were allowed to settle and the solution was decanted to obtain a saturated solution.

Working standard sulphide solution - About 5 ppm sulphide solution was prepared by diluting 25.00 ml of -20 ppm sulphide solution (see section 2D (i) above) to 100 ml in a volumetric flask with distilled water.

Buffer solutionslH朽-A number of buffers of different pHs were prepared

as summarized in Tables 1-1 below. The exact pHs of the final solutions were

measured using a pH meter with a combined pH electrode. The pH meter was

calibrated using standard pH buffer solutions, with pH 4.00 and 7.00 for solutions

having pH values equal to or below 7.00 and with pH 10.00 and 7.00 for those

with pH values equal to or above 7.00.

Tables 1-1 Tables showing the preparation of the buffers used

Table 1-1 a Table showing the preparation of acetate buffers with pH 4-5

pH Volume of 0.2 M sodium Volume of 0.2 M acetic acid

acetate solution (ml) solution (ml)

4.0* 9.0 £ijO

5.0* 35.2 14^

stock 0.2 M sodium acetate 0.2 M acetic acid

prepa- 27.21 g of sodium acetate 11.55 ml of glacial acetic

ration trihydrate (Beijing Chemical acid (E. Merck) were diluted

Works) were dissolved and to 1 liter with distilled

diluted to 1 liter with distilled water.

water.

*The mixtures were diluted to 200 ml using distilled water.

27 Table 1-1 b Table showing the preparation of citrate buffers with pH 4-5

pH Volume of 2 M sodium Volume of 2 M citric acid (ml)

hydroxide solution (ml)

4.0*

5.0* 21.1 10.0

stock 2 M sodium hydroxide 2 M citric acid

prepa- 8 g of sodium hydroxide 42.0 g of citric acid ration pellets (BDH Chemical Ltd.) monohydrate (BDH Chemical were dissolved and diluted Ltd.) were dissolved and to 100 ml with distilled diluted to 100 ml with distilled water. water. •

*The final solutions were diluted to 100 ml using distilled water.

Table 1-1 c Table showing the preparation of pH 6 to 8 phosphate buffers

pH Volume of 0.2 M sodium Volume of 0.2 M disodium

dihydrogen phosphate hydrogen phosphate solution

solution (ml) (ml)

6.0* 87^

6.5* 68.5 31^

7.0* 39^

7.5* 84^

8.0* 5.3 94.7

stock 0.2M sodium dihydrogen 0.2 M disodium hydrogen

phosphate solution phosphate solution

prepa- 31.2 g of sodium 28.39 g of dried disodium ration dihydrogen phosphate hydrogen phosphate (BDH dihydrate (BDH Chemical Chemical Ltd.) were dissolved i=t^) were dissolved and and diluted to 1 liter with diluted to 1 liter with distilled water. distilled water.

*The mixtures were added up to 200 mi.

28 Table 1-1 d Table showing the preparation of pH 9 to 10 buffers

pH Volume of solution A (ml) Volume of 0.1 M sodium

hydroxide solution (ml)

9.0* 10.0* 50.0 43.7

stock solution A (mixture of boric 0.1 M sodium hydroxide

acid and potassium chloride) solution

prepa- 6.184 g of boric acid (E 10 ml of 1 M sodium ration Merck) and 7.455 g of hydroxide solution (prepared potassium chloride (Riedel-de above) were diluted to 100

Haen) were dissolved and ml with distilled water, diluted to 1 liter with distilled

water.

*The mixtures were diluted to 100 ml using distilled water.

Table 卜1e Table showing the preparation of phosphate buffers with pH 11-12

pH Volume of 0.1 M sodium Volume of 0.05 M disodium

hydroxide solution (ml) hydrogen phosphate solution

(ml) 11.0* 4J ^ 12.0* 26.9 50.0

stock 0.1 M sodium hydroxide 0.05 M disodium hydrogen

solution phosphate solution prepa- 10 ml of 1 M sodium 7.10 g of disodium hydrogen

ration hydroxide solution (prepared phosphate (BDH Chemical

above) were diluted to 100 Lt^) were dissolved and

ml with distilled water. diluted to 1 liter with distilled

L water. *The final mixtures were made up to 100 ml to get the desired pH buffer solution.

29 Table 1-1 f Table showing the preparation of buffers with DH 11-12

pH Volume of solution B《ml) Volume of 0.1 M sodium

hydroxide solution (ml)

11.0 51^

12.0 35 65

stock Solution B (mixture of glycine 0.1 M sodium hydroxide and sodium chloride solution) solution

prepa- 7.505 g of glycine (E. Merck) 10 ml of 1 M sodium ration and 5.85 g of sodium chloride hydroxide solution (prepared (Beiiing Chemical Works) were above) were diluted to 100 ml dissolved and diluted to 1 liter with distilled water. with distilled water.

Procedure

Varying amounts of the standardized sulphide solution (-5 ppm) were added to the 50-ml volumetric flasks such that the resulting solutions contained 0 (reagent blank), 0.25, 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, 10.0 ^ig of sulphide respectively.

1.00 ml of 0.75m M p-HMB solution was added to each of the volumetric flasks, following 10.00 ml of pH 7.00 phosphate buffer and 5.00 ml of saturated sodium chloride solution. The mixture was then diluted to the mark with distilled water. After mixing the content of each volumetric flask thoroughly, the mixtures were poured into separatory funnels separately and 2.00 ml of dithizone solution was added to each separatory funnel, which was stoppered and shaken gently for 2 minutes. Time

(two minutes) was allowed for the phases to separate. The lower organic layer was drained out and the upper aqueous layer was used for spectrophotometric measurement as described in section 2D《iii) below (p. 31).

30 (iii). Spectrophotometric determination of sulphide

(a). Construction of the calibration graph

Apparatus

Hitachi U-2000 double beam spectrophotometer and a pair of matched 1-cm

quartz cells

Procedure

After setting instrumental zero at 237 nm with the blank solution (by placing

the blank solution in the quartz cells), the cell for the sample was rinsed with a small

amount of the sample solution several times and was then filled with the sample

solution. The absorbance of standard sulphide solution at 237 nm was measured. A

calibration graph of absorbance at 237 nm against the amount of the respective

standard sulphide (in p.g) was constructed.

(b). Determination of sulphide in the samples

From the calibration graph constructed, the concentration of sulphide in the

sample could be found from the absorbance of the sample measured at 237 nm after the treatment of samples in section 2D (iv) (p. 31) below.

(iv). Treatment of samples

The samples, which were preserved as in section 2A (p. 18) and pretreated as in section 2B (p. 19), were treated as the procedure below and the extraction

procedure in section 2D (ii) above (p. 30) were then followed. If purging or distillation in section 2C (p. 19) was required as described below, equal number of milli-moles of 1 M HCI was added to the sodium hydroxide obtained from the separation after 1.00 ml of 0.75m M p-HMB was added in the extraction procedure.

(0.50 ml of 1 M HCI was added for in purging while 1.00 ml of 1 M HCI was used for distillation.) The determination for each sample was done in triplicate.

31 (a). Treatment of the water samples

A sample, which was shaken homogeneously, with a volume up to 30 ml was used for the spectrophotometric determination if separation was not incorporated. Purging and distillation in section 2C (p. 19) were done before the spectrophotometric determination for samples with low concentrations of sulphide or high concentrations of interfering ions.

(b). Treatment of the beer samples

The beer samples were purchased from a local supermarket, and were preserved and pre-treated as in the water samples except that more sodium hydroxide solution was added to the samples to ensure that it was alkaline. Purging procedure was adopted before spectrophotometric method. Addition of 1 drop of silicone anti-foaming agent, which was an aqueous emulsion containing 30% w/w silicone, to the samples before purging was needed to reduce the foams of the samples during purging,

(c). Treatment of the orange juice samples

The orange juice sample was purchased from a local supermarket. Fresh juice sample was prepared by hand-squeezing several oranges using a fruit squeezer. The treatment procedure was the same as described in section 2D (iv) (b) above (p. 32).

(E). Spectrophotometric determination of sulphide by standard

methylene blue method as the counter-check method 14

Apparatus

Hitachi U-2000 double beam spectrophotometer, a pair of matched cells and

10-ml volumetric flask

Reagents

Sulphuric acid solution - Concentrated sulphuric acid 《95-98o/o} (Beiiing

Chemical Works) was mixed with distilled water in a proportion of 1 to 1.

32 Amine-sulphuric acid stock solution -- 2.42 g of N,N-dimethyl-p- phenylenediamine dichloride (E. Merck) were dissolved in a cooled mixture which was prepared by mixing 5 ml of concentrated sulphuric acid with 3 ml of distilled water. When the mixture was cooled, it was diluted to 10 ml with distilled water and stored in a dark glass bottle.

Amine-sulphuric acid reagent ~ 2.5 ml of amine-sulphuric acid stock solution were diluted with 97.5 ml of sulphuric acid solution and stored in dark glass bottle.

Ferric chloride solution ~ 100 g of ferric chloride hexahydrate (Riedel-de

Ha6n) were dissolved in 40 ml distilled water.

Diammonium hydrogen phosphate solution - 40 g of diammonium hydrogen phosphate (BDH Chemical Ltd.) were dissolved and diluted to 100 ml with distilled water.

Procedure

(i). Construction of the calibration graph

0 (reagent blank), 0.25, 0.50, 0.75, 1.00, 2.00, 3.00, 4.00 and 5.00 ml of

1 ppm sulphide solution were added separately to 10-ml volumetric flasks following

0.5 ml of amine-sulphuric acid reagent and 3 drops of ferric chloride solution. 1.6 ml

of diammonium hydrogen phosphate solution were then added 5 minutes after the

appearance of the blue color. Distilled water was added to the mark of the volumetric

flasks. The solution should be mixed on each addition of reagent. The mixture was

allowed to stand for 3 to 15 minutes before spectrophotometric measurements of

the developed colour were made at 670 nm with the reagent blank as reference. A

calibration graph of absorbance at 670 nm versus the amount of sulphide used (|ig)

was constructed.

33 (ii). Treatment of samples

The samples were treated the same as described in section 2D (iv) (a), (b) and (c) above (p. 32) except that 7.50 ml of the water samples was used for the spectrophotometric measurements. The procedure in section 2E (i) (p. 33) was then followed.

(iii). Determination of sulphide in the samples

The amount of sulphide in the samples or sodium hydroxide (from the

separation procedure) was then deduced from the calibration graph and the

absorbance of sample solutions at 670 nm. The determination was done in triplicate.

Dilution of the sample (using distilled water) was needed when the concentration of

the sample was higher than the standard.

3. Results and Discussions (A). Optimization of the proposed spectrophotometric method

The effects of the amount of the various reagents used, the pH, the

extraction medium, the wavelength for the quantitative measurement and other

experimental conditions on the spectrophotometric method were studied. The

investigation for the optimal conditions for each experimental parameter was carried

out using the same experimental conditions except the one being investigated. Blank

was used as the reference.

(i). Choice of reagent used

Two reagents, namely, para-hydroxy{mercuri)-benzoate (p-HMB) and ortho-

hydroxy(mercuri)-benzoate (o-HMB), were tested for the possibility to be used for the

spectrophotometric measurements. The reagents were reacted with sulphide and

extracted with dithizone in chloroform. From the wavelength scans of the respective

34 aqueous phases, the use of o-HMB as a reagent exhibited no quantitative absorption peak within the wavelength region of 200-700 nm whereas that of p-HMB had an absorption peak at about 240 nm. Therefore, p-HMB was used as the reagent for the spectrophotometric measurements.

(ii). Selection of suitable wavelength for the measurements

The spectrum of the solution after the extraction was scanned to fix the wavelength for the quantitative measurement for sulphide. From the spectrum, the peak of the solution was at 237 nm. The spectrophotometric measurement was made at this wavelength. Figure 1-9 showed the spectrum of the spectrophotometric system after the extraction for various concentrations of sulphide.

f\ .•I ‘ 1.11.1 — — Amount of sulphide used _ 圓 0. 000 P^^^^。二、麵一 I , mi 220 240 260 230

Figure 1-9 UV spectrum of the reaction system at various concentrations of sulphide after the extraction .

35 (iii). Selection of a suitable DH for the extraction and the volume of the buffer used

In the presence of the buffers at pH 4.0 and 5.0, p-HMB was found to be precipitated out and a much lower absorbance was obtained compared with that obtained at pH 7.00. At or above pH 11.5, the extractant, dithizone, dissolved in the aqueous layer to a greater extent due to the formation of dithizonate ion. Moreover, it was reported that the higher the pH, the lower would be the extractability of the complex formed between carboxyphenylmercury cation and dithizone into tetrachloromethane due to the formation of water-soluble complex anions as shown in Figure 1-4 above. The percentage extraction of p-carboxyphenylmercury cation by dithizone was found to be 100.0 % at pH 6.82 and was decreased above pH 7.70 a pH range of 6 to 10 was being tested for the suitability of the optimal pH for the spectrophotometric measurements. The test was carried out using same volume but buffer solutions at different pH's with other experimental conditions unchanged.

From the results in Table 1-2, it could be seen that there was no great difference in the absorbance of the solution within the pH range from 6.00 to 9.00, but a great decrease in the absorbance was observed at pH 10.00. Although the extraction of the complex between dithizone and p-HMB was reported to be not complete at alkaline pH, good linearity between absorbance and the amount of sulphide at pH 8 or 9 was still found in the experiments. It might be due to the fact that the extraction of the complex between dithizone and p-HMB was reported to be quantitative up to a pH of 9.5.70 Different types of buffers with the same pH were also assessed to see if there was any effect due to the nature of the pH buffer rather than the pH of the solution. This was performed at higher pH's where behaviour of the spectrophotometric system was not the same as that around neutral. The compositions of the buffers used were shown in the Experimental Section (pp. 27-

30). Similar results were obtained at the same pH for different compositions of the

36 buffers. A neutral pH, that was, 7 was chosen for the spectrophotometric measurements.

Table 卜2 Effect of pH on the spectrophotometric method*

pH Absorbance at different amounts of

sulphide 5.82 卯 23.28 卯

6.0 0.269 0.938

6.5 0.286 1.067

7.0 0.291 1.083

7.5 0.282 1.094

8.0 0.286 1.080

9.0 0.299 1.031

10.0 0.091 0.876

*Each experiment was carried out using a 25.00-ml solution, containing 5.82 or 23.28 ng of sulphide, 5.00 ml of buffer, 1.80 ml of Im-M p-HMB, which was extracted with 5.00 ml of dithizone solution (containing 0.0144g in 100.00 ml of chloroform).

In deciding the volume of the buffer used, preliminary studies had been made to find the volume of the buffer needed to maintain the pH of the resulting solution to within ±0.10 of the pH of buffer. Three volumes of buffer, namely, 0.50 ml, 1.00 ml and 3.00 ml, added to a final volume of 25.00 ml. 3.00 ml of buffer was found to satisfy the above requirement.

In the separation, sodium hydroxide was employed for absorbing hydrogen sulphide, and was neutralized with hydrochloric acid, afterwards, however, it was better to add more buffer than 3.00 ml/25 ml of final solution in order to have better control of the pH of the solution. Therefore, for a final volume of 50.00 ml, 10.00 ml of buffer solution were used.

37 (iv). Separation of the phases and the volume of the solvent used for the extraction

Chloroform was used as the extracting solvent on the dissolution of dithizone. Practically, the time of the separation of the aqueous and the organic phases was longer for smaller volume of the organic phase used. In addition, saturated sodium chloride solution was added to the aqueous portion to see if there was any shortening in the waiting time for the separation of the two immiscible phases. The results were shown in Table 1-3, where it could be seen that there was a significant shortening in the time for the separation of the phases. Moreover, the addition of saturated sodium chloride solution affected the absorbance when the amount of sulphide was relatively small, however, a better linear relationship of the calibration graph should be obtained.

