Comparison of Three Separation Tecnniques for Arsenic (III) and Arsenic (V) in Sea Water

Separation and determination of arsenic species in sea water is an attractive area of current research primarily due to the effects the different oxidation states of the element have on its bioavailability and toxicity. Many separation procedures for the arsenic species in sea water prior to their determination by graphite furnace -, hydride generation atomic absorption spectrophotometry or neutron activation technique have been reported. Evaluation of three of these separation procedures based on (1) solvent extraction, (2) ion-exchange, and (3) thiol cotton is reported in this dissertation. The evaluation is based on the analytical parameters: Detection limits, Sensitivity, Reproducibility, Precision, Recovery, Accuracy, Cost and Time of analysis. The separation procedure based on solvent extraction was found to be superior to the other two procedures for routine analysis of sea water samples.

ACKNOWLEDGEMENTS I am grateful to Professor James L. Fasching, my dissertation advisor, for his direction and encouragement. I wish to thank W.E. Johnson, Andrew Kocsi and C.L. Strate for their technical assistance. Finally, many thanks go to Cheryl Blanck who typed this manuscript.
The large number of publications on the various techniques for separating and determining As(lll) and As (V) makes it important to evaluate these techniques before one makes a choice for his or her work.
In writing this dissertation on the comparison of arsenic speciation techniques, the standard form was adopted.
The dissertation consists of four chapters and each chapter has subsections. There are two appendices and these are:

1.
Equations and formulae utilized in the dissertation and blank analysis.

2.
Characteristics of the sea water reference material (SRM) and the sea water from the Narragansett Bay.
iv   The choice of techniques for . the separation and 1. determination of arsenic(lll) and arsenic(V) in sea water is rather limited due to the complex nature of the matrix and the low levels of arsenic species in this matrix (~0.06 -2 ppb) see Table 1. 7 The techniques are reviewed below and   summarized in Tables II, III and IV    showed that there is a differential absorption of the elements depending upon their oxidation states on the thiol cotton.
Recently, Yu and Liu 82 have reported the determination of the various oxidation states of arsenic, antimony, selenium and tellurium by hydride generation after separation with thiol cotton. The method has excellent detection limits with good sensitivity. The method was, therefore, adopted for the comparative work reported in this dissertation.

1.2
Arsenic Speciation: Speciation of an element as has been defined by Florence 30 13. is the determination of the individual physico-chemical forms of that element which together make up its total concentration in a sample. This definition implies that in order to completely speciate an element in, for example, sea water sample, the various chemical forms in all sea water phases (liquid, colloidal and particulate) will have to be characterized.    is (3) therefore, Arsenious acid may also be formulated as H 3 As(OH) 6 or As(OH) 3 (oH 2 ) 3 as has been reported by Voronova et. a1. 46 The dissociation equilibria of this form is depicted below, and The dissociation constants for these equilibria can be evaluated using the arguments discussed above. These 18. equilibria however do not suggest the formation of arsenious ion As 3 + in 'slightly' acidic solution.
Arsenic acid, H~Aso 4 , unlike ars~nious acid As(OH) 3 is almost exclusively acid in its behaviour. The dissociation equilibria of arsenic acid is shown below, H  These equilibria indicate that arsenic acid is essentially some ionic form at analytical pH range of 4-8.
Organic Compounds of Arsenic.
The occurrence of two organic acids of arsenic in sea water has been reported by M.O. Andrea 9 • These acids are in methyl arsenic acid and dimethyl arsinic acid. These acids are formed by microbial transformation of the inorganic 19. acids. Many reactions which produce organic arsenic compounds 25 from inorganic arsenic compounds have been reported. The conversion of inorganic to methylated arsenic compounds by microorganisms is well established. The mechanism as has been proposed by Challenger 20 Recently, trimethylarsoniumlactate and its derivatives and arsenobetaine have been found to be present in some sea t . 14 wa er organisms.
Two o.ther organo-arsenic species, 0-phosphatidyltrimethylarsoniolactic acid and arsenic-containing sugar have also been isolated from the sea water organisms, Chaetoceros concavicornis and Ecklonia zadiata, respectively. 26115 The structures of these compounds are shown in Figure 2.
The arsenic cycle showing the transformations of arsenic compounds in sea water environment is shown in Figure 3.
Organo-arsenic compounds occur in very low concentration The methods which are commonly used to speciate these organo-arsenic compounds is based on reduction with sodium borohydride followed by separation and detection of the arsines produced. Table VII   The ratio of arsenic(lll) to arsenic(V) also depends on 3-+ -~ the redox potential of the system, Aso 4 + mH + ne ~ H2AS03 + H20 RT a(As 3 +) where E 0 = -0.67 volts (As(V)/As(lll)) base. m = # of moles of hydrogen ions n = # of electrons. a(As 3 +) and a(As 5 +)are the activities of As 3 + and A 5+ s • The equation above indicates that As 3 + to Ass+ ratio is a pH dependent. If concentration constants are used instead of activity constants, then the ratio will vary as the ionic strength. Since salinity is a measure of ionic strength, changes in salinity will affect this ratio. The effect of salinity on As 3 + to Ass+ ratio in sea water has however not been reported~

