JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, OCTOBER 1987, VOL. 2 699

Flow Injection Ion-exchange Pre-concentration for the Determination of Aluminium by Atomic Absorption Spectrometry and Inductively Coupled Plasma Atomic Emission Spectrometry

M. R. Pereiro Garcia, M. E. Diaz Garcia and Alfredo Sanz Medel Analytical Chemistry Department, Faculty of Chemistry, University of Oviedo, Oviedu, Spain

A flow injection ion-exchange pre-concentration procedure, using a micro-column loaded with Am berlite IRA-400, is outlined for the simple and quantitative determination of trace amounts of aluminium in the ng 1-1

range by atomic absorption and/or inductively coupled plasma atomic emission spectrometry. The optimisation of the operating conditions and figures of merit are given and a possible retention mechanism for aluminium in the anion exchanger is discussed. Serum aluminium is so tightly bound by proteins that it is not retained by the resin in the flow procedure. The method can be used, however, for the rapid determination of free aluminium in waters and haemodialysis fluids. Keywords: Aluminium determination; pre-concentration; flow injection; atomic absorption spectrometry and inductively coupled plasma atomic emission spectrometry; biological materials, dialysis fluids and waters

A variety of deleterious physiological effects have been observed related to the presence of aluminium in patients with chronic renal failure; encephalopathy ) anaemia, osteomalacic osteodistrophy and cardiotoxicityl are disorders related to aluminium intoxication in haemodialysis patients. Toxic effects of aluminium have also been observed in aquatic environments. In fact, it is now believed that dissolved aluminium, released from sediments and suspended matter by acid rain, could account for the serious decline recently observed in fish populations which has been attributed to acid rain.2.3

The necessity for control in dialysis patients and research into A1 toxicity explains the present demand for analytical techniques to determine aluminium at trace and ultra-trace levels in waters, dialysis fluids and blood serum.

Studies carried out in our laboratory over the past four years to control A1 in waters, dialysis fluids and the serum of renal failure patients48 compared three atomic methods) namely) atomic absorption spectrometry (AAS), graphite furnace atomic absorption spectrometry (GFAAS) and inductively coupled plasma atomic emission spectrometry (ICP-AES) . Our experience has demonstrated that if the A1 content of the sample is below 30 pg 1-1, GFAAS is the technique of choice, on the grounds of its exceptional sensitivity, in spite of its traditional pitfalls.

For the less sensitive techniques such as conventional AAS or ICP-AES, pre-concentration methods can overcome the sensitivity limitations and the well known analytical advan- tages of AAS and ICP-AES techniques can be achieved for A1 determinations in such samples.

However, as for the other trace metals, the potential impact of A1 on the environment, animals and man can only be properly assessed when the nature and concentration of each individual species in the sample is known. The toxic A1 species is considered to be the dihydrolysed cation, AI(OH)Z+, because this is the kinetically favoured form in the reaction with the target compounds (usually proteins); however, diffusion of the relatively small Al(OH)2+ ion through the dialysis membrane into the bloodstream (in those patients undergoing haemodialysis) should be easier than that of bulkier polymerised species.

The combination of flow injection analysis (FIA) with flame AAS or ICP-AES provides an efficient sample introduction technique with several important advantages over conven- tional aspiration techniques,gJO allowing the use of flow injection liquid - liquid or ion-exchange techniques for

pre-concentration, speciation or conversion11 prior to AAS or ICP-AES detection.12 Numerous FIA designs have been developed using mini-columns for the determination of heavy metals in sea water by AAS'3-I5 and for increasing the sensitivity of ICP-AES.11J6

In this work we investigated the use of a miniaturised column with an FIA system to enhance the sensitivity of AAS and ICP-AES for the determination of A1 in waters and dialysis fluids. The analytical potential of this approach to speciate aluminium in biological fluids has also been assessed.

Experimental Apparatus and Reagents

A Perkin-Elmer Model 2280 atomic absorption spectrometer and a Perkin-Elmer ICP/5000 inductively coupled plasma atomic emission spectrometer were employed for the absorp- tion and emission measurements) respectively. The peak heights were measured in both instruments on a Perkin-Elmer Model 56 recorder connected to the spectrometer. Atomic absorption measurements were carried out in an N20 - C2H2 flame. pH measurements were made with a WTW pH meter and a Radiometer GK-2401-C combination glass - saturated calomel electrode.