The volume of saturated sodium chloride solution added did not have any effect on the absorbance of the spectrophotometric system. Therefore, a volume ratio between saturated sodium chloride solution to final solution of 0.1 was used.

It was found that the volume of chloroform used for extraction also had no influence on the spectrophotometric system. (1.00, 2.00, 5.00, 10.00, 15.00,

20.00, 25.00 ml chloroform having different concentrations but the same amount of dithizone were tested.) In order to save the solvent used, less chloroform should be used. However, the separation time of the organic and the aqueous phases would be shorter for greater volume of the organic phase used. Fortunately, the short separation time could be achieved for small volume of the organic phase by using saturated sodium chloride solution in the aqueous phase. The separation time was more or less the same for 5.00 to 25.00 ml of chloroform but slightly slower for a volume of 1.00 ml, and 2.00 ml when saturated sodium chloride solution of 2.50 ml/25 ml of final solution was used. Hence, 2.00 ml of chloroform and 5.00 ml of

38 saturated sodium chloride solution were used in the separation when 50.00 ml of the aqueous phase was used.

Table 1-3 Effect of the addition of saturated sodium chloride solution to the reaction system on the absorbance measured and the time of separation of phases reauired 蚕

Amount of Absorbance in the presence or absence of sat. NaCI

sulphide {|ig) volume of sat. NaCI added (ml) without sat. NaCI

0.50 1.00

1.04 0.131 0.132 0.162 2.08 0.260 0.266 0.296 4.17 0.525 0.530 0.535

6.25 0.785 0.790 0.803

8.34 1.044 1.044 1.061

Time (min.) ** 2 2 30

*Each experiment was carried out using a 10.00-ml solution, containing different amounts of sulphide, 0.72 ml of Im-M p-HMB, 2.00 ml of pH 7.00 buffer, with or without saturated sodium chloride solution, which was extracted with 2.00 ml of dithizone solution (containing 0.0072 g in 50.00 ml of chloroform).

* *This was the time for complete separation of phases after shaking for the extraction.

(v). Choice of extracting medium (solvent used for dissolution of dithizone)

As dithizone was soluble in tetrachloromethane and chloroform, these two solvents were tested to see if there was any difference in the extraction. The same amount of dithizone was dissolved in the two solvents with other experimental conditions unchanged. From the results in Table 1-4, it could be seen that basically no significant difference was observed for the two solvents used for the extraction.

Chloroform was used as the solvent in the experiment.

39 Table 1-4 Effect of the extracting medium to the sDectrophotometric method*

Amount of Absorbance with different solvents

sulphide (ppm) CCU CHCh

0.230 0.287 0.290

0.459 0.586 0.578

0.918 1.125 1.112

*Each experiment was carried out using a 25.00-ml solution, containing various amounts of sulphide, 1.80 ml of Im-M p-HMB, 10.00 ml of pH 7.00 buffer, which was extracted with

10.00 ml of chloroform or tetrachloromethane solution (containing 0.0038 g in 50.00 ml of the solvents separately).

(vi). Stability of the complex formed

The stability of the product formed was investigated by measuring the absorbance of the aqueous portion after extraction throughout a period of 24 hours.

Time was measured after shaking for extraction. From the results shown in Table 1-5, the product formed was stable over the period studied.

40 Table 1-5 Effect of time on the spectrophotometric measurement after extraction*

Time Absorbance at different amounts of sulphide

(hourmin.) 0.234 ppm 0.931 ppm

0:15 0.283 1.093

0:30 0.285 1.095

0:45 0.284 1.094

1:00 0.285 1.095

1:30 0.283 1.095

1:45 0.287 1.093 2:00 0.286 1.093

2:30 0.285 1.093

3:00 0.284 1.093

3:30 0.287 1.095

4:00 0.286 1.095

4:30 0.283 1.094

5:00 0.287 1.095

6:00 0.285 1.095

7:00 0.286 1.095

8:00 0.284 1.097

24:00 0.283 1.095

Relative standard 0.5 0.1

deviation (%)

*Each experiment was carried out using a 25.00-ml solution, containing 0.234 or 0.931 ppm of sulphide, 1.80 ml of Im-M p-HMB, 5.00 ml of pH 7.00 buffer, which was extracted with 5.00 ml of dithizone solution (containing 0.0144 g in 100.00 ml of chloroform).

‘ 41 (vii). Effect of the amount of the dithizone used on the extraction

The amount of dithizone used for the extraction was varied to see if there was any effect on the extraction of excess reagent. As dithizone and p-HMB formed a 1:1 complex, mole ratios of 1.2, 1.5, and 2.0 were tested by changing the amount of dithizone used. From the results in Table 1-6, there was only a slight decrease in absorbance with increasing amounts of dithizone (or mole ratio) used in the extraction of the excess p-HMB after reaction with the same amount of sulphide used. A mole ratio of 1.2 was used and too much dithizone should not be used for the extraction so as to save reagent used.

Table 卜6 Effect of the amount of dithizone used on the spectrophotometric method*

Amount of sulphide Absorbance at different mole ratios Relative

(ppm) of dithizone to p-HMB standard

1.2 1.5 2.0 deviation (%)

0.211 0.264 0.265 0.253 3

0.422 0.524 0.527 0.513 1

0.844 1.065 1.057 1.042 1

*Each experiment was carried out using a 25.00-mI solution, containing different amounts of sulphide, 10.00 ml of pH 7.00 buffer, 1.80 ml of Im-M p-HMB, which was extracted with

10.00 ml of dithizone solution (containing different amounts in 50.00 ml of chloroform).

(viii). Effect of the amount of the mercury reagent used on the method

The amount of p-HMB used was varied to see if there was any effect on reaction with sulphide and absorption measurements. From the experimental results in Table 1-7, there was a slightly decrease in the absorbance when the amount of the reagent used increased. This was probably due to the incomplete removal of p-HMB

42 as the amount of dithizone used was limited. For the reason of saving reagent, 1.80

ml of 1 m M p-HMB solution was used for subsequent measurements.

Table 1-7 Effect of the amount of the mercury reagent used on the spectrophotometric method*

Amount of Absorbance at various amounts (volumes) of sulphide (ppm) comp exing agent used (ml)

1.80 2.25 3.00

0.227 0.284 0.286 0.265

0.681 0.811 0.807 0.780

*Each experiment was carried out using a 25.00-ml solution, containing different concentrations of sulphide, different volumes of Im-M p-HMB solution, 10.00 ml of pH 7.00

buffer, which was extracted with 10.00 ml of dithizone solution (containing 0.0180 g in

250.00 ml of chloroform).

(ix). Order of addition of the reagents

it had been reported that the lower the pH value of the solution, the higher the amount of sulphide existed as hydrogen sulphide, and at pH 7.0, the amount of

hydrogen sulphide was 0.33 of the amount of the dissolved sulphide in the water

(under a temperature range of 20。to 30®C and a dissolved mineral content below

2000 mg per liter).18 The addition of sample containing sulphide to the solution

containing the buffer solution only would result in the generation of hydrogen

sulphide gas that might have the possibility of the loss of the sulphide if sulphide

was not reacted in a short time. Hence, sample containing sulphide should not be

added before the addition of complexing agent, p-HMB. Six orders of addition of

reagents had been tested as shown below:

43 Sequence code Order of addition

1 p-HMB — sulphide buffer — sat. NaCI

2 p-HMB -> sulphide — sat. NaCI -> buffer

3 p-HMB — buffer sat. NaCI sulphide

4 p-HMB -> sat. NaCI — buffer -> sulphide

5 p-HMB — buffer -> sulphide -> sat. NaCI

6 p-HMB -> sat. NaCI -> sulphide — buffer

From the results in Table 1-8, it could be seen that there was no significant difference in the absorbance using different orders of addition of the reagents.

Table 1-8 Effect of the order of addition of the reagents on the spectrophotometric method*

Sequence Absorbance at various amounts of sulphide (|ig)

Code 2.00 6.00 10.00

1 0.099 0.295 0.492

2 0.099 0.294 0.495

3 0.100 0.297 0.492

4 0.099 0.298 0.493

5 0.100 0.296 0.490

6 0.100 0.295 0.493

Relative standard 0.6 0.5 0.3

deviation (%)

*Each experiment was carried out using a 25.00-ml solution, containing different concentrations of sulphide, 0.72 ml of Im-M p-HMB solution, 2.50 ml of saturated sodium chloride solution, 5.00 ml of pH 7.00 buffer, which was extracted with 1.00 ml dithizone solution (containing 0.0072 g in 25.00 ml of chloroform).

44 (B). Optimization of the separation/preconcentration methods

The optimization of the separation/preconcentration methods for sulphide from the aqueous sample was done by changing the experimental conditions and then determining the recovery of sulphide using the proposed spectrophotometric method.

Known amount of sulphide solution was added to the experimental set-up.

The sulphide content of the sample and the one with known sulphide addition were then found from the calibration graph. The amount of sulphide in these solutions should be within the limits of the calibration graph. The percentage recovery could be found as follows^^:

(Measured concentration in fortified material - 一 Measured concentration in unfortified material ) % Recovery = ： — ： XI00% Knwon increment in concentration

The amount added should be a substantial fraction of, or more than, the amount present in the unfortified material. Obviously, the nearer the value of percentage recovery to 100, the better would be the method.

(i)- Separation involving purging the acidified samples with an inert gas

The Pasteur capillary pipette was used to reduce the size of the gas bubbles coming out from the apparatus, which, in turn, increased the surface area for the absorption of hydrogen sulphide by sodium hydroxide. The effect of purging rate and purging time of nitrogen, and the amount of sodium hydroxide used on the recoveries of sulphide were studied,

(a). Purging time and purging rate

The effects of purging time under the same purging rate (7 ml/min) and purging rate under the same purging time (25 minutes) on the recovery of sulphide

45 using the apparatus described by Lindell, et ajJ^ (except a 125-ml wash bottle was used instead of a 25-ml wash bottle) were assessed. 1 ml of 1 M sodium hydroxide was used as the absorbing agent. The results were shown in Tables 卜9 and 1-10.

Table 1-9 Effect of purging time at a purging rate of 7 ml per minute on the recovery of S.OOug of sulphide in 80 ml of sample solution using the set-up of Lindell. et alJQ

Purging time (minutes) Average Recovery* (%)

^ 20.3 士 3.5

40 25.1 士 5.5

§0 35.5 ±3.2

60 40.0 ±3.5

70 50.6 土 6.5

SO 45.3 ±4.5

^ 55.6 士 7.8

*Average of 3 determinations

Table 1-10 Effect of purging rate with a purging time of 25 minutes on the recovery of 5.00 UQ of sulphide in 80 ml of sample using the set-up of Lindell. et ^JQ

Purging rate (ml/minute) Average Recovery* (%)

20 13.2 ±2.8

50 15.6±3.9

100 20.5 土 5.8

35.6 ±4.6

^ 37.9 ±5.6

43.6 ±4.5

^ 50.6 ±5.3

350 65.3 ±3.5

*Average of 3 determinations

46 From the results in Tables 卜9 and 1-10, it could be seen that the recoveries of sulphide obtained under the tested conditions fluctuated greatly and hence were not good. These were unexpected as the volume of nitrogen gas passing into the experimental set-up was larger than the air space in the experimental set-up and hence, all hydrogen sulphide should have been driven out of the apparatus into the absorbent.

The poor recovery might be due to the effect of mixing of nitrogen gas with hydrogen sulphide gas (mixed gas) inside the set-up, since the diameter of the outlet tube was too small, and consequently the chance of hydrogen sulphide being purged out from the set-up was reduced. Another possibility was that the absorption of hydrogen sulphide was not good enough possibly because the amount or the concentration of the absorbing agent used was not right or the size of the Pasteur capillary pipette was too great for effective gaseous absorption. However, it was unlikely that poor recovery was due to inefficient absorption because there was an increase in the recoveries of sulphide at a high flow rate of nitrogen gas.

Hence, the experimental set-up was modified by increasing the internal diameter of the outlet from 1 mm to the original size of outlet of the Dreschel bottle head (i.e. 5 mm). This modified experimental set-up was shown in Figure 1-7. The effect of purging rate under the purging time of 25 minutes using the modified experimental set-up was investigated to see if there was any improvement in the recovery of sulphide. Blank determination was also made. 1 ml of 1 M sodium hydroxide was used as the absorbing agent.

From the results in Table 1-11, the increase in the internal diameter of the outlet resulted in a significant reduction of purging rate (with other experimental conditions remained unchanged) needed to achieve a similar recovery in comparison with the previous set-up employed. The recoveries were good when the nitrogen flow rate was attained at and higher than 200 ml/min.

47 Table 1-11 Effect of purging rate for 25 minutes of purging on the recovery of 5.00 u

Q of sulphide in 80 ml of sample using the modified experimental set-up

Purging rate (ml/minute) Average Recovery (%)

^ 33.1 ±5.3

^ 50.9 ±7.2

75.4 土 6.5

86.3 土 3.5

200 95.8 ±0.6

^ 98.6 土 1.9

^ 97.2 土 1.6

The effect of purging time under the purging rate of 250 ml/min was performed using the modified experimental set-up and the recoveries of sulphide were determined. 1 ml of 1 M sodium hydroxide was used as the. absorbing agent.

Blank determination was also made. From the results in Table 1-12, 15 minutes were needed for good recovery of sulphide from the solution. Hence, nitrogen gas flow rate of 250 ml/min for 15 minutes purging was used in subsequent experiments.

Table 1-12 Recoveries of 5.00 ug of sulphide in 80 ml of solution using the modified experimental set-up at different purging time, the flow rate being 250 ml/min

Purging time (minutes) Average Recovery (%)

5 59.6 士 5.9

— 10 86.5 ±4.3 15 98.1 ±1.2

20 96.5 土 1.5

^ 98.6 ±1.9

48 (b) Amount of sodium hydroxide used

Different concentrations and volumes of sodium hydroxide solutions were assessed for the effectiveness to absorb hydrogen sulphide under a 250 ml/min of nitrogen gas flow rate with purging time of 15 minutes using the modified experimental set-up as shown in Figure 1-7 and the recoveries of sulphide were determined. Blank determination was also made. In the experiment, 10-ml, 25-ml and

50-ml measuring cylinders were used to accommodate the absorbing agent. From the results in Tables 1-13 and 1-14, the recoveries were good with no significant difference throughout the range of the volumes and the concentrations of the absorbing solutions under study. Hence, 0.10 M sodium hydroxide solution was used for the absorption process. In using volumetric flask as container for the absorbing agent, more reagents were needed to cover the outlet of Pasteur capillary pipette owing to the wider base of the flask and 5.00 ml of absorbing agent were used for

50-ml volumetric flask.

Table 卜13 Effect of the volume of 0.10 M sodium hydroxide solution used for the absorption of sulphide generated from 80 ml of solution containing 5.00 |xq of sulphide

Volume of NaOH used (ml) Average recovery (%)

1:00 97.2 ±1.2

98.6 土 1.3

96.5 土 0.9

10.00 96.9 ±1.5

15.00 98.1 土 1.4

20.00 97.0 ±0.6

49 Table 1-14 Effect of the concentration of 1.00 ml of sodium hydroxide solution used for the absorption of sulphide generated from 80 ml of solution containina 5.00 ug of sulphide

Cone, of NaOH used (M) Average recovery (%)

g^ 96.3 士 1.5

qJO 98.2 士 1.0

g^ 97.9 ± 1.8

q^ 97.2 土 0.7

97.2 ±1.2

(c). Recovery of different concentrations of sulphide from the samples

The separation/preconcentration using purging with the optimized procedure were tested with different concentrations of sulphide and the recoveries of sulphide were determined. Blank determination (using sample containing all reagents employed in the procedure but without sulphide) was also made following the same procedure.

From the results in Table 1-15, the recoveries of sulphide using the purging procedure were satisfactory.