1.4
Toxicity Of Arsenic Compounds.
Interest in the study of arsenic partly stems from its toxicity. The toxicity of arsenic compounds may be defined by one of the following modes of action as has been reported by The use of dithiophosphates in the study of arsenic speciation has seldom been reported. The determination of arsenic(lll) using ammonium-sec-butyl dithiophosphate 19 (ASBD) and diethyldithiophosphoric acid (HDEDTP) 54 has been 28. reported. The structures of these compounds are shown below: The dithiocarbamates form with arsenic(lll) analytically useful complexes of the general type as has been elucidated by et a1. 21 The formation of these complexes which are pH-dependent can be represented by the equation.  Table VIII. Divinylbenzene is used as the crosslinking agent. The stoichiometry for the exchange The acetate form of the ion-exchange was used for the 31. separation of arsenic(lll) from arsenic(V). Arsenic(V) is retained whereas arsenic(lll) passes through without retention in a solution of pH range 2-6.
A possible reason for the retention of As(V) but not As(lll) is probably due to the fact that in the pH range of 2-6 arsenic acid is in some ionic form whereas arsenious acid is not. This can be seen from the solution equilibria of the two acids shown below and.which has been discussed earlier on in this thesis.
The gaseous arsine produced is swept into a pre-heated quartz furnace at a temperature of approximately 900°c by a carrier gas, usually argon or helium. Sometimes nitrogen or hydrogen gas is used.
In the furnace, AsH 3 is decomposed into arsenic and hydrogen atoms. Atomization followed by absorption of arsenic resonance radiation makes it possible to measure the amount of arsenic present in the sample. Two mechanistic theories have been put forward to explain the process that goes on in the quartz furnace. The first theory, proposed by Akman et al. 3 , is that the formed AsH 3 is decomposed on the quartz surface before the atomization temperature is reached and the metallic arsenic is vaporized as As 4 , which is then decomposed to As 2 dimers and atomized . by gas-phase dissociation. This is shown schematically below AsH 3 (g) ~As (s)~As 4 (g) ~ As 2 (g) ~As (g) The other theory has been proposed by Welz  The possibility for the formation of the dimer is according to the equilibria: Under mild acidic conditions, arsenic(V) is not reduced by borohydride solution. This is probably due to the fact that arsenic(V) is exclusively in the arsenate form. The arsenate ion might, however, by very slightly ionized into Ass+ ions in very strong acidic conditions and a reduction to arsine by borohydride solution might then take place · according to the following equilibria: H3AS04 + 5 H+ # Ass+ + H20 Ass+ + 2H-~ As 3 + 2H+ The carrier gas used was argon and the flow rate was regulated by a flowmeter. Figure 6 shows the assembly of the system.  An®onium Acetate Buffer pH 6.0. 470ml ammonium hydroxide were added to 430ml glacial acetic acid. The pH was then adjusted to pH 6.0 with ammonium hydroxide or acetic acid.
Potassium iodide was used as purchased.
One gram APDC was dissolved in lOOml demineralized water. To purify the solution, it was extracted with chloroform. The purified solution was prepared as needed.
The organic solvent, chloroform, was used as purchased.
Each stock standard solution was diluted to give an appropriate concentration before use.
Perchloric acid (70%) and sulphuric acid 18M were used as purchased. Nitric acid was redistilled in a pyrex distilling kit.
Potassium iodide (Fisher) was used in the solid form.
Whatman # 4 paper, which was pretreated with 1 molar nitric acid and sufficient deionized water to render it acid-free, was used in the dry ashing. water. This was filtered through an 0.45 m filter membrane followed by extraction with chloroform.
pyrrolidine was used as purchased from Fisher.
Two molar HCl was prepared from the concentration solution.
Solvent Extraction: All glassware was cleaned by washing several times in 4 molar nitric acid and then rinsing in demineralized water until it was neutral to litmus paper.
Synthetic sea water or natural water was spiked with an appropriate concentration of arsenic(lll) and arsenic(V) solution. This procedure was used in the various studies.
Natural sea water was collected from Narragansett Bay.
It was filtered through a 0.45~m filter membrane to remove any particulate matter. Analysis of the filter membrane showed no retention of arsenic. The sea water was stored at a pH of 2 in a polyethylene bottle. The pH of 2 was attained by using hydrochloric acid. It was assumed that the As(lll) I As(V) ratio did not change at that pH value. Total arsenic was determined by reducing arsenic(V) to arsenic(lll) in one batch of sea water using potassium iodide. The sea water was acidi fie d to acidic pH using hydrochloric acid before potassium iodide was added. To each liter sea water sample 2g 42. potassium iodide was added. The added potassium iodide did not interfere with the extraction procedure. This is probably due to the chemistry of the reduction reaction which can be In order to determine the optimal pH for the complexation reaction between the APDC and arsenic(lll), and the minimal amount of APDC for optimal results separate experiments were carried out using standard solutions. The experimental method followed is the same 1 as that used for the speciation study.
For the pH study As(lll) concentration was fixed at lppb. The experiment was repeated at different pH values (range of 3.5 -6.0) in the APDC preconcentration step.  (1) and ( 2) shows that an increase in the concentration of dithiocarbamate, as well as a decrease in hydrogen ions, will shift the equilibrium to the right.
Consequently, the second equilibrium will also be shifted to the right and more of the arsenic (111·) species will be extracted. While a decrease in H+ ion concentration favors more extraction and stability of the dithiocarbamate, an increase in H+ concentration favors the availability of As(lll) ions which are required for complexation with APDC.
The pH dependence of As(lll) availability can be illustrated by th f 11 . · 1 . b . 63 e o owing equi 1 ria As(OH)3 ~ As03 3 -+ 3H+ As(OH)3 ~ As 3 + + 30Hand according to mass action law  3-clear ly indicates that the ratio of As(lll) ions to Aso 3 47. ions is dependent on the hydrogen ion concentration of the solution; therefore, As(lll) is available for complexation with APDC in acidic solution as has been discussed earlier on in this thesis.
The dependence of the stability of APDC on pH is also illustrated by the following equation (14):