A complete description of the optimum ICP operating conditions for A1 has been given elsewhere.6

Analytical reagent-grade chemicals were employed for the preparation of all solutions. Freshly prepared ultrapure water (from Milli-Q) was used in all experiments. The preparation and handling of solutions and containers were carried out as described elsewhere5 to minimise any possible risk of A1 contamination.

A 1000 pg ml-1 aluminium stock solution was prepared by acid dissolution of the pure metal. The following buffer solutions (pH 7) were used: 0.05 M NH3 - 2-(N-morpholino) ethanesulphonic acid (MES) (Sigma), NH3 - piperazine-N,N- bis(2-ethanesulphonic acid) (PIPES) (Sigma), AcNH4 - NH3 (Merck) and triethanolamine - NH3 (Merck). The strongly basic anion-exchange resin Amberlite IRA-400 (25-50 mesh) was used in the chloride form (Fluka).

Flow Injection Pre-concentration Procedure

The FIA layout used is shown in Fig. 1. A 0.05 M MES buffer solution at a flow-rate of 1.5 ml min-1 is used as a carrier into which the sample is injected through a septum (B in Fig. 1).

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700

0.250

0.200 E

2 Q: 0.100

2 0.150 P

0.050

JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, OCTOBER 1987, VOL. 2

(a)

-

-

-

- 0

f - l

Fig. 1. Flow diagram of the system used for the determination of aluminium: A, peristaltic pump; B, septum; C, injection valve; D, mini-column; and E, detector

The sample and carrier mix by flowing through a mixing coil (150 cm) and are propelled through the mini-column (where the A1 should be deposited). The volume injected is usually 1 ml and 2 min are allowed for the pre-concentration step. After pre-concentration , aluminium is stripped from the mini-column by injecting a 75-p1 plug of 1 M NaOH through the injection valve (C in Fig. l), which is passed into the flame (for AAS) or plasma (for ICP-AES). The resulting transient signal is recorded at 309.6 nm for absorption and at 396.15 nm for emission measurements.

Mini-column Preparation

The column body consisted of a 35 mm length of 3 mm i.d. silicone rubber tubing. The column end-caps were formed by fitting 20 mm of 0.8 mm i.d. tubing into a 10 mm piece of 1.5 mm i.d. PTFE tubing and a small piece of nylon netting was put between them to prevent movement of the resin particles by the carrier stream. One end-cap of the column was sealed with a suitable adhesive (Araldite) and the column was then filled with a slurry of Amberlite IRA-400 by aspiration with a syringe. The other end-cap was then fitted, resulting in a mini-column with a total volume of about 0.25 ml and containing ca. 0.108 g of resin.

Because of its unacceptably high aluminium content, Amberlite IRA-400 resin was carefully purified prior to packing. A 6-g portion of resin was placed in a glass column supported by coarse cotton plugs. A 1 M NaOH solution was fed through the column for 3 d. The collected effluents were analysed for A1 by AAS until no A1 signal was obtained. The resulting anion-exchange resin, now in the hydroxy form, was re-converted into the chloride form by washing it with de-ionised water to remove the excess of NaOH, treated several times with 1 M HCl solution and stored in 1 M HC1 for extended periods of time in polyethylene bottles (it is recommended that this “clean” resin should be washed with fresh 1 M HC1 solution and tested for A1 prior to use).

We also investigated the effect of placing the mini-column in the loop position of the injection valve (so that the direction of carrier flow at sorption and elution could be reversed and the dispersion of A1 through the column minimised). Experiments showed that the observed sensitivity improvement was margi- nal and the more straightforward single-line FIA system of Fig. 1 was eventually adopted.

Results and Discussion Metal Pre-concentration as a Function of pH

The effect of pH on the aluminium deposition and elution was studied by using buffer solution carriers of the desired pH. Preliminary experiments demonstrated that aluminium reten- tion on the column occurred around pH 6-8 and a more detailed study was then carried out using various buffer solution carriers in this pH range.