With the use of the modified experimental set-up and the corresponding procedure, a shorter purging time with a good recovery could be attained compared with the literature^

The pre-concentration achieved using purging could only enable samples containing down to 0.005 ppm of sulphide to be determined. With samples containing sulphide at a level of 0.005 ppm or lower, pre-treatment of the sample to concentrate sulphide as described in section 2B (p. 19) was needed.

50 Table 1-15 Recoveries of sulphide using purging as the separation method

Cone, of Vol. of Amount of Recovery (%) Average sulphide (ppm) sample (ml) sulphide (^g) Recovery (%)

0-1 40 4.00 94.8, 97.5, 97.8 96.7 ±1.7 0-04 100 4.00 94.3, 95.1, 95.1 94.8 ± 0.5

0-02 2.00 93.8, 97.3, 97.8 96.3 士 2.2

Q-Q1 100 103, 102, 100 102 士 2 0.005 igo 0.50 94.2,98.0,100 97.4 ± 2.9 0.0025 100 0.25 102,98.0,94.0 98.0 士 4.0

(丨丨)• Separation involvina distilling the acidified samples under an inert atmosphere

The effect of the volume of distillate collected on the recoveries of sulphide was investigated using the distillation set-up as shown in Figure 1-8. The results were shown in Tables 1-16 and 1-17. Three trials were made for each determination. Blank determination was also made.

From the results in section 3B (i) (p. 49), 0.1 M sodium hydroxide solution was used for the absorption of hydrogen sulphide in purging. Owing to the fact that the distillate collected would dilute the concentration of the absorbing agent, a relatively concentrated sodium hydroxide solution, 1.0 M, with a volume of 1.00 ml was used for the absorption of sulphide.

As the volume of the distillate collected was affected by the heating rate of the heater, it would be more accurate and meaningful to measure the volume of the distniate collected rather than the distillation time, which of course, could be used for . reference. A blank determination was made.

51 Table 1-16 Recovery test for the distillate collected at different period of time for 100

ml of sample solution containing 4 ua of sulphide (0.04 ppm sulphide solution)

Volume of the Time needed to collect Recovery (%) Average distillate (ml) the distillate *(min.) Recovery (%)

5 10 32.5, 30.9, 29.6 31.0 ± 1.5

10 15 76.2, 72.3, 70.8 73.1 ±2.8

15 19 101, 101, 102 101 土 1

20 22 101,104,103 103 士 2

25 ^ 103, 103, 102 103 士 1

*Timing was started when the solution in the flask started to boil.

Table 1-17 Recovery of the distillation procedure in couple with the proposed

spectrophotometric method utilizing 100 ml of sample of various concentrations of

sulphide for the collection of about 15 ml distillate

Cone, of Amount of Recovery {%) for the Average sulphide (ppm) sulphide (卯} three determinations Recovery (%)

0.04 101, 98.2, 103 101 ±2

0-02 96.8, 94.9, 97.8 96.5 ±1.5

0.01 99.4, 98.4, 102 99.9 ±1.9

0.005 g^ 101, 103, 96.6 100±3

0.0025 0.25 101, 104, 96.8 101 土 4

From the results in Table 1-16, a distillate volume of 15 ml was needed for good recovery of sulphide from the sample and was smaller than that reported in the literature^' 46. jhe recoveries of sulphide using various concentrations of the sulphide were then determined. The results were shown in Table 1-17 where it could be seen that the recoveries were good for all concentrations under study. Blank

52 determinations were performed using the same experimental conditions except the samples containing no added sulphide. No sulphide was found in the blank.

For samples containing low concentrations of sulphide, a pre-treatment of sample described in section 2B (p. 19) or a larger sample volume could be used in order to obtain a sample with a higher amount of sulphide for the distillation.

(C). Construction of the calibration graph for the spectrophotometric determination of sulphide and determination of the molar absorptivity coefficient

The data for the construction of calibration graph (absorbance at 237 nm versus conc. of sulphide) for the determination of sulphide using the proposed spectrophotometric method were shown in Table 卜 18. The correlation coefficient obtained from least square plotting was 0.99998 indicating the plot was linear. The calibration graph was shown in Figure 1-10.

Table 1-18 Data for the calibration graph for the determination of sulphide using para- hvdroxv(mercuri)-benzoate as a reagent

Concentration of sulphide (|ig) Absorbance at 237 nm 0.006 0.012

L^ 0.026

0.050

0.099

0.148

^ 0.197

10-00 0.246

correlation coefficient 0.99998

slope (卯-1) 0.0246

y-intercept 3.47 x 10-3

linear range (|ig) 0.25 to 10

53 0.35 r

0.3

0.25 Z

0.2 /

Absorbance

0.15 Z

0.1 Z

0.05 /

0 Z -—

-0.05 0 2 4 6 8 10 12 Amount of sulphide (ng)

Figure 1-10 Calibration graph of sulphide with absorbance at 237 nm plotted against the amount of sulphide _

Beer's law was obeyed in the range of 0.25-10.0 ^g. The molar absorptivity coefficient of the reaction product at 237 nm could be calculated from the slooe of calibration graph and was found to be 39.4x1l.mol"''cm'''

(D). Precision and detection limit of the proposed spectrophotometric method

From ASTm58, precision is defined as the degree of agreement of repeated measurements of the same property, expressed in terms of dispersion of test results about the arithmetical mean result obtained by repetitive testing of a homogeneous sample under specified conditions. The precision of a test method is expressed quantitatively as the standard deviation computed from the results of a series of a

54 controlled determinations. According to A0AC77, the precision is expressed as

relative standard deviation, which is the standard deviation divided by the mean

value of the absorbance (multiplied by 100% for the result expressed in percentage).

Hence, the precisions for the proposed spectrophotometric method were deduced

from ten replicate measurements of the absorbance of several standard sulphide

solutions with the concentrations of sulphide in the linear range of the calibration

graph. The data and computed results were summarized in Table 1-19, where it could

be seen that the precision of the proposed spectrophotometric method was good.

Table 1-19 Precision test for the determination of sulphide

Absorbance at different amounts of sulphide (}j,g)

Trial 1.00 2.00 4.00 6.00 8.00 10.0

1 0.026 0.050 0.099 0.148 0.196 0.246

2 0.025 0.050 0.099 0.148 0.197 0.246

3 0.025 0.049 0.099 0.149 0.197 0.246

4 0.025 0.049 0.098 0.148 0.196 0.246

5 0.025 0.050 0.098 0.148 0.196 0.246

6 0^5 0.050 0.099 0.149 0.196 0.247

Z 0.099 0.148 0.197 0.246

8 0.025 0.049 0.099 0.148 0.197 0.246

9 0.025 0.050 0.099 0.148 0.197 0.246

10 0.025 0.050 0.099 0.148 0.197 0.247

Average 0.197 0.246

Standard Deviation 3x10.4 5x10'^ 6x10.4 4x10.4 5x10-4 4x10-4

Rel. std. Dev. (%) 1 1 0.6 0.3 0.3 0.2

The detection limit, which was expressed as the concentration required to

give a signal equal to three times the standard deviation of the blank^l deduced from ten replicate measurements of the blank, was found to be 0.049 ^ig.

55 (E). Interferences studies of the proposed method

The potential matrix interferences for the determination of sulphide in water samples using the proposed spectrophotometric method was investigated using synthetic samples containing sulphide and various concentrations of ions. The

Interference was represented as the tolerance limit, which was the ratio of the concentration of the ion to that of sulphide with which the absorbance for the samples containing sulphide with that ion was not changed by more than 土 5% compared with solution containing the same amount of sulphide but without that ion.

Throughout the test, the amount of sulphide used was 4 }ig (20 ml of 0.2 ppm sulphide). The results of the study were shown in Table 1-20.

From Table 1-20, it could be seen that copper (II), cadmium and iodide showed serious interferences whereas iron, nickel, lead, zinc, nitrite, thiosulphate and thiocyanate showed milder interferences. Other ions did not show interferences when present at or below the levels under study. The interferences due to the metal ions might be due to the formation of the respective metal sulphides at pH and

retarded the formation of the complex with p-HMB. The metal ions might complex with dithizone to some extent and were extracted into the chloroform layer. In the

presence of ions at or above the tolerance limits, purging and distillation separations

were used to separate sulphide from the interfering ions before spectrophotometric

measurements using the proposed spectrophotometric method.

The interference studies were further carried out using solutions containing 4

|ig of sulphide with different concentrations of various ions having low tolerance

limits which affected the proposed spectrophotometric method. The results were

summarized in Tables 1-21 and 1-22.

56 Table 1-20 Effect of some common ions in water samples on the determination of 4 jj, g of sulphide (20 ml of 0.2 ppm sulphide solution) using the proposed spectrophotometric method but without puraina or distillation

Ions Cone, of the Tolerance limit (the ratio ions (ppm) of the conc. of ion in ppm to that of sulphide) Cations copper (II) 0.05

cadmium ^J iron (II)

lead (II) OA 1

zinc q^

nickel (II) OJ 3

calcium** 750

ammonium 350 1750

magnesium, potassium, > 2000* > 10000* sodium Anions iodide OJ

nitrite, thiocyanate, 1.5 7.5

thiosulphate

nitrate 6 ^

sulphate ^

oxalate ^ 150

carbonate, phosphate

sulphite 1500

citrate ^ 2000

acetate, bromide, chloride > 2000* > 10000* •Higher concentration of the ion had not tested.

** Precipitation was observed at concentration at and above 170 ppm of calcium ion.

57 Table 1-21 Effect of some ions on the determination of 4 ug of sulphide (40 ml of 0.1

ppm sulphide solution) using the proposed SDectroDhotometric method with purging

Ions Cone, of the Tolerance limit {the ratio of the conc. ions (ppm) of ion in ppm to that of sulphide) Cations copper (II) 0.005 0.05 cadmium, lead, iron (II), nickel 100* > 1000*

zinc 200* > 2000* Anions nitrite 20 400 .

iodide, thiocyanate, thiosulphate 100* > 1000*

* No more measurement was made beyond 100 ppm.

Table 1-22 Effect of some ions on the determination of 4.00 叫 of sulphide (100 ml

of 0.04 ppm sulphide solution) using the proposed spectrophotometric method with

distillation

Ions Conc. of the Tolerance limit {the ratio of the conc.

ions (ppm) of ion in ppm to that of sulphide) Cations copper (II) 0.003 0.075 cadmium, lead, iron 40* > 1000* (II), nickel

zinc 200* > 5000* Anions nitrite, thiosulphate M ^

thiocyanate ^ i_5

iodide 40* > 1000*

‘*No concentration beyond this was tested.

58 Possibly, the greater the volume of distillate collected in the distillation, the

greater the chance the interfering ions being distilled over and collected. Hence, the

volume of the distillate collected was limited to 15 ml.

From the results in Tables 1-21 and 1-22, the tolerance limits of the heavy

metal ions except copper (II) were greater after purging and distillation. No

improvement was observed for copper (II). It had been reported that copper (II) ion

formed acid-stable compound with sulphide and precipitation of sulphide with copper

(II) ion still occurred in 18 M sulphuric acid.78 with samples containing copper (II)

ion, sulphide determination was limited to include acid-hydrolyzed sulphide

compounds.16 |n addition, some metal sulphides, including cadmium sulphide, were

partially precipitated in 1.5 M sulphuric acid.78 Therefore, concentrated acid was

added in the separation to hydrolyze the metal sulphides and to generate hydrogen

sulphide.

‘ For the anions, a greater tolerance limits could be found with the

incorporation of purging to the proposed spectrophotometric method (as shown in

Table 1-21). Among the anions, nitrite ion had a comparatively greater interfering

effect on the proposed method. This might be due to the greater ease of purging

nitrite ion from the apparatus than the other ions under the experimental conditions.

While the tolerance limit for iodide was much greater with the incorporation

of distillation (as shown in Table 1-22), the tolerance limits of the anions,

thiocyanate, thiosulphate and nitrite ions, were only slightly better than the results

obtained without distillation. The comparative lower tolerance limits for the anions

just described might be due to the greater ease in distilling these anions under

heating from the apparatus.

The tolerance limits using respective purging and distillation incorporated

with the spectrophotometric measurements were compared in Table 1-23.

59 Table 1-23 Comparison of the tolerance limits of various ions obtained using respective purging and distillation before spectrophotometric measurements

Ions Tolerance limit {the ratio of the concentration of ion in ppm to that of sulphide) Purging Distillation Cations copper (II) 0.05 0.075

cadmium, lead, iron (II), > 1000 > 1000 nickel

zinc > 2000 > 5000 Anions nitrite ^ 10

thiosulphate > 1000 10

thiocyanate > 1000 15

iodide > 1000 > 1000

Concentration of sulphide 0.100 0.04

used in the experiment (ppm)

On the whole, the separation using purging was a better method in reducing

the interferences. Moreover, the time needed to complete one

separation/preconcentration was shorter using purging compared with distillation.

However, the purging method used much more nitrogen than distillation.

(F). Spectrophotometric determination of sulphide in water 、

The methylene blue method was used as the counter-check method.

Separation techniques were incorporated to the proposed spectrophotometric method

in the determination in the water samples. Equal moles of 1 M hydrochloric acid was

added to sodium hydroxide solution for neutralization after the separation and the

addition of p-HMB such that the pH of the mixture could be controlled by the buffer.

60 All the determinations were performed in triplicate. The results were shown in Table

1-24, where it could be seen that the results obtained using the proposed method agree well with those using the standard methylene blue method except for the waste water obtained from the primary treatment of the sewage treatment work. The later method gave a relatively higher result. It was worthy to note that the precision of the proposed methods was better than those obtained with the methylene blue method.

Table 1-24 Determination of sulphide in water samples using the proposed spectrophotometric method and with purging and distillation procedure and the methylene blue method

Sulphide found ()j.g/ml)

Location of the Proposed Method Methylene blue

collected sample Purging Distillation method

Shing Mun River 0.0509 土 0.0007 0.0517 士 0.0001 0.050 土 0.003

Sewage Treatment Works: (i). Final effluents

1. Sha Tin 0.0099 ± 0.0001 0.0100 ± 0.0001 0.010 ±0.001 2. Shek Wu Hui 0.0116 土 0.0002 0.0118 ± 0.0008 0.012 ±0.001 3. Tai Po 0.0140 土 0.0004 0.0141 土 0.0002 0.014 士 0.001

(ii). Wastewater after the primary treatment:

1. Shek Wu Hui 2.17 土 0.03 2.25 土 0.05 2.59 ± 0.08

(iii). Wastewater after the secondary treatment:

1. Shek Wu Hui | 0.0507 ± 0.0006 | 0.051 9 ± 0.0008 0.0513 土 0.0015

Results for the recovery test of sulphide from the samples were shown in

Tables 1-25 and 1-26. The recoveries using purging procedure were in the range of

95-100 % while those using the distillation procedure were all 101 %.

61 Table 1-25 Recovery test for the determination of sulphide ion in the water samples using the proposed spectrophotometric method with purging

Location of the sulphide sulphide Recovery Average

collected sample added _ found (%) Recovery (%)

Shing Mun River 4.00 3.95,3.86, 98.7, 96.5, 97.4 土 1.2

3.88 97.0

Sewage Treatment Works:

(i). Wastewater after the primary treatment

1. Shek Wu Hui 4.00 3.93,3.99, 98.3,100, 98.6 ±1.3

3.90 97.5

(ii). Wastewater after the secondary treatment -

1. Shek Wu Hui 2.00 1.92,1.93, 96.1,96.6, 97.2 ± 1.6

1.98 99.0

(iii). Final effluents .

1. Sha Tin 1.00 1.00,1.00, 100,100, 100 士 1

0.99 99

2. Shek Wu Hui 1.00 0.99,0.97, 99,97,96 97 ± 2

0.96

3. TaiPo . 1.00 0.98,0.95, 98,95,91 95 士 3

0.91

*This was the amount of sulphide after deducting the amount of sulphide found in the sample

from the amount of sulphide found in the sample with known addition of sulphide .