From equation (1) an increase in APDC concentration
should result in a shift of the equilibrium to the right.
consequently, more of the APDC-arsenic(lll) complex will be extracted. The optimum concentration of APDC necessary to bring about maximum extraction of the complex was studied.
The sea water matrix contains traces of metals such as Cu, Ni, Fe, Pb, Co, Zn, Cd, and Hg. It has been reported that APDC forms complexes with all these elements 58149 and that during the extraction and determination of arsenic, these elements are likely to be present in the sample solution.
Severe interference by Cu and Ni in the hydride-generation analysis of arsenic has also been reportea. 23 Studies were, therefore, carried out to investigate the effect of traces of Cu(ll) and Ni(ll) on the determination of arsenic using the hydride-generation technique. Studies were also conducted to Results And Discussion.

I. Trace Element Interference:
The results for the study are shown in Table IX. They indicate that Ni(ll) and Cu(ll) did not interfere below 5 parts per million levels. Since these elements occur in sea water below Sppm (1) one should not worry about their interferences when using a hydride generation technique to analyze for arsenic in sea water matrix. There were no   APDC or pyrrolidine to the arsenic(lll) solution before analysis as shown in Table ~     collected on a pretreated Whatman #4 filter paper. After drying it in air the paper was dry ashed in a Low Temperature Asher. At the end of the ashing, the ash residue was dissolved in 2 molar hydrochloric acid and the solution was made up to 25ml with hydrochloric acid in a volumetric flask.
Ten ml aliquots of this solution were analyzed for arsenic by atomic absorption-hydride generation technique.  donors and it is the donated oxygen atom which destroy the organic matter.
Because of the oxidizing property of these acids, arsenic(lll) is oxidized to arsenic(V) during the process of ashing. The following equation illustrates the oxidation of arsenic(lll) to arsenic(V) by nitric acid.
However, arsenic(V) is not reduced to arsine by borohydride solution. There is therefore the need to reduce arsenic(V) to arsenic(lll) before analysis by the hydride generation technique. The best system for the reduction was found to be potassium iodide among other reducing systems tried and this method was used in this experiment.