1 I I I I I

6.0 6.5 7.0 7.5 8.0 PH

Buffer molecule length-

Fig. 2. (a) pH influence on the aluminium retention using different buffer systems: A , ammonium acetate; A, triethanolamine; 0, MES; and B, PIPES. ( b ) Influence of the nature of the buffer on the retention of 3.0 pg aluminium at pH 7: 0, phosphate; 0, Tris - HC1; A , AcNH4, A, triethanolamine - HCl; 0, MES - NH,; and B, PIPES - NH3

The various pH - retention graphs obtained are shown in Fig. 2(a). As can be seen, the maximum retention of aluminium was obtained at a critical pH of 7, whatever the buffer used. Nevertheless, the nature of the buffer greatly influenced the aluminium sorption: the more bulky the buffer molecules the higher the retention observed [see Fig. 2(b)l. Therefore, we selected a 0.05 M MES - NH3 buffer for subsequent studies (PIPES produces unwanted precipitates at pH < 4). The aluminium retention was virtually unchanged when using MES - NH3 buffer in the range 3 x 10-2-0.1 M .

Stripping Agents

The elution of aluminium from the mini-column was tested by using NaOH, HN03 and HCl solutions, at various concentra- tions, as stripping agents. The results obtained showed incomplete elution of aluminium for all acid solutions. The use of 50-100 pl of 1 M NaOH, however, was sufficient to recover the resin-retained aluminium and a 75-pl volume of 1 M NaOH was used throughout.

Influence of Salt Content

The effect of adding increasing amounts of NaCl and Na2S04 to the injected sample on the A1 signal (1 ml of 1 pg ml-1 A1 injected for AAS measurements and 1 ml of 0.2 pg ml-1 A1 injected for ICP-AES measurements) was investigated. Fig. 3 shows the effect of NaCl (a fundamental component in serum and haemodialysis fluids); the aluminium signal increased with increasing NaCl concentration, up to ca. 3000 pg ml-1 C1- and then remained constant. A final concentration of about 3500 pg ml-1 of Cl- in the injected sample was subsequently used.

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JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, OCTOBER 1987, VOL. 2

A - 70 1

I800 0.080 t / 0.060

0 0.040 1 / 'i 0.020

{ 400

/*oo

1500 3000 4500 6000 7500 Cl-ivg ml-1

Fig. 3. Influence of the chloride concentration on aluminium pre-concentration: A, ICP atomic emission measurements for 0.2 pg Al; B, atomic absorption measurements for 1 pg A1

The addition of sulphate (up to 5000 pg ml-1) did not disturb the expected aluminium signal obtained by AAS or ICP-AES .

Flow Parameters and Retention Efficiency of the Mini-column

The recovery of aluminium was practically unchanged at carrier flow-rates from 0.75 to 1.75 ml min-1, but dropped significantly above this rate. The flow-rate used for analysis was kept constant at 1.5 ml min-1, a value close to the aspiration rate of our AAS and ICP nebulisers.

The effect of column parameters on the aluminium pre- concentration was also examined. Five columns were pre- pared with lengths of 15, 20, 30, 35 and 40 nm, all with the same internal diameter of 3 mm. The results obtained for each column by injecting the same amount of aluminium (1 pg) indicated that peak shapes were almost the same for the 30-40 mm columns, but elution was slightly more efficient, with less dispersion, for smaller columns, Dispersion mainly occurs between the eluent-releasing injection valve and the column, and between the column and the nebuliser. Columns of 35 mm length and 20 mm of connecting tubing upstream and downstream from the column were eventually selected and used. Under such conditions, the observed response was independent of the sample volume injected, at least in the interval tested (from several p1 up to 2 ml), which allows a favourable enrichment.