62 Table 1-26 Recovery test for the determination of sulphide ion in the water samples using the proposed spectrophotometric method with distillation

Location of the sulphide sulphide Recovery Average collected sample added _ found _* W Recovery (%)

Shing Mun River 4.00 4.02,4.02, 101,101, 101 ± 1

3.99 100

Shek Wu Hui Sewage Treatment Work:

(i). Wastewater after 4.00 4.06,4.04, 102,101, 101 士 1

the primary treatment 4.02

(ii). Wastewater after 2.00 2.04, 2.02, 102, 101, 101 土 1

the secondary treatment 2.01 101 •

(iii). Final effluent 1.00 0.98, 1.03, 98, 103, 101 士 3

1.02 102

*丁his was the amount of sulphide after deducting the amount of sulphide found in the sample from the amount of sulphide found in the sample with known addition of sulphide .

(G). Spectrophotometric determination of sulphide in beers

As beer contained a lot of foam, especially when shaken or purged, the foams were carried over to the absorbing agent during purging. The dissolved gases

in the preserved beer sample were attempted to be removed by degassing in an

ultrasonic bath for over an hour. (Preservation of the beer sample was important

because the pH of the sample was tested to be around 3 by pH paper.) However,

quite a lot of foam still remained when the beer was purged. Another method to

reduce the amount of foam was to add an anti-foaming agent, which was a

commonly used, thermally stable organosilicon oxide polymer. Addition of 1 drop of

this silicone anti-foaming agent to the beer sample was found to be adequate and

basically no foam was found when the beer sample was purged with nitrogen or

under heating. Preliminary test using synthetic sample solution containing sulphide

63 with the silicone anti-foaming agent showed that the addition of anti-foaming agent did not affect the results using proposed spectrophotometric method with distillation and purging. All the determinations were done in triplicate.

From the preliminary experiments using the methylene blue method, and the proposed spectrophotometric method with purging and distillation, the results obtained for the amount of sulphide in beer differently widely. The highest results were obtained with the proposed method with distillation procedure, which might be attributed to some water soluble organic matters in beer (or compounds that were decomposed during the heating process in the distillation) having UV absorption that were carried over to the distillate during distillation. Another possibility was the decomposition of sulphur-containing compounds in beer samples giving hydrogen sulphide as one of the products in the heating during distillation. Either or both factors would affect the determination and gave inaccurate results. No improvement in the results was obtained by incorporating a trap (replacing the still head) to the distillation set-up.

When the methylene blue method was applied directly to the beer samples, relatively higher results were obtained compared with those found using the proposed method with purging. This implied that some substances in the beer samples interfered with the determination, however, they might probably not be carried over during purging of the samples. Hence, in the quantitative determination of sulphide in beer samples using the methylene blue method, the beer samples were purged before analysis to reduce the interferences present in the beer samples.

The results for the concentrations of sulphide found in the beer samples were shown in Table 1-27. The results obtained with purging for both the proposed method and the methylene blue method were comparable. The relatively high results obtained using distillation for separation coupled with the proposed spectrophotometric method might be due to some organic matters being carried over

64 as remarked above. From the standard deviation of the concentrations of sulphide found in the samples, the precision of the proposed method appeared to be better than the standard methylene blue method.

Table 1-27 Determination of sulphide in beer samples

Sulphide found (}ig/ml) Brand names of beer Proposed Method Methylene blue used for analysis With purging With distillation method*

San Miguel 0.0218 土 0.0004 0.0441 土 0.0213 士 0.0006 0.0002 Pabst Blue Ribbon 0.0102 土 0.0002 0.0414 土 0.0098 士 0.0004 0.0002

*The samples were purged before analysis.

The recoveries of sulphide from the samples were determined as shown in

Table 1-28. The recoveries of sulphide using the proposed spectrophotometric method with purging were satisfactory. From the good recovery of sulphide, the use of purging as the separation procedure with the proposed spectrophotometric method was expected to obtain a reliable result because the proposed method involved a separation procedure that removed the analyte, sulphide, from the complex sample matrix.

65 Table 卜28 Recovery test for the determination of sulphide ion in beer samples using the proposed spectrophotometric method with Duraina

Brand names of beer sulphide sulphide Recovery Average used for analysis added _ found _* (%) Recovery (%)

San Miguel 4.00 3.98, 3.87, 99.6, 96.8, 97.3 土 2.1

Pabst Blue Ribbon 3.00 3.01, 2.94, 100,98.0, 98 ± 2

2.91 97.0 *丁his was the amount of sulphide after deducting the amount of sulphide found in the sample from the amount of sulphide found in the sample with known addition of sulphide .

(H). Spectrophotometric determination of sulphide in orange juices

Anti-foaming agent was added to the juice samples before purging because some foams were formed on purging. The pHs of the orange juice sample were about 2 (estimated from pH paper) and so enough sodium hydroxide solution was added to the juices to prevent the loss of sulphide as hydrogen sulphide. From the literature, 19, 23-24 heating food would induce sulphide (or hydrogen sulphide) from some sulphur-containing compounds. Hence, only purging was used for the separation of sulphide from orange juice samples and the distillation technique were not used in this part of the experiment. For the methylene blue method, purging was incorporated to the analysis to reduce the interferences due to the presence of the large amount of suspended matters in the orange juices. The results were shown in

Table 卜29.

From the results in Table 1-29, the concentrations of sulphide found in the samples using purging plus the proposed spectrophotometric method and the standard methylene blue method were comparable. From the standard deviation of the concentration of sulphide found in the samples, the precision of the proposed

66 method seemed to be better than the methylene blue method. The recoveries of sulphide from the samples were determined. The results were shown in Table 1-30 and the recoveries using the proposed spectrophotometric method with purging were good.

Table 卜29 Determination of sulphide in orange juice samples with purging as the separation/preconcentration for both the proposed spectrophotometric and the methylene blue method

Sulphide found (fig/ml)

Brand name and type of juices Proposed Method Methylene blue

used for analysis With purging

"Sunkist" fresh orange juice 0.0480 土 0.0007 0.0461 土 0.0012

"Mr. Juicy" (Sunkist) processed 0.0396 土 0.0008 0.0379 土 0.0010

orange juice

Table 1-30 Recovery test for the determination of sulphide ion in orange juice samples using the proposed spectroDhotometric method with purging

Brand name and type of sulphide sulphide Recovery Average juices used for analysis added _ found _* (%) Recovery (%)

"Sunkist" fresh orange 4.00 3.96,3.91, 99.0,97.8, 97.7 ±1.4

iuice 96^

Mr. Juicy (Sunkist) 4.00 3.94, 3.84, 98.5, 96.0, 96.8 土 1.5

processed orange juice 3.83 95.8

替丁his was the amount of sulphide after deducting the amount of sulphide found in the sample from the amount of sulphide found in the sample with known addition of sulphide .

67 4. Conclusions

An alternative spectrophotometric method for the determination of sulphide with separation/preconcentration techniques has been developed. The proposed spectrophotometric method involves a direct measurement of the absorbance of the complex formed with sulphide and p-HMB. However, the method is subjected to interferences due to some anions and cations, so that a separation/preconcentration step is suggested, resulting in longer analysis time. The separation step removes most of the interferences such as ions and suspended particles that are present in the sample. Nevertheless, the determination is expected to determine samples with lower sulphide concentration because the separation step also serves as a concentration step. The proposed spectrophotometric method coupled with purging or distillation step, provides a useful tool for the determination of sulphide in water samples. The separation utilizing purging seems to be more suitable for the removal of interferences from the food samples in the determination of sulphide as heating for the distillation might generate hydrogen sulphide from some sulphur-containing compounds.

The instrument required for the proposed method is a simple UV-VIS spectrophotometer, which is relatively cheap in comparison with other instruments and is available in most laboratories.

Table 1-31 showed the comparison of the proposed spectrophotometric

method and the methylene blue method where it could be seen that the proposed spectrophotometric method was more sensitive.

68 Table 1-31 Comparison of the methylene blue method and the proposed spectrophotometric method

Method Methylene blue Proposed spectro-

method* photometric method

Molar Absorptivity (l.mo|-l .cnrr'') 34,000 39,400

Range of Beer's Law (^ig/ml) 0.02-20 0.008-0.33

Precision (%) not reported < 1

*The data were based on reported literature. 18, 46

Although the precision of the methylene method was not reported, it could be seen by comparing the results that the proposed spectrophotometric method had a better precision. The accuracy in measuring low concentrations of sulphide using the methylene blue method was not good.

For further study, as hydrogen sulphide is one of the aroma of foods (as discussed in the introduction) such as hydrogen sulphide in the beer, investigation of the concentration of this compound using the proposed method in some other food samples may be tried.

For the separation of hydrogen sulphide by purging with nitrogen, a further study can also be made. The air-space inside the sample container can be reduced by using a larger amount of sample or adding distilled water such that the purging time or purging rate or both can be further reduced in order to achieve a good recovery of sulphide from the sample solution.

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(72). H.-H. Perkampus, "UV-VIS Spectroscopy and its Applications", Springer- Verlag, New York, 1992, pp. 3-9 and 68-69. (73). C. N. R. RAO, "Ultra-violet and Visible Spectroscopy: Chemical Applications", Third ed., Butterworths & Co (Publishers) Ltd., London, 1975, pp. 16-19 and 206- 209.

73 (74). R. C. Weast, M. J. Astle and W. H. Beyer, "CRC (Chemical Rubber Company) Hanbook of Chemistry and Physics Sixty-seventh ed,, CRC Press, Inc., Boca Raton, 1986, p. B-130, no. s257.

(75). D. D. Perrin, Australian Journal of Chemistry, 1963, Vol. 16, pp. 572-578. (76). D. D. Perrin and B. Dempsey, "Buffers for pH and metal ion control", Chapman and Hall, Ltd., London, 1974, Chapter 9 (pp. 117-122) and appendix II (pp. 129-

156)

(77). K. Helrich, "Official Methods of Analysis of the AOAC (Association of Official Analytical Chemists)", Fifteenth ed., AOAC, Inc., Arlington, 1990, Vol. I, Appendix (pp. 673-684).

(78). L Erdey, "Gravimetric Analysis", Pergamon Press, Ltd., London, 1963, Vol. I, pp. 178-184.

74 Part II Determination of fluoride General Introduction

All forms of living matter require inorganic elements (or minerals) for the maintenance of their normal life processes and fluoride is one of the trace essential elements needed by the living organisms J A study in mice shows that a deficiency of fluoride in the diet causes impairment of the reproduction capacity, decrease in growth rates and anaemia J' 6-8 Fluoride is beneficial when present in small amounts and is needed for calcification in living organisms. It is necessary for the development of bones and teeth with good quality as it reduces the chance of dental caries and osteoporosis (porosity of bones) J' 3-4, 9-12

On the other hand, fluoride is a cumulative poison and an excess intake of fluoride is toxic (fluorosis)」-5' 9-10' "13-14 no ill-effect is observed when a small amount of fluoride is consumed because two protection systems are present in the animals to deal with the increase in the level of fluoride in the bodies. They are the excretion of fluoride in urine and the deposition of fluoride in the bones and teeth.

When the bones and teeth become saturated with fluoride, accumulation of fluoride in the tissues occurs and causes adverse effects to the normal functioning of the bodies.1_2, 4, 10, 13-14 Mottling of teeth (chalky white patches with the secondary infiltration of yellow to brown staining), weakening and losing of the enamel in the teeth, changing the size, shape and orientation of teeth, osseous lesions, abnormal outgrowth of bone (called exostoses), thickening and ankylosing of joints due to the mineralization of tendons occur」9-10, 13-14 pgin appears in the joints and the animals become stiff and disabled, resulting in problem in locomotion J' 4' 10, 13

Owing to the occurrence of pain in teeth during eating or drinking, secondary effects such as poor appetite, and loss of body weight will develop. 1, 4, 5, 10, 14 other effects, including gastroenteritis, decrease in lactation, degenerative change in the organs and soft tissues such as weakness in the muscles, clonic convulsions,

75 pulmonary congestion and failure in the respiratory and cardiac functions, will also appear when a larger amount of fluoride is taken J' 12-14 丨门 extreme cases, serious diarrhea, cachexia, emaciation, metabolic disturbances and even death of the animals will result.I-2' 4-5, 13

The major sources of excess intake of fluoride in animals were the contamination of feeds by fluoride, feeds of animal origins, feeds containing high amount of phosphate (as rock phosphate, which contains a high concentration of fluoride, is one of the mineral phosphates that uses as a dietary supplement) and high fluoride content in water. 5

Fluoride is not equally toxic to all species of animals. Moreover, the tolerance levels of fluoride to the same species will be different with the age of the species, the chemical form of fluoride ingest, length of time and the continuity of fluoride intake, the components of diet, the environmental differences and the grazing conditions of the animals expose, etcJ'

Other types of levels have also been suggested, e.g. the performance tolerance level {which is the level without clinical interference from normal performance) and the pathology tolerance level (at which level of intake wni cause pathological changes) J ^

Excessive intake of fluoride is of more concern than the deficiency in livestock and poultry5' 10 because of the toxic effect of high levels of fluoride and fluoride is only required in trace amount in the diets of the animals. The margin of the safety (beneficial and toxic) intakes of fluoride happened to be small 1 and the determination of the fluoride therefore becomes important. As a result, the level of fluoride in the animal feeds should be monitored. The suggested tolerance levels of fluoride to some species, including livestock and poultry are shown below.

76 Suggested tolerance of fluoride ion to various kinds of animals^

Animals Tolerance level Definitely unsafe

(F ppm in diet) (F ppm in diet)

Breeding animals

Beef or calves ----- 40 and above

Beef or dairy heifers 30-40 50 and above

Mature beef or dairy cattle 40-50 60 and above

Ewes ^ 70 and above

Horses 40-60 80 and above

Sows 100-150 160 and above

Laying hens 4£0 440 and above

Animals to be slaughtered

Beef or dairy calves ^ 65 and above

Beef or diary heifers ^ 80 and above

Growing chickens (broilers) 300 340 and above

Mature beef or dairy cows 120 and above

Feeder lambs 170 and above

Finishing pigs 200 and above

77 spectrophotometric determination of fluoride 1. Introduction (A). Review of the reported methods for the determination of fluoride

From the literature, many methods were used for the determination of fluoride in various kinds of samples with the use of different separation methods for the reduction of interferences. 16-39 por samples such as foods and animal feeds, destruction of the organic matter in samples by ashing at high temperature is used as a pretreatment before the separation methods.17-19, 23 Distillation is the traditional and common separation method of fluoride from the interfering substances in the samples.1other less commonly reported separation methods using volatilization technique are micro-diffusion^®' 19-21, 30-32 and pyrohydrolysis^19-21, 27, 33-

34. Another separation method is ion exchange.16, 19-21, 27, 35-39 Commonly used methods for the determination of fluoride ion are spectrophotometry^ 6-27, 40-

and potentiometric method using a fluoride ion selective electrode (abbreviated as

F|se)16-18, 26-27, 32, 45-47. jhe development of ion chromatography^^, 48-50 increases its application for the determination of fluoride. Other less commonly used quantitative methods are gas chromatography^^' 51-53, polarography54-55, atomic

and molecular absorption spectroscopy^^' 56-57, gravimetryl 6' 19/ 58-60, titrimetry16, 18-19, 61-62 and enzymatic indicator method63-64, etc.

Classically, distillation^ is incorporated into different analytical methods to separate fluoride ion from the interfering substances in the samples as HaSiFs or

SiF4 under a highly acidic medium containing silica. Preliminary distillation is needed

to reduce the interfering substances in the sample and a large volume (-150 ml) of

distillate is required in the final distillation. Moreover, there are quite a number of

precautions in the experimental procedures, including attention of the analyst to

neutralize the acidic distillate using alkali during the distillation and a special cleaning

procedure for the apparatus after each determination to prevent contaminating the

78 next determination. A disadvantage of the distillation method is that fluoride at low concentrations will be hold back in the distillation apparatus and does not distill out.