Separation By Ion-Exchange.
Experimental: The ion-exchange columns were made from 50ml burettes with a diameter of lOmrn. The burettes were cut to 30cm long.
A separatory funnel was used as a reservoir. see Figure 13.  Glass wool was treated with 4 molar nitric acid before use. Procedure: A small amount of the pretreated glass wool was inserted to the bottom of the burette column. This was necessary to prevent the ion-exchange or other particles from passing through the column. The ion-exchange was then slurry packed into the column to fill it to about lOcm in length.
Pretreated glass wool was inserted onto the surf ace of the ion-exchange. The column was washed several times with deionized demineralized water.
In order to convert the chloride form to the acetateform, 15ml of 1 molar sodium hydroxide was passed through the column. This was followed by 15ml of demineralized water.
Each addition was allowed to drain through the column at the Experimental.
The thiol cotton columns were of the same dimensions as that of the ion-exchange. A separatory funnel was used as a reservoir. Reagents: High quality cotton was used for the separation. Acetic anhydride, glacial acetic acid, sulphuric acid, methyl thioglycollate and dithioglycollic acid were used as purchased. Procedure: Preparation of Thiol Cotton.
A solution mixture of methyl thioglycolate (30ml), acetic anhydride (25ml), glacial acetic acid (15ml), 67. concentrated sulphuric acid (0.2ml) and water (Sml) was 14. Wait for a minimum of 15 seconds to allow the system to be purged free of air when the quartz cell has reached its optimal temperature for atomization •• For As the baseline falls as air is purged from the system; wait until the baseline is stable before actuating the plunger. Recovery experiments were also conducted to assess the accuracy of the methods. The results are shown in Tables XI I -xv. Synthetic sea water were spiked with known amounts of arsenic(lll) and arsenic(V). The recovered amounts were expressed as percentages.

Sensitivity And Detection Limits:
Sensitivities were calculated from the slopes of the calibration curves of each method. In practice, the values obtained correspond to the concentration of arsenic that will give an absorbance value of 0.0044.
-*Detection limits were calculated from the formula D.L. = X + 3 ~ where X is the average value of ten blanks determination, ais the standard deviation and 3 is a probability factor at the 99% confidence level.
Results for the sensitivity and detection limits studies are given in Tables XVI -XVIII. 72.

5.
As (111) added (         Accuracy of analytical methods is usually determined by two methods, (a) Reference method and (b) Recovery experiments. Both methods were applied to establish the accuracy of the three separation methods used in this study.
The results for the accuracy study is shown in Table   XII  The results for precision studies is given in Table XIX -XXI and the means and standard deviations for the three methods are tabulated in

Conclusions:
The solvent extraction method of separation was found to be superior to the other methods with respect to detection limits, accuracy, precision and reproducibility. The method was, however, inferior with respect to cost and time of analysis. The cost difference would, however, not overshadow the better analytical variabilities.

104.
Although all the three separation methods possess analytical features for the separation of As(lll) and As(V), solvent extraction method is recommended for routine use in the separation and determination of As(lll) and As(V). The  ,6LANK ANALYSIS.
To correct for non-specificity, blank analysis is required. To compensate for the reading given by substances other than the analyte, which are present in the sample, synthetic sea water was used as the blank in all the three separation methods studied.
The average value for ten blank analysis of each method was substracted from the analyte reading. Collection was with 12 litre GO FLO samplers (General Oceanics). These PVC samplers were internally coated with teflon. They were modified at the Bedford Institute of Oceanography, Dartmouth, N.S. in order to reduce trace metal contamination. The samplers were acid leached and rinsed with ultrapure water prior to deployment.
The sea water was acidified to pH 1.6 with high purity nitric acid solution immediately upon collection. It was transferred to 50-litre acid leached polypropylene carboys conditioned with ultrapure water acidified to pH 1.6.
Previous storage experiments indicated that the integrity of a sea water sample with respect to total trace metal contents could be maintained for at least two years using this procedure.
The The natural sea water sample used in this experiment was Narragansett Bay water. The level of arsenic concentration in this water as has been determined by Johnson et al. is l.65ppb by neutron activation method.
The water sample was collected from the Bay behind the Narragansett Marine Laboratory. The salinity as determined by Mohr titrimetric method is ia 0 1oo· Collection was done using I-litre acid leached polypropylene bottle. The water was immediately acidified to pH of 2 using redistilled hydrochloric acid. The sea water was filtered through 0.45Mm filter membrane to remove any