The presence of high concentration of aluminium and silica will decrease the volatilization of fluoride and retard the distillation of fluoride. Moreover, the distillate is not free from anions such as sulphate, halides and nitrate, etc. Other disadvantage includes the loss of hydrogen fluoride by reaction with glass vessels. A rather new and relatively rapid distillation method at room temperature is suggested, where a volatile compound formed in the reaction between hexamethyldisilazane and fluoride is purged and absorbed in an alkaline solution.^^ However, the absorption is affected by the pH of the absorbing solution and therefore acid gas {such as carbon dioxide) generated from the samples (containing carbonate) influences the result；

Separation methods such as microdiffusion and pyrohydrolysis require special apparatus.16' 19-21, 27, 30-34 Moreover, the former requires considerable time (2-

24 hours) whereas the later needs attention and care during heating at high temperature {~1000®C). Although the recovery of fluoride using pyrohydrolysis is good, it is too specialized and is not suitable for general laboratory use.

The concentration and separation of fluoride from the interfering substances in the sample matrix using anion exchange resins pose problems. Formation of negative metal-fluoride complexes in the presence of interfering metal cations and tailing of fluoride eluted from the resin】®' 19-21, 24, 27, 35-36 result in a large volume of eluate in order to collect fluoride completely. The use of chemically modified ion exchange resins37-38 requires preparation to adhere the chemical, Zr, on the support for the adsorption of fluoride, but is interfered by ions including phosphate that can complex with Zr adsorbed. The reported ion exchange spectroscopy,39 jn which a spectrophotometric system is adsorbed on the resins, enables concentration and determination of fluoride in the samples, however, the

79 method is affected by the general problems associated with the spectrophotometric

method, which are discussed below.

A number of spectophotometric methods, including colorimetric and

fluorometric, have been used for the determination of fluoride. 16-27, 40-44 Most of

the methods are based on the inhibitory or decomposition action of fluoride ion on

the complexes formed between multi-valent metals and organic complexants, which

result in the formation of more stable metal ion-fluoride complexes. The decrease in

the absorbance due to the decomplexation of metal-complexant is related to the

fluoride concentration. A few methods involved the measurement of absorbance on

the released complexant. Some reported spectrophotometric systems are Zr with

erichrome cyanine r19-23, 25, alizarin red s16' 19, 24, 26, sodium 2-

(parasulfophenylazo)-1,8-dihydroxy-3,6-naphthalene disulfonate (SPADNSp 9-22, 26-

27, 40, xylenol orange20-21, soiochrome cyanine R^l or trypan blue^^.八| with 8-

hydroxyquinolinelB, erichrome cyanine r19 or chloranilic acid''®' 20-21; La with

, alizarin complexonel 6-17, 20, 22-23, 27, 43 or chloranilatel 6, 20-21, 丁卜 with

alizarin 5^8-19, 23, 44 。「 2-(1,8-dihydroxy-3,6-disuIpho-2-naphthylazo)-

phenoxyacetic acid20-21. Obviously, these methods are complicated by other

cations or anions in the samples that form stable complexes with the organic

chelates or the metal cations in the spectrophotometric systems.16, 19 Also,

considerable time (30 minutes to 1 hour) is required for most of the

spectrophotometric systems to complete the reaction before making any

measurement. Although the reported La-alizarin complexone system involves the

measurement of the complex formed, the chance of possible interferences is higher “

compared with other metal-complexant systems (e.g. Zr-complexant and AI-

complexant) as La had a relatively lower combination tendency with fluoride

compared with other cations such as Al and Zr, etc.Yet, some of the metal-

80 organic dyes used are unstable and will decompose on standing. All these factors will contribute to give inaccurate result.

The procedures of potentiometric method using a FISE are simple J

27, 32, 45-47 However, this method suffers from the errors due to the signal shift, temperature variation, high noise level and chemical interferences in the sample

matrix. Memory effect of the electrode will affect the accuracy of results when

measurement of sample with low fluoride concentration is made after immersing the

electrode in a solution with a relatively high fluoride concentrations. The activity of

the ion, rather than the concentration of the ion in the solution, responded by the

FISE is influenced by the ionic strength of the solution which requires that the ionic

strength in all the standard and sample solutions should be similar. Other

experimental parameters such as the time for recording potential reading after

immersing the electrode into the sample solution (the time for equilibrium), the

absence of the bubbles on the surface of membrane in the electrode especially during

stirring, pH of the solution (due to the response of electrode to hydroxide ion) and

the purity of the reagents used should be aware throughout the experiment for

obtaining reliable results. The use of lanthanum fluoride membrane in the electrode is

limited to a certain level of fluoride (-10"^ M) due to the dissolution of fluoride from

the membrane at low fluoride concentrations, giving a background signal J ®

The chromatographic determinations, including ion chromatography^^' 48-50

and gas chromatography^ ^ allow the separation and analysis of fluoride from

simple and complicated matrices. In ion chromatography, a number of preparations

are required before and after the experiment, including filtration of sample, elution of

the analytical column with the mobile phase for several hours to assure a low

background and equilibrium of the column, and regeneration of the suppressor

column for several hours after use. These will increase the time of analysis.

Empirically, the separation of fluoride from samples containing organic acids requires

81 the column to be rather new. The use of base as the eluent will affect the accuracy of determination using conductivity as the eluate has a low conductance after neutralization though the suppressor. Moreover, the anions in the eluent will affect the resolution of ions in the sample.^^ The poor elution of fluoride from the column also results in difficulty in quantitation and detection limit.Although the variation in the rate of elution of anions can be overcome by using potentiometric and conductometric detectors^^, the method becomes more complex. The method without using suppressor column developed can decrease the apparatus used, however, the sensitivity is comparatively lower than that using this column. The procedures in the determination of fluoride using gas chromatography have a number of shortcomings. Different derivatizing agents for fluoride impose different disadvantages either on the reaction conditions or in the separation step in the column such as long reaction time, low temperature reaction condition and low boiling point of the complex formed (that requires a cold environment to reduce the evaporation), etc.

Although the lower limit of the polarographic method54-55 jg iqw and the sensitivity is good, electroactive substances having neighbour electroactive potentials with fluoride present in the sample will interfere with the determination seriously.

Another disadvantage is the long analysis time.

Atomic and molecular absorption spectroscopy have been employed for the determination of fluoride.56-57 The procedure in the atomic absorption spectroscopy involves the measurement of redissolved metal from precipitated metal fluoride after filtration. Errors such as those described below concerning the gravimetric determination also appear in this kind of determination and affect the precision and accuracy of the results. Fluoride determination using the absorption due to the diatomic metal-fluoride molecule formed such as aluminium-fluoride is retarded by interferences in the sample matrix. Extraction helps to improve the

82 cationic tolerances in the interferences but the interfering ions including chloride (due to the formation of aluminium-chloride) and sulphate (due to the volatilization of HF through pyrohydrolysis reaction) are still significant.^^

Precipitations of fluoride as calcium fluoride, lead chlorofluoride (PbCIF), lead bromofluoride (PbBrF), lithium fluoride, triphenyltin fluoride and lanthanum fluoride for the quantitative determination of fluoride^®' 58-60 are affected by coprecipitation of other ions in the solution during the course of precipitation. Each analysis is time-consuming for the need to wait for complete precipitation, evaporation of the mother liquor and drying before making any quantitative measurement. The method is only suitable for samples with high fluoride content and the precipitation depends on the experimental conditions. Moreover, there are other drawbacks of the precipitation method such as lack of sensitivity, re-dissolution of the precipitate formed, variation in composition of the precipitate, incomplete precipitate, gelatinous precipitate, etc.

The titrimetric methods for the determination of fluoride^ 18-19, for example, thorium (IV) or zirconium solution as the titrant with alizarin red S as an indicator, are not accurate and precise enough in comparison with other instrumental methods. This is because the end-point depends on a visual detection, which can be interpreted and performed differently by different experimenters. Also, titrimetric methods are only suitable for samples containing a considerate amount of fluoride in the sample solution. Apart from the normal titration using visual end-point,

photometric and potentiometric titration were employed for end-point detection.61 _

Problems present in spectrophotometry and the use of ion selective electrode can

be found in the corresponding method, respectively. A disadvantage is that the end-

points of the titrations are not definite and has to be located by extrapolation.

83 The use of enzyme sensor for the determination of fluoride63-64 jg too specialized for ordinary chemical laboratories. Moreover, extra biological techniques are required.

(B). General description of the proposed scheme for the spectrophotometric determination of fluoride in animal feeds

The objective of this project was to develop a relatively convenient and

general procedure for the determination of fluoride content in animal feeds compared

with those using inconvenient separation methods such as distillation. Basically,

there were two major considerations: conversion of the sample into an aqueous

solution and elimination of interfering substances in the sample solution.

In the conventional analysis of fluoride content in animal feed samples,

calcium hydroxide was used as a fixing agent for fluoride in the ashing J 65-66

Distillation of fluoride in the ash in the form of hydrogen fluoride under strongly

acidic condition was then followed due to the insolubility and low solubility of

calcium fluoride in water and in slightly acidic condition, respectively. It was also

used to reduce the interfering substances in the sample solution. A number of

disadvantages could be found in the distillation and had been discussed in the review

above. Some reported the direct ashing of samples without the use of the fixatives if

the sample was alkaline enough.67-68 八 series of reagents including hydroxides and

carbonates of the alkali metals and oxides and hydroxides of the alkaline earth metals

were reported as the additives or fixatives to prevent the loss of fluoride during

ashing.A simpler method was the dissolution of animal feed sample with 1 M

hydrochloric acid rather than the use of ashing.70

Preliminary tests to the above ashing and dissolution methods of the animal

feed samples had been done in this project. The samples were only sparingly soluble

in 1 M and 6 M hydrochloric acid accompanied by stirring overnight using a magnetic

84 stirrer. There might be some bound fluorides in the insoluble residues. An ashing process under a high temperature was needed to remove the organic compounds in the sample before the determination of the total fluoride content of the sample. eq

Porcelain crucibles were contaminated with small amounts of fluorine In the glaze.

Nickel crucibles were used in the ashing process. The recovery of fluoride by known addition of fluoride to the animal feed sample before ashing at SSCTC for 3 hours was found to be low (-30 %) without the use of any fixative in ashing (although the ashing was found to be complete). The amounts of fluoride in the sample solution in the preliminary tests were determined by potentiometric method using a FISE owing to the presence of interferences for the spectrophotometric method, as wHI be

discussed in section 3B below.

After literature survey and preliminary tests, the procedures of the analysis in

this project were carried out to include the following steps: ashing the animal feed

sample to remove the organic matter in the sample with an alkaline reagent to reduce

the loss of fluoride, leaching of fluoride in the ash with hot deionized water to aid

dissolution of the cake, acidifying the solution to slightly alkaline (pH 8), filtering the

residue that separated the insoluble residue from the solution, and determining the

amount of fluoride in the filtrate with a spectrophotometric method after removing

the interfering substances in the sample solution.

The aim of pH adjustment to 8 was to precipitate some cations, including the

interfering cations such as iron that interfered the spectrophotometric method, as

their oxides if the concentrations of these ions were not too high. The precipitate

were then removed by filtration.^Solution containing 62-1720 ppm Fe could be

reduced to below 0.05 ppm.71 This could reduce the interferences to the

spectrophotometric method by the lowering the amount of interfering cations that

can complex with fluoride.

85 Anion exchange resin was used for the separation of fluoride ion from the interfering substances. Concentration of solution to a smaller volume relative to the

sample solution used might be possible. Alternatively, a batch method with cation

ion exchange resin was used to remove the interfering ions from the samples by

adding the resin directly to the sample for the removal of interfering cations without

extra work for cleaning, or preparing a column to hold the resin.

Fluoride in the sample solutions, after removing the interfering substances,

was then determined using the Zr^ + .SPADNS system^^, 40 by measuring the

decrease in the absorbance owing to the inhibitory action of fluoride ion to the

spectrophotometric system. The structure of SPADNS is shown in Figure 11-1. OH OH _

Na 0 S 0 3Na

Figure 11-1 Structure of SPADNS

The main reason for using this spectrophotometric method is due to the short

reaction time compared with other spectrophotometric methods. Measurements can

be made immediately after mixing the +-SPADNS solution with the sample

solution. This method is widely used for the determination of fluoride in portable

waters and is used as a standard method for water analysis. Also, the stability

constant of the complex between fluoride and Zr was found to be the greatest

among analogous complexes with other metal cations. 19' 28 jhe procedure of the

method is simple and no special manipulations other than the measurements of the

volume of the reagent and the samples during the preparation are necessary. The

reagents and the instrument used are readily available and the method is suitable for

most chemical laboratories.

The principle of analytical technique, namely, UV-VIS spectrophotometric

method, used in the project was referred to the previous descriptions, (p. 15)

86 2. Experimental Section

The quantitation of fluoride content in animal feed was carried out by ashing the sample followed by removal of the interfering substances in the sample solution

using anion exchange before the spectrophotometric measurement of fluoride.

Potentiometric measurement using a FISE was used as the counter-check method for

the determination of fluoride.

The 'deionized water' used in the present work was doubly deionized water

prepared by passing the deionized water obtained from Barnstead D4742 nanopure

ion-exchange system through Millipore Milli-Q^® ultrapure water system. All the

solutions prepared were stored in plastic bottles especially solutions containing

fluoride which could be adsorbed on glass.奶’乃

(A). Ashing of the animal feed samples

Apparatus

70-ml nickel crucibles, 250-mI plastic beakers and Lindberg Hevi-Dutv muffle

furnace

Reagents

Sodium hydroxide pellets (BDH Chemical Ltd.) and silica (BDH Chemical Ltd.)

Procedure

0.500 or 1.00 g of the animal feed, which was previously grounded in a

mortar and dried in an oven at 110 overnight, was weighed and placed in a 70-ml

nickel crucible. About 2.5 times the amount of the animal feed of sodium hydroxide

(1.25 and 2.50 g of NaOH for 0.50 and 1.00 g o于 sample respectively), and 0.05 g

of silica were added to the crucible. A small amount of deionized water was added to

cover the sample. The crucible was partially covered with a lid (to allow the gases to

escape during ashing) and was placed in a cooled muffle furnace. The sample in the

crucible was preashed at 300®C for 1 hour in the furnace, and the temperature of the

furnace was then raised to 550®C for two and a half to three hours. The crucible

87 was then removed from the muffle furnace and cooled. Hot deionized water was added to cover the cake in the crucible to aid the dissolution of the cake and the contents were heated occasionally on a hot plate with stirring and rubbing with a rubber policeman until nearly all the solids were dissolved. (The alkaline residue in the nickel crucible was difficult to remove if it was just dissolved in deionized water.)

The solution was poured into a 250-ml plastic beaker and the crucible was

rinsed several times with deionized water, and the rinsings were combined with the

solution in the plastic beaker. The use of the plastic beaker was to reduce the loss of

fluoride as it was adsorbed on glass.45' 73 Hot deionized water was again added to

the nickel crucible and the crucible was heated gently for 10 minutes in order to

remove all the materials from the crucible. The hot solution in the crucible was

transferred into the plastic beaker. The crucible was rinsed with deionized water

several times and the rinsings were poured into the crucible. The cooled solution was

adjusted to a pH value of 8 with concentrated hydrochloric acid (2.60 and 5.20 ml of

acid for 1.25 and 2.50 g of NaOH used respectively). The pH adjustment should be

done with care because some fluoride would be lost during neutralization if the

solution was heated at or above a temperature of 60°C in an acidic medium.The

solution was filtered with a Whatman no. 41 filter paper into a 100-ml volumetric

flask with the aid of a funnel to remove the residue. The plastic beaker and the filter

paper were rinsed several times with a little amount of distilled water and the

rinsings were also transferred for filtration. The solution was then made up to the

mark with deionized water and the flask was shaken thoroughly. If the analysis was

not started immediately, the solution was stored in a plastic bottle.

88 (B). Reduction of interferences in the ashed sample solutions using an anion exchange resin

Apparatus

A 50-ml burette (1.00 cm i. d.), glass wool, a column (2.5 cm i. d.) closed at one end with sintered glass, and a stopwatch

Reagents

Amberfite IRA-400 {Cl_} (Fluka)

1.5 M sodium hydroxide solution - 60 g of sodium hydroxide pellets (BDH

Chemical Ltd.) were dissolved and diluted to 1 litre with deionized water.

0.15 M sodium hydroxide solution - 6.0 g of sodium hydroxide pellets (BDH

Chemical Ltd.) were dissolved and diluted to 1 litre with deionized water.

Procedure

(i). Pretreatment of resin^^

If the resin was new, the resin should be stirred and washed thoroughly with

deionized water in a beaker. The supernatant was decanted and washing was

repeated until the supernatant was clear and colourless.

(ii). Conversion of resin to hydroxide form75

The Amberlite IRA-400 resin was converted from its chloride form to

hydroxide form by passing approximately threefold amount of 1.5 M sodium

hydroxide solution (relatively to the amount of resin used) through the resin in a

column with 2.5 cm internal diameter within 30 to 40 minutes by adjusting the flow

rate. The resin was then washed with deionized water to remove the excess alkali

until the eluate had the pH of deionized water.

(iii). Filling of the column with resin^^

A glass wool pad, soaked with deionized water, was transferred to the

bottom of the column (a 50-ml burette) using an iron wire. The resin, together with

the deionized water, was transferred to the water-filled column (to prevent trapping

89 of air bubbles between the resins during transfer) with a funnel. The water in the column was drained out to allow the resin to settle to the bottom of the column.

More deionized water was passed through the column until there was no downward movement of the resin in the column. The transfer of resin to the column was continued until the desired length of resin was reached.

It was important to keep the solution above the resin and to prevent any air bubble trapped in the resin.

(iv). Separation of fluoride from the interfering substances usino the packed column

The sample solution {50 and 100 ml for 1.00 and 0.50 g of sample respectively) obtained from ashing was passed through the column using a Pasteur capillary pipette for transfer. The flow rate through the column was adjusted to 1.5 ml/min. 30 ml of deionized water were then allowed to pass through the column.

0.15 M sodium hydroxide solution was added to the column. The eluate was collected in a 100-ml measuring cylinder and after 75 ml of eluate were collected, a

50-ml volumetric flask was then used for the collection of the eluate and about 48 ml were collected. It was important to keep the solution level above the resin to within

3 to 10 cm during the passage of solution.

(v). Regeneration of the resin used75

After each experiment, the used resin was regenerated to its hydroxide form following the procedure in section 2B (ii) (p. 89). A 50-ml burette might be used.

(C). Preparation of solutions for the calibration graph and the ashed sample solutions for the spectrophotometric measurement^Q

Reagents

Standard fluoride solution - 0.221 g of sodium fluoride (Riedel-de Haen). which was previously dried at 110®C for two hours, was weighed accurately,

90 dissolved and made up to 100 ml in a volumetric flask with deionized water. This solution contained 1000 ppm of fluoride ion.

2.5 and 10 ppm fluoride solutions -- 2.5 and 10 ppm of fluoride solutions were prepared by transferring 2.50 and 10.00 ml of standard fluoride solutions to 1- litre volumetric flasks separately, which were made up to the mark with deionized water.

SPADNS solution -- 1.198 g of sodium 2-(parasulfophenylazo)-1,8-dihydroxy-

3,6-naphthalene disulfonate (SPADNS) (Sigma Chemical Co.), were dissolved and diluted to 500 ml in a volumetric flask with deionized water.

Zircony I-acid reagent -- 0.14 g of zirconyl chloride octahydrate (BDH

Chemical Ltd.). ZrOCIz.SHsO, was dissolved in about 25 ml of deionized water and was transferred to a 500-ml volumetric flask. The solution was added with 350 ml of

37 % hydrochloric acid and diluted to the mark with deionized water.

-SPADNS solution - Equal volumes of SPADNS solution and zirconyl- acid reagent were mixed.

6 M hydrochloric acid - 125 ml of 37 % hydrochloric acid (Beiiing Chemical

Works) were diluted to 250 ml in a volumetric flask with deionized water.

Phenolphthalein -- 1 g of phenolphthalein (E. Merck) was dissolved in 100 ml of ethanol (E. Merck) and 100 ml of deionized water were added. The insoluble residues were filtered.

Solution A - AO Q of sodium hydroxide (BDH Chemical Ltd.) were dissolved in 800 ml of deionized water and 84 ml of 37 % hydrochloric acid were added. The solution was diluted to 1 litre with deionized water.

Procedure

(i). Preparation of calibration graph:

Solutions containing 0.05, 0.10, 0.20, 0.40, 0.60, 0.80, 1.00, 1.20 and

1.40 ppm of fluoride were prepared by pipetting 1.00, 2.00, 4.00, 8.00 ml of 2.5

91 ppm fluoride solutions and 3.00 4.00, 5.00, 6.00, 7.00 ml of 10 ppm fluoride solutions, respectively, to 50-ml volumetric flasks separately. Each flask was added with 7.50 ml of solution A and diluted to the mark with deionized water. 20.00 ml of these fluoride solutions were added to 25-ml volumetric flasks separately and 4.00 ml of Zr4 + -SPADNS solutions were added. The solutions were mixed thoroughly,

(ii). Preparation for the sample solutions obtained from the anion exchange resin

The eluate collected from the anion exchange resin [from section 2B (iv) (p.

90)] was adjusted to pH 8 with 6 M hydrochloric acid using phenolphthalein as an indicator, and diluted to 50 ml with deionized water, and 20.00 ml of this sample solution were mixed thoroughly with 4.00 ml of Zr4 + -SPADNS solution in a 25-ml volumetric flask.

(D). Spectrophotometric determination of fluoride in the ashed samples

Apparatus

Hitachi U-2000 double beam spectrophotometer and a pair of matched glass cells

Reagent

Reference solution -- 10 ml of SPADNS solution were mixed with 100 ml of deionized water, followed by the addition of 10 ml of a diluted hydrochloric acid solution (7 ml of 37 % hydrochloric acid were diluted to 10 ml with deionized water).

Procedure

(i). Construction of the calibration qraoh^^

The absorbance at 570 nm was adjusted to zero by placing the reference

solution in both cells. Each fluoride solution prepared in section 2C (i) (p. 91) was

used to rinse the sample cell several times before the absorbance of the solution was

92 found. A calibration graph of absorbance at 570 nm against the respective concentration of fluoride (ppm) was constructed,

(ii). Determination of fluoride in the samples

The absorbances of the sample solutions at 570 nm, which were obtained in section 2C (ii) (p. 92), were measured, and their fluoride concentrations were deduced from the calibration graph.

The concentrations of fluoride in the samples (mg/kg) were then calculated from the mass of samples used and the concentrations of fluoride in the sample solutions.

(E). Potentiometric determination of fluoride in the ashed sample solutions by the FISE method as the counter-check method

Apparatus

Orion Model 96-09 Combination Fluoride Electrode, Orion 720A meter,

Labinco 532 hot plate/magnetic stirrer, a magnetic stirrer bar, a 100-ml plastic beaker and a stopwatch

Reagent

Total ionic strength adjustment buffer (TISABp'^ -- 58 ml of glacial acetic acid (E. Merck) and 12 g of sodium citrate dihydrate (Beijing Chemical Works) were added to 300 ml of deionized water. The solution was adjusted to a pH of 5.0 to 5.5 with 25 % sodium hydroxide (BDH Chemical Ltd.) before making up to 1 litre with deionized water.

Procedure

(i). Preparation of diluted fluoride solutions

Solutions with 0.05, 0.10, 0.20, 0.40, 0.60, 0.80, 1.00, 1.20 and 1.40 ppm of fluoride were prepared by pipetting 1.00, 2.00, 4.00, 8.00 ml of 2.5 ppm fluoride solutions and 3.00 4.00, 5.00, 6.00, 7.00 ml of 10 ppm fluoride solutions,

93 respectively, to 50-ml volumetric flasks separately. Each flask was added with 7.50

ml of solution A (p. 91) and diluted to the mark with deionized water.

(ii). Construction of the calibration araoh^^

Equal volumes {10.00 ml : 10.00 ml) of TISAB and diluted fluoride solution

were pipetted and mixed in a plastic beaker. The combination fluoride electrode,

which was connected to Orion 720A meter, was immersed in the mixture with

constant stirring using a magnetic stirrer for 4 minutes. The potential reading of the

solution was then taken. The electrode was then withdrawn from the solutions,

rinsed with deionized water and blotted dry. The procedure was repeated for other

fluoride solutions. A calibration graph of potential vs. log Cp where Cp was the

concentration of fluoride (ppm) used, was constructed.

(iii). Determination of fluoride in the samples

The experimental procedure in section 2E (ii) above was repeated for the

ashed sample solutions. The potential of each sample solution was measured and the

concentration of each sample solution was deduced from the calibration graph. The

concentrations of fluoride in the samples (mg/kg) were calculated from the mass of

samples used and the concentrations of fluoride in the sample solutions.

3. Results and Discussions (A). Ashing conditions of the animal feed samples

The determination of fluoride in the solution from the sample in this part of the

project was performed by potentiometric measurement using a FISE due to the

presence of interferences in the sample solution which interfered with the

spectrophotometric determinations. The sample solutions, obtained after ashing a rat feed sample at 550°C for 3 hours, were filtered after adjusting to pH 8 with sodium

hydroxide solution unless specified and were used in the experiments.

94 A recovery test for fluoride was done by adding a known amount of fluoride to the sample before ashing. A low recovery of fluoride (-30%) in the sample solution was found by ashing the sample alone without the use of additive.

An X-ray fluorescence (XRF) spectrum taken with a Spectrace 5000 Tracor X-

ray, energy dispersive type, on a rat feed sample was shown in Figure 11-2.

• t 1 1 . o ^

;r—. r— .一• — . ••• . r— —• » a - ^― t—. _ J u J r— • i —-一- :-：f-'Li！ riHL-c. 1 r-ii n-LM'iinfM i ：二‘ COWNT^RRW . A

TUSH VCLTAGS ： 2G 5IV ptt Tiy-p rrcgn . 'TTT-rv TUBE CURflE2?T • o • 05 r TTrv^Tiro • 20O SZC A . A T"0

^ * * «•« Wte • _ _ ^^^ • • •丨一 •、 口 S. •“ 2JI I…丨丨丨m I丨,“丨丨w丨“丨丨丨丨m丨i M丨丨丨丨m h m i 丨丨丨丨丨I m 丨丨丨I 丨丨丨i t f h丨丨I 丨丨丨j 11 “丨丨j丨f ” ； m 11丨丨f “丨m丨！ f j “ i • ！ ！ .i i ！ ！. ； i i ！ i i ！ i ： A J ‘ r I : i : _!i I： Ii 3 : I！ ！;)•_!' i. 「li i I i i i I i � i. i i :t i ！ 丨丨i ii J 丨：J il ！ ‘. i i i 「 j 丨 I i '!i 丨 ||| !! ！ ！ ij il i i ' ii J r i ！ f ii" j f |,i .! ！1 i- 1 ！ I -! J L I i' ！ ！ i/- i ！ r» ！

I 幼. ！! J i- 1 ！ I J i r # I ！ I ！ K I -I \i L ！二丨- - . i n a. n .jf I . S > 1 ！ f I 」f^Z C F P 2 乂 ！ 1 •sn i< 5. a a n — i i I ‘ -kr I . \ iJ I' }'. i ,、一、八j u . J U•一、.:' U>、、.>一乂'， I …m⑴丨 ⑴丨Hi、丨 uu 丨I⑴ |m i|im!imi"i lim 丨i⑴…m、…⑴ «'»»»»«»»» t I I t I I I ； I 1 J i 2 3 4 5 6 7 S 9 le 11 12 13 14 15 16 17 IS 19 22 vwr

Figure 1卜2 XRF spectrum of a rat feed sample

95 From the XRF spectrum, the presence of calcium in the sample was indicated.

Although the amount of calcium could be reduced by filtering the sample solution after adjusting to pH 8 (a solution with 5-650 ppm of calcium could be reduced to below 10 ppm), the sample might still contain a high amount of calcium that might interfere with the determination as it formed water-insoluble calcium fluoride.

As calcium fluoride was soluble in strong mineral acids, acidification of the ashed sample solution (without the use of additive in the ashing) before filtration (the filtrate was adjusted to pH 8 afterwards) resulted in a higher recovery of fluoride

(-65%). This indicated the loss of fluoride as insoluble fluoride. However, the

acidification might result in loss of fluoride as hydrogen fluoride.

The effectiveness of sodium hydroxide as an additive was then assessed.

Sodium hydroxide was chosen because a relatively low temperature range (450。C to

600°C) had been reported.的 It was added to solubilize the insoluble fluoride under

alkaline medium. The addition of sodium hydroxide to the ash (with ashing the animal

feed alone) resulted in a higher recovery {-70%) of fluoride than without the addition

of sodium hydroxide. This indicated the solubilization of fluoride in the ash. A higher

recovery of fluoride (-80%) was found when sodium hydroxide was added to the

feed sample before the ashing compared with the cases when without this alkaline

agent and with its addition after the ashing. Based on the results, sodium hydroxide

pellets were added to the sample before the ashing process.

Empirically, the larger the amount of sodium hydroxide used, the better the

ashing within a fixed interval of time but the easier the spillage of the sodium

hydroxide melt from the crucible during the high temperature ashing. After trial and

error, 0.500 or 1.00 g of the samples with 2.5 times the sample weight of sodium

hydroxide was used in order to achieve good ashing and prevent the spillage of the

sodium hydroxide melt during the ashing process. In addition, a pre-ashing at 300°C

for an hour was found to eliminate the spillage of sodium hydroxide from the

96 crucible. If a higher mass ratio of sample to sodium hydroxide was used, a longer ashing time was required. For example, the use of 2.0 of sample and 2.5 g of sodium hydroxide would result in incomplete ashing (a significant amount of carbon particles remained in the ash) even after ashing for 6 hours at 550®C.

As the loss of sodium fluoride was significant at or over a temperature of 600

the ashing temperatures studied were from 450®C up to 550®C, The time required to achieve good ashing at different temperatures with 1 g of sample and 2.5 g of sodium hydroxide were shown in Table 11-1. The lower the ashing temperature, the longer the time required to complete the ashing. The cake after ashing was heated with deionized water on a hot plate and rubbed with a rubber policeman to facilitate the dissolution.

Table 11-1 Time required for ashing 1 g of sample with 2.5 a of sodium hydroxide at different ashing temperatures

Ashing Temperature (°C) Time required for good ashing (hours) — I I I I • —I _ _|_圓|, •_ I .1 i • •» i| _•• il ••! •丄•••• ^ 12 . 500 6 ^ 3

In order to achieve good ashing in a relatively short interval of time, ashing temperature at 550®C was employed.

However, the recovery of fluoride, which was described above, was not

satisfactory. This might be due to the loss of fluoride in one or more of the

experimental steps:

1. volatilization of fluoride in the ashing process;

2. heating the cake in the crucible after ashing;

97 3. pH adjustment of ashed sample solution to 8;

4. filtering loss;

5. interferences in the sample affecting the measurement of fluoride.

Experiments were performed to investigate which step(s) was (were) responsible for the loss of fluoride. Addition of known amount of fluoride prior to performing of the corresponding experimental step was done to see whether fluoride was lost through that experimental step. The recovery of fluoride in the sample was then determined. A recovery near 100 % of fluoride was found in steps 2, 3 and 4

above but a relatively lower recovery (-80%) was found in step 1. The relatively

lower recovery of step 1 indicated the probable loss of fluoride in the ashing process.

Besides the use of sodium hydroxide, a number of additives were tested in

order to establish a better ashing condition and to improve the recovery of fluoride.

The additives tested were silica, calcium oxide and alumina and it was hoped that

they could interact with fluoride to prevent the loss of fluoride during ashing. (Note

that small amount of silicon, calcium and aluminium might be removed as their

oxides by pH adjustment of the ashed sample solution followed by filtration.71-72)

In this experiment, they were added to the sample separately before ashing. The use

of small amount of silica with sodium hydroxide improved the recovery to over 90

%. However, the use of the other two additives resulted in a recovery of fluoride

below 20 %, and hence the use of these reagents were excluded.

The amount of silica used was studied with 1 g of sample and 2.5 g of

sodium hydroxide as a basis. No difference in the amount of fluoride and recovery

was found with 0.05, 0.1, 0.2 and 0.4 g of silica used.

Fusion of sparingly soluble fluorides with silica and alkali yielded sodium

silicofluoride (SiFe^" and/or SiF4) which was more readily soluble. Different reactions

could be found as shown below.

98 NasSiFe + 4 NaOH -> Si(0H)4 + 6 NaF (at pH 8.5)17

SiF4 + 2 H2O -> 4 HF + SiOa”’ 28

Silicofluoride was converted to free fluoride in the dissolution with deionized water after ashing. The alkaline medium prevented fluoride to exist as hydrogen fluoride.

(B). Removal of the interferences in the ashed sample solutions

The determination of fluoride in the solution from the ashed sample was performed initially using both the chosen spectrophotometric method (Zr4 +-SPADNS mixture) and potentiometric method. However, the results showed that the amount of fluoride found, which were obtained after ashing the feed sample with sodium hydroxide with and without the addition of silica, using the spectrophotometric method did not agree with those obtained using the FISE method, but with much higher fluoride found using former method than those using the latter method. The potentiometric measurements on the diluted sample solutions yielded a comparatively lower amount of fluoride than those using the undiluted one. However, the determination of fluoride in the diluted and undiluted sample solutions did not yield significantly different results using the spectrophotometric method.

A recovery of about 96 % of fluoride by known addition of fluoride to the sample solution, which was obtained after ashing, was found using the potentiometric method. These indicated that the matrix interferences, which affected the spectrophotometric measurement of fluoride ion in the sample solution, were not significant to the FISE method. The relatively high result of fluoride ion found in the sample solution using the spectrophotometric method might be due to the presence of interfering substances in the sample solution. The presence of the interfering substances made the determination of fluoride in the feed sample using the spectrophotometric method difficult.

99 Hydroxide ion and metal ions, which complexed strongly with fluoride ion, interfered with the potentiometric measurement of fluoride using a FISE. TISAB (total ionic strength adjustment buffer) was added to the sample before measurement for the following purposes: to free the fluoride from complexes as the electrode measured free fluoride ion only (reduce the complexation of fluoride with interfering cations); to minimize liquid junction potential and to maintain a constant and high ionic strength of the sample solution such that it was similar in sample and standards. Ions only interfered if their concentrations were high in the sample solution. Moreover, most of the buffers kept the pH within 5.0 to 5.5, which eliminated the interfering effect of hydroxide ion. A pH lower than 5.0 was undesirable because fluoride would complex with proton to form undissociated HF and /or HF。"". Basically, matrix interferences using the FISE method were not great.

The spectrophotometric method was interfered by anions such as phosphate

{>16 ppm) and sulphate (>200 ppm) based on a fluoride error of 0.1 ppm at a fluoride level of 1.0 ppm.40 Although no phosphorus and sulphur compounds in the

rat feed sample were detected from the XRF spectrum, the sample might contain a

certain amount of these compounds that were not detected by XRF. As discussed

before, the spectrophotometric measurement was based on the decrease in

absorbance of the coloured complex -SPADNS system due to decomplexation

by fluoride. The Zr4 +-SPADNS complex was also decomposed by phosphate and

sulphate, which decreased the absorbance of the solution, and hence would cause a

large positive error. The higher amount of fluoride found from the spectrophotometric

method than that from the FISE method might indicate the presence of anionic

interferences.

Other than calcium, iron was detected from the XRF scanning on the rat feed

sample. Considerable amount of iron (>10.0 ppm)40 jp the sample would affect the

spectrophotometric determination of fluoride. As discussed before, the interference

100 due to iron could be reduced by filtering the sample solution after adjusting to pH

8.71

Apart from the presence of interfering anions in the sample, sample solution might contain high concentrations of interfering cations that were not reduced to below interfering levels by the pH adjustment^ ^ and affected the spectrophotometric determination of fluoride. Separations were attempted to eliminate such interferences before spectrophotometric determination. As the determination of fluoride using the

FISE method was found not affected by these interferences, the efficiencies of the separation methods were monitored using the FISE method.

Two separation methodologies were tested: (1) the use of anion exchange resins and (2) the addition of cation to remove interferences through precipitation followed by the use of cation exchange resin.

When the sample solution was passed through a column of an anion exchange resins, the cations in the sample solution were removed because they were not adsorbed by the resin in the column and were eluted away. The anions in the sample solution were adsorbed by the column. Fluoride ion was the first anion eluted from the adsorbed column as reported from the literature.75 Further, it was also possible to concentrate fluoride in this step.

The second separation method removed the anionic interferences by precipitation and filtration after the addition of precipitating solution. The cations in the filtrate, including the interfering cations originally present in the sample solution and the cation added to eliminate the anionic interferences, were then removed by cation exchange resin. The resin was removed by filtration and the amount of fluoride ion in the filtrate was then determined.

Assessment of the effect of matrix on the measurements as well as the effectiveness of the two separation methods were described below.

101 (i). Effect of matrix on the measurements

As sodium hydroxide was used as an additive in ashing and an eluent in the anion exchange, and hydrochloric acid was used for the pH adjustment, tests were carried out to see the effect of the different matrices, containing different concentrations of the mixture of sodium hydroxide and hydrochloric acid, on the determination of fluoride using the FISE and spectrophotometric methods.

The absorbances of the spectrophotometric system were lower for fluoride solutions containing the mixture of sodium hydroxide and hydrochloric acid compared with those without this mixture for the same concentration of fluoride. The higher the concentration of the mixture, the greater the decrease in the absorbance, but the slopes of the calibration graphs (with absorbance versus concentration of fluoride) were similar, i.e., there were shifts in the calibration graphs. In order to outweigh the matrix effect on the spectrophotometric reading, solution A (p. 91) was added to the calibration standards so that there would be a similar concentration of sodium hydroxide and hydrochloric acid in the fluoride solutions compared with the sample solution.

For the potentiometric measurement, the potential measured was increased

with an increase in the concentrations of the mixture of hydroxide and chloride used.

However, the potential and the amount of fluoride found were not affected to a great

extent. Using the fluoride solutions without the mixture of hydroxide and chloride as

a reference, a relative error was less than -5 % for measuring the same concentration

of fluoride with 0.10, 0.15 and 0.20 M of the mixture, but was larger than -5 %

with 0.25 M, and was higher with higher concentrations of mixture although TISAB

was used in the experiment to provide a high and constant ionic strength of the

solution. In order to achieve a good accuracy, a similar amount of solution A (p. 91)

with respect to that used was added during the preparation of the diluted fluoride

solutions to correct for the effect due to the difference in the matrix.

102 (ii). The use of anion exchange resin to reduce interferences in the ashed sample solutions for spectrophotometric measurements

In the experiments below, the ashed sample solution was prepared by ashing

0.5 g of chicken feed sample with 1.25 g of sodium hydroxide and 0.05 g of silica at

550°C for 3 hours. 100 ml of the ashed sample solution were used to pass through the anion exchange resin and the amount of fluoride in the eluate under different experimental conditions was determined. From the literature^^, flow rates within 0.2 to 2.0 ml/min were suggested in column separation. A flow rate of 1.5 ml/min was used in all the experiments.

Experiments were carried out for finding the best conditions for the separation of fluoride as described below:

1 • A column with a certain amount (length) of resin was packed and the sample solution was transferred to the packed column using either a funnel inserted at the top of the column or a Pasteur capillary pipette with the help of deionized water (to complete the passage of sample through the column) according to the procedure in section 2B above (p. 89).

2. The eluate was collected during the passage of sample solution through the column and the amount of fluoride in the eluate was then determined after pH adjustment with hydrochloric acid using phenolphthalein as the indicator (pH range:

8.3, colourless, to 10.0, pink}76 by the FISE method. This was to see whether fluoride ion in the sample solution was completely adsorbed or not by the resin in the column. If fluoride ion was detected in this solution, a column with a longer resin length should be packed until no fluoride was found in the eluate. The resin should be regenerated as in section 2B above before starting another trial.

3. After all fluoride ion in the sample solution was found to be adsorbed, sodium hydroxide solution was added to the column to elute fluoride ion from the column.

103 The eluate was then collected in 25-ml volumetric flasks and a total volume of 200 ml was collected.

4. The amount of fluoride ion in each portion of the solution collected was found using spectrophotometric and FISE methods after pH adjustment to 8 with concentrated hydrochloric acid using phenolphthalein as the indicator. The results

(the amount of fluoride in each portion and the total amount) obtained from both

methods were then compared.

5. Then, different concentrations of eluent, sodium hydroxide, were used to find out the proper condition for the separation of interferences from the sample matrix.

In order to out find the suitable volume of eluate, separate portions of

solution were collected rather than the collection of solution in one portion.

Tests had also been carried out to compare the spectrophotometric results

with and without the addition of phenolphthalein. The results showed no difference

among each other.

In the preliminary test, anion exchange resins, Amberlite IRA-400, in the

chloride and the hydroxide forms were tested. From the results, the anion exchange

resin in the hydroxide form did adsorb fluoride ion in the sample solution. However,

the chloride form did not adsorbed fluoride ion even a longer column (two times the

column length) relative to that used in hydroxide form for the complete adsorption of

fluoride in the sample solutions under other conditions being the same. Therefore,

the anion exchange resin in hydroxide form was used for the reduction of

interferences.

Preliminary tests had been carried out on the suitability of columns with

different diameters (1.00, 2.00 and 2.50 cm) for the separation of fluoride from the

interfering substances in the sample. It was found that with the use of 0.5 M sodium

hydroxide solution as the eluent, the minimum amount of resin to adsorb fluoride in

the sample solution was different when the diameters of the column used were

104 different. From the results of the tests as shown in Table 11-2, the column with 1.00 cm diameter was the most suitable one among the three owing to the smaller volume of solution to elute fluoride for columns with smaller diameters. Also, a 50-ml burette was readily available in most of the laboratories. In the experiments followed, the 50- ml burette was used.

Table H-2 Effect of the diameters of the column on the adsorption and elution of fluoride from the ashed sample solutions with fluoride determined using the FISE method

Successive 25-ml portion of Concentration of eluted fluoride (ppm)

solution collected determined by the FISE method

1 g^ q^ 0.01

2 0.06

3 gjo 0.53 • • 0.40

4 g^ 0.45 5 qm 0J2 0.25 6 g^ q^ 0.05 7 qm 0.01 8 0.01 0.01 0.01

Amount of fluoride found _* ^ ^ 30

Cone, of sodium hydroxide (M) ^ ^ 0.5

Diameter of column (cm) 2.5

Length of resin in the column (cm) 41 22 15

*This was the total amount of fluoride in eight portions of solution, excluding those portions of solution with 0.01 ppm of fluoride.

From Table 11-2, small amounts of fluoride (0.01 ppm) were found in some portions of the solutions collected, indicating that trace amount of fluoride was found in the eluate collected.

105 The effect of the amount of resin used on the elution of fluoride and the

separation of interferences in the ashed sample solution was investigated with fluoride determined using the FISE and spectrophotometric methods, which were

shown in Tables 11-3 and 11-4. The concentration of sodium hydroxide used was 0.15

M. "Trace" amounts of fluoride were detected in the some portions of solution by

the spectrophotometric method, however, the amount of fluoride detected was

probably below the detection limit of the spectrophotometric method.

With a longer column, the elution of fluoride ion was delayed for the same

concentration of eluent as shown in Table II-3. The total amounts of fluoride found

using the FISE method were the same when columns with different lengths were

used.

From Table I卜4, the total amounts of fluoride ion found using the

spectrophotometric method were smaller using longer resin lengths compared with

those using shorter resin lengths. For the 50-cm and 53-cm resin lengths, the results

obtained with the spectrophotometric method were closer to those obtained using

the FISE method although the total amount found from spectrophotometric method

using 50-cm resin length was still higher than that of the FISE method. The results

obtained from the spectrophotometric method using the other three shorter resin

lengths were much higher than those obtained from the FISE method, which

indicated that interfering substances were co-eluted.

Further comparisons could also be made to each corresponding portion of the

solution collected by considering the amount of fluoride found using the FISE and

spectrophotometric methods in Tables 11-3 and 11-4. It was noted that for the 50-cm

resin length, no fluoride ion was found by the FISE method in the 8th portion

whereas the spectrophotometric reading indicated the presence of high concentration

of fluoride (or interfering substances) in this portion. These results indicated that the

interfering substances for the spectrophotometric method were eluted in this later

106 portion of the solution. This might probably reveal the possibility to separate fluoride from the interfering substances completely for the spectrophotometric method under this condition.

From the results above, the conditions were chosen such that the length of

resin in 1-cm i. d. column was 50 cm long and sodium hydroxide used for elution

was 0.15 M. Under such conditions, fluoride was found in the fourth and fifth

portions and these two portions were collected for subsequent determinations.

Table 11-3 Effect of the amount (length) of the resin used in the separation of the

interfering substances in the ashed sample solutions with 0.15 M sodium hydroxide

as the eluent with fluoride determined using the FISE method

Successive 25-ml Concentration of eluted fluoride (ppm) portion of solution determined by the FISE method collected

1 0.01 0.01 0.01 0.01 0.01

2 0.99 0.06 0.01 0.01 0.01

3 0.15 0.96 0.28 0.01 0.01

4 0.01 0.14 0.87 0.30 0.01

5 0.01 0.01 0.01 0.88 0.17

： 6 0.01 0.01 0.01 0.01 0.64

7 0.01 0.01 0.01 0.01 0.35 8 0.01 0.01 0.01 0.01 0.01

Amount of fluoride ([i 29 29 29 30 29 91： Length of resin in the 41 44 47 50 53 column (cm)

*This was the total amount of fluoride in eight portions of solution, excluding those portions of solution with 0.01 ppm of fluoride.

107 Table li-4 Effect of the amount (length) of the resin used in the separation of the interfering substances in the ashed sample solutions with 0.15 M sodium hydroxide as the eluent with fluoride determined using the spectrophotometric method

Successive 25-ml portion Concentration of eluted fluoride (ppm)

of solution collected determined by the spectrophotometric method

1 trace trace trace trace trace

2 1.85 0.04 trace trace trace

3 1.25 1.15 0.29 trace trace

4 0.60 1.63 0.89 0.27 trace 5 0.29 1.52 1.70 0.89. 0.17 6 trace 0.12 1.12 trace 0.67

7 trace trace 0.60 trace 0.36

8 trace trace trace 0.80 trace

Amount of fluoride (|ig)* 99 112 115 30

Length of resin in the 41 44 47 50 53

column (cm) ,

N.B. "Trace" referred to the determination smaller than the blank.^^

The effect of the concentrations (0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.40 and 0.50 M) of sodium hydroxide on the elution of fluoride from the same column

(50-cm resin length) was then carried out with fluoride determined using FISE method. The results were shown in Table 11-5. It could be seen that fluoride appeared in earlier portions for higher sodium hydroxide concentrations. Also, the eluents being tested could concentrate fluoride ion within 50 ml except for 0.10 M NaOH, where a relatively larger volume {> 50 ml) was required for the collection of fluoride and 0.05 M NaOH did not elute fluoride within 200 ml and their uses were excluded.

108 Table 11-5 Effect of the concentrations of sodium hydroxide on the elution of fluoride from a column with a resin length of 50 cm with fluoride determined using the FISE method

Successive 25-ml Concentration of eluted fluoride (ppm) portion of solution determined by the FISE method collected 1 g^ 0.01 0.01 0.01 2 g^ 0.01 0.01 0.45

3 g^ 0.14 0.75

4 0.08 0.30 0.99 “ 0.05

5 q^ 0.88 0.02 0.01

6 qj2 q^ 0.01

Z 0.01 0.01 0.01 8 0.01 0.01 0.01 0.01

Amount of fluoride {|i 32 30 29 31

911 ：

Cone, of sodium 0.10 0.15 0.20 0.25 hydroxide (M)

*This was the total amount of fluoride in eight portions of solution, excluding those portions of solution with 0.01 ppm of fluoride.

Table 11-6 showed the suitable length (amount) of resin corresponding to some eluent concentrations required for the separation of fluoride from the interferences using the spectrophotometric method.

109 Table 11-6 Suitable lengths of resin required for the separation of fluoride with the use of different concentrations of sodium hydroxide solution

Concentration of sodium Suitable length of

hydroxide (M) resin (cm)

guo ^ gj5 ^

g^ 54

^

Based on the volume of solution needed to collect fluoride ion, the amount of

resins and the reagents used, 0.15 M sodium hydroxide solution with a resin length

of 50 cm in a 50-ml burette (1.00 cm diameter) was used as the proper condition for the separation of fluoride from the interfering substances in the sample solution and

elution of fluoride. For the collection of eluate for the analysis, about 50 ml of eluate,

which were the 4th and 5th of the 25-ml portions of solution, were collected after

sample solution was eluted through the column and the first 75 ml of eluate on

passing the eluent were discarded as the collection of the earlier portions would

result in dilution of fluoride collected.

(iii). The addition of cation to remove anionic interfering substances and the

use of cation exchange resin to separate the catioic interfering substances in

the ashed sample solutions for spectrophotometric measurements

Ashed sample solutions obtained from ashing the chicken feed sample with

sodium hydroxide and silica were used in the experiments.

The cations employed for the test were based on the values of the solubility

product of their phosphates and sulphates, which were the interfering ions in the

spectrophotometric method. Phosphate and sulphate compounds with low solubility

110 products^^' 77-78 were chosen in the test. The cations selected were barium, cadmium, copper (II), lead, nickel, strontium and zinc. All these cations had low solubility products for their phosphates while barium also had low solubility product for its sulphate. Chloride salts of the cations were chosen for the test because the spectrophotometric method had a very high tolerance level of chloride. Preliminary test showed that a 0.4 ppm fluoride solution containing 8m M barium, cadmium, lead, strontium or zinc solutions did not affect the absorbance of the solution measured. When the coloured nickel or copper solution was used as the precipitating agent, it was later removed with Amberlite IR-120 (H + ) cation exchange resins having 20-50 mesh size after filtration.

It was found that addition of small amount of precipitating agent did not improve the spectrophotometric determination of fluoride. When the concentration of the precipitating solution was increased, the amount of fluoride found in the sample

solution using the spectrophotometric method was closer compared to that obtained

using the FISE method. However, the amount of fluoride found using

spectrophotometric method was still 25-50 % higher than that using the FISE

method, although recoveries ranging from 90-120 % were obtained using

spectrophotometric determination. Also, the amount and recovery of fluoride found

using spectrophotometric method fluctuated greatly.

In addition to the use of Amberlite IR-120 (H + } (E. Merck). Dowex 50-X8

(Na + ) (BDH Chemical Ltd.) had also been used to test if there was any effect of the

use of different resins. Similar results were obtained with these two resins.

As a summary, the results were not good and satisfactory with the use of

precipitating solution to remove anionic interferences. The use of zinc and cadmium

as the precipitating solutions seemed to get the best results among the cations

tested, and the results obtained for these two cations were closer to the results from

the FISE method. The closer fluoride results between the use of precipitating solution

111 for the spectrophotometric determination and the FISE method also gave information that phosphate was probably one of major interferences for the spectrophotometric method.

(C). Construction of the calibration graph for the spectrophotometric determination of fluoride

The calibration graph for Zr^^-SPADNS system with absorbance at 570 nm plotted against concentration of F" (ppm) was shown in Figure 11-3 and the corresponding data in Table I卜7.

0.4 —

0.3 \-

1 考 0.2 -

‘ 0.1 - \

0 ' 1 0 0.5 1 1.5 Concentration of fluoride (ppm)

Figure 11-3 Calibration graph of fluoride with absorbance at 570 nm plotted against the concentration of fluoride (ppm)

112 Table 11-7 Data for the calibration graph for the spectrophotometric determination of fluoride

Concentration Absorbance of F" (ppm) at 570 nm

0 0.352 Correlation coefficient -0.999

0.05 0.343 Slope (ppm'” -0.175

0.10 0.331 y-intercept 0.348

0.20 0.313

0.30 0.295

0.40 0.276

0.60 0.239

0.80 0.203

1.00 0.172

1.20 0.139

1.40 0.109

(D) Precision of the spectrophotometric method

According to AOAC^ the precision is expressed as the relative standard

deviation, which is the standard deviation in absorbance from ten replicate

measurements of the absorbance of the fluoride solutions divided by the mean value

of the absorbance (multiplied by 100% for the result expressed in percentage). The

precisions of the Zr4 +-SPADNS spectrophotometric method in measuring 0.10, 0.60

and 1.20 ppm of fluoride solutions were found to be 0.3, 0.3 and 0.2 %,

respectively.

113 (E). Spectrophotometric determination of fluoride in animal feeds

(i). Effectiveness of the anion-exchanqe step

The ashed sample solution of each feed sample was separated in two portions, with one spiked with known amount of fluoride ion and the other unspiked.

The recovery of the anion exchange step was found by determining the fluoride ion concentration in ashed sample solutions and the corresponding solutions with known fluoride addition. The amounts of fluoride in these solutions were within the concentration of the calibration graph. The percentage recovery could be found by the following equation^ ^；

(Measured concentration in fortified material - Measured concentration in unfortified material )、， % Recovery = ： ： ： X100 % Knwon increment in concentration

The amount added should be a substantial fraction of, or more than, the amount present in the unfortified material. Obviously, the nearer the value of percentage recovery to 100, the better would be the method. Four animal feed samples shown in Table I卜8 were tested and the results were shown in Table 11-9.

Table 11-8 Animal feed types and their sources

Sample no. Sample type Source

a rat feed Biochemistry department

b chicken feed local bird shop

c rabbit feed local bird shop

d bird feed local bird shop

114 The experimental conditions for the anion exchange were as follows:

Column used: 50-ml burette; Column length: 50 cm;

Flow rate: 1.5 ml/min; Eluent and its concentration: 0.15 M sodium hydroxide

From the results in Table H-9, it could be seen that good recoveries of

fluoride were found using anion exchange resin to separate fluoride from the

interfering substances in the sample solutions.

Table H-9 Recoveries of fluoride in the ashed sample solutions determined bv the

spectrophotometric method after the anion exchange clean-up

Sample Fluoride (fig) Recovery

门 0. Added Foimd (%)

a — 1.43 —

97.0

b --- 12.1 ---

24^ 99.2

c — 11.5 —

12.0 23.6 101

d — 2.40 — ‘

3.00 5.38 I 99.3

(ii). Determination of fluoride in animal feeds

The amounts of fluoride in the sample solutions were determined using the

calibration graph constructed and the absorbances of the sample solutions measured.

The determination of fluoride in each feed sample was done in triplicate. The results

were summarized in Table 11-10. The recoveries of the total analytical procedure and

the precision in measuring the samples were also shown in Table 11-10. The amounts

of fluoride in the animal feed samples were shown in Table 11-11.

115 Table 11-10 Analysis of animal feed samples by the proposed method and results for the recoveries of added fluoride

Sample Fluoride (^ig) Standard Relative standard Average

no. Added Found* deviation deviation (%) Recovery (%)

a — 1.48 0.06 4 —

2.00 3.32 0.08 2 92.0

b — 11.9 0.3 3 — 12.0 23.2 0.8 3 94.2 c — 11.4 0.3 3 ---

12.0 22.6 0.7 3 93.3

d — 2.36 0.06 3 — 3.00 5.15 0.10 2 93.0 •Average of three replicate analyses.

Table 11-11 Determination of fluoride content in animal feed samples using the proposed method

Sample no. Fluoride content ()ig/g)

a

b ^

c

d U^

The reason for the recovery of fluoride less than 100 % might be caused by the equilibria below.79

SiFe^- SiF4 + 2 F

SiF4 + 2 H2O 4 HF + Si02

The dissociation of fluorosilicate ion to fluoride was reported to be at least 95 % at the concentration for the fluoridation of portable water supplies (1 ppm fluoride). The

116 higher the fluoride concentration, the lower would be the percentage dissociation.

However, it was also reported that the degree of dissociation was affected by the

ionic population of the water and 100 % dissociation in tap water was reported for fluoride concentration up to 30 ppm. Although the concentration of fluoride

determined in the sample solution was about 1 ppm, the matrix in the sample

solution might affect the equilibria which resulted in the incomplete dissociation to

fluoride.

(F). Potentiometric determination of fluoride in animal feeds by the

FISE method as the counter-check method

A number of buffers including TISAB had been reported. They were

incorporated with various complexing agents to free fluoride from the interferences.

Table 11-12 showed the composition and preparation of various buffers?^' 80-82

According to the manual of the fluoride ion selective electrode, the

potentiometric method using a FISE could measure the potential of fluoride ion in a

solution within a linear range as low as 0.02 ppm.8Q |t had also been reported that

calibration graph in TISAB was linear up to 100 |igdm'^ of fluoride.^^

In the experiment, preliminary studies for seven buffers (Nos. 1 to 7)

reported were performed to find out the suitability for the determination of fluoride

ion in a solution by considering the linearity (correlation coefficient, r) of the

calibration graph of potential versus the log value of the concentration of fluoride (log

Cp). Fluoride solutions for the calibration were prepared as shown in section 2E

above (p. 93). Equal volumes of buffer and fluoride solution were used with the

equilibrium (immersion) time of electrode in the mixture of 4 minutes.

117 Table 11-12 Composition and preparation of buffers for the FISE method

Buffer Composition and Preparation

1 58 ml of glacial acetic acid (E. Merck) and 12 g of sodium citrate dihydrate (Beijinq Chemical Works) were added and dissolved in 300 ml of deionized water. The solution was adjusted to a pH of 5.0 to 5.5 with 25 % sodium hydroxide

(BDH Chemical Ltd.) and diluted to 1 litre with deionized water(TISAB) 2 58.8 g of sodium citrate dihydrate (Beiiina Chemical Works) and 20.2 g of potassium nitrate (BDH Chemical Ltri.、were added and dissolved in 800 ml of deionized water. The solution was adjusted to a pH of 5.0 to 5.5 with (1+1}

hydrochloric acid and diluted to 1 litre with deionized water.72 3 57 ml of glacial acetic acid (E. Merck) and 58 g of sodium chloride (Beijing

Chemical Works) were added and dissolved in 500 ml of deionized water. The

solution was adjusted to a pH of 5.0 to 5.5 with 5 M sodium hydroxide (BDH

Chemical Ltd.) and diluted to 1 litre with deionized water.80 “

4 42 ml of 37 % hydrochloric acid (Beijing Chemical Works). 121 g of

TRIS(hydroxymethyl)aminomethane (E. Merck) and 115 g of sodium tartrate

dihydrate (Beiiina Chemical Works) were added and dissolved in 250 ml of

deionized water. The solution was made up to 500 ml with deionized water.80, 81 (TISAB IV)

5 57 mi of glacial acetic acid, 58 g of sodium chloride and 0.30 g of sodium citrate

dihydrate were added and dissolved in 500 ml of deionized water. The solution

was adjusted to a pH of 5.0 to 5.5 with 5 M sodium hydroxide and diluted to 1

litre with deionized water.(TISAB I) 6 30 g of citric acid monohydrate (BDH Chemical Ltri.) 105 g of sodium citrate

dihydrate (Beijing Chemical Works), 26.75 g of ammonium chloride (Beiiina

Chemical Works) and 33.5 ml of ammonium hydroxide (E. Merck) were added and

dissolved in 250 ml of deionized water. The solution was made up to 500 ml with

deionized water.^^ 7 1 g of ethylenediaminetetra-acetic acid disodium salt (BDH Chemical Ltd.). 57 ml

of glacial acetic acid (E. Merck) and 58 g of sodium chloride (Beijing Chemical

WedSi) were added and dissolved in deionized water. The solution was adjusted

to a pH of 5.0 to 5.5 with 50 % sodium hydroxide (BDH Chemical Ltd.) and

diluted to 1 litre with deionized water.82

118 Three buffers (nos. 1, 3 and 5) were found to have good r (r>-0.999). For buffers no. 2 and 7, the calibration graph deviated from linearity at low fluoride concentration (r was -0.997 but r>-0.999 without the inclusion of 0.05 ppm fluoride). The other two buffers had r values of -0.990 and hence were not suitable.

As the electrode also responded to hydroxide ion (due to the similarity in its ionic radius and charge to fluoride)84, buffers no. 4 and 6 were not suitable for the measurement of low concentrations of fluoride as they were alkaline. Deviation from linearity in measuring low fluoride concentrations using buffers no. 2 and 7 might be due to the contamination of the reagents used.27' 83 Although the time to establish equilibrium for the electrode at low fluoride concentrations was longer than those for higher fluoride concentrations, no improvement in r value was found by increasing the stirring time from 4 to 8 minutes before the potentiometric measurement.

Among the three buffers (nos. 1, 3 and 5), buffer 1 was preferred because it contained citrate which could form complexes with the interfering cations such as aluminium and iron, and more citrate were found in buffer 1 than in buffer 5. Buffer

1 was chosen in the determination.

The precisions of the FISE method from ten replicate measurements of the potentials of 0.1 and 1.0 ppm of fluoride solutions were found to be 1 %. The results obtained using the FISE method were shown in Table 11-13 where the results using the proposed spectrophotometric method were also included for comparison.

There was close agreement obtained using the two methods.

119 Table 11-13 Amount of fluoride in animal feed samples found using the FISE method

with the inclusion of the results obtained from the proposed spectrophotometric

method

Sample Fluoride content (|ig/g)

no. FISE method proposed method

a

b 57^

c 57J

d LLS

For the measurement of a large number of samples, the calibration graph

should be re-constructed due to the shift in the potential with time.

4. Conclusions

An ashing procedure for the determination of fluoride content in animal feed

samples has been developed. Fluoride can be determined after ashing using

spectrophotometry. However, a clean-up by anion exchange was found necessary to

reduce the effect of interfering substances. It took about 120 minutes for the anion

exchange separation. Nevertheless, the reaction between fluoride and the reagents

was immediate.

Alternatively, the fluoride ion can be determined after ashing using

potentiometric measurement with a fluoride ion selective electrode (FISE). Although a

total ionic strength adjustment buffer (TISAB) is sufficient to reduce the interferences

in the sample solution and hence no separation step is necessary, yet the time

required to establish equilibrium for each measurement was 4 minutes. The time

required for measurements of a sample (in triplicate) plus the standard solutions for

the calibration graph was around 70 minutes.

120 In conclusion, the potentiometric method for the determination of fluoride is more convenient, however, it requires a good potentiometer and a fluoride ion selective electrode. For poorly equipped laboratory, the spectrophotometric method is preferred because spectrophotometers are readily available in most laboratories.

Although it takes around 120 minutes for the ion exchange separation, the total time required for the spectrophotometric determination for a sample plus the standard solutions for the calibration graph is within 10 minutes.

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126

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