In order to determine the efficiency of aluminium uptake, 0.108 g (dry mass) of Amberlite IRA-400 resin was used for packing and a 1 ml sample containing 1 pg of aluminium and adjusted to the optimum pH value was injected. After elution with sodium hydroxide solution the collected effluents were analysed for aluminium by GFAAS to obtain the break- through retention efficiency of the column for the metal. The percentage of aluminium recovered at a flow-rate of 1.5 ml min-1 was 73 k 3.5%, which is equal to 0.025 mmol per gram of resin. This relatively small retention capacity of the Amberlite IRA-400 column is not a problem here if the minute amount of aluminium present in the samples to be analysed is considered.

Analytical Performance Calibration graphs for AAS prepared from the results of triplicate 1-ml injections of aluminium standard solutions were linear from 0.1 to 5 pg ml-1 of A1 (correlation coefficient 0.9999). The detection limit calculated as twice the standard deviation of the blank signal was 0.020 pg ml-1 and the relative standard deviation (n = 10) at 0.5 pg ml-1 was +2.7%. The sensitivity and detection limit observed were both about 15-fold better than those obtained by direct aspiration.

~

Table 1. Effect of foreign ions on the recovery of 0.5 pg of aluminium

Ions Cations:

CU" . . . . . .

Zn" . . . . . .

CrVI . . . . . .

CrrrI . . . . . .

Fe"' . . . .

Na+, K+, Ca2+, Mg2+

Anions: Acetate . . . . . . Carbonate . . . . Hydrogen carbonate Sulphate . . . . Phosphate . . . .

Fluoride . . . .

EDTA . . . . . .

. .

. .

. .

. .

. .

. .

. .

. .

. .

. .

. .

. .

. .

. .

. .

. .

. .

. .

1 .

. .

. .

. .

. .

. .

. .

. .

Mass ratio, ion : aluminium Recovery, %

200 20

200 20

200 20

200 20

200 40 8 4

200

8000 2000 4000 6000 2000 200

10 4 1

100.0 100.0 104.0 97.5

103.0 98.0 32.0 97.8 0.0

48.0 57.0 81 .O

100.0

96.0 97.0

102.0 100.0

0.0 105.0 46.0

105.0 23.0

Calibration graphs for the ICP-AES determination of aluminium by flow injection pre-concentration (from trip- licate 1-ml injections of standard aluminium solutions of 10, 25, 50, 75, 100, 200 and 300 pg 1-1) were linear with a correlation coefficient of 0.9997. The detection limit, calcu- lated as above, was 0.003 pg ml-1 and the relative standard deviation (n = 10) at the 0.1 pg ml-1 level was k 6 % .

The influence of various ions usually present in serum, waters and dialysis fluids on the recovery of aluminium is shown in Table 1. As can be seen, the biologically relevant trace metals, Zn, Cu and CrVI, did not interfere with the determination of aluminium, in spite of the fact that all were retained by the resin under the conditions described. The effect of FeI" and CrIrl can be ascribed to the formation of hydroxide suspensions at pH 7.

We verified that Fe"' is retained on the mini-column but is not released by 1 M NaOH, as expected for Al(OH),; only when using 2.5 M HC1 as the releasing eluent were iron signals obtained in the spectrometer. Hence, in the presence of Fe"', aluminium seems to be trapped in the column by the iron hydroxide and is unable to be solubilised and released by 1 M NaOH solution.

Alkali and the alkaline earth metals present in serum are not retained by the resin and did not interfere in the determination of A1 (Table 1).

Anions that form complexes with aluminium, namely, fluoride, phosphate and EDTA, interfered in the determina- tion. Therefore we have a system which is very sensitive (see effect of F-) to the presence of ligands for AP+.

Retention - Elution Mechanism for Aluminium

Useful information on the speciation of aluminium in the solution can be gained by studying the mechanisms of its retention and elution on the mini-column used here. In fact there are some striking features in the observed uptake of A1111 by the anionic-exchanger Amberlite IRA-400. The retention pH is very critical and is at an optimum around pH 7 whatever the buffer used; the nature of the buffer is also critical and chloride ions markedly influence the retention (Figs. 2 and 3). All the above observed aspects should be related to the physico-chemical form of the aluminium species formed under the conditions of the general procedure. In aqueous solutions

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702 JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, OCTOBER 1987, VOL. 2

several A1111 species have been reported to exist depending on the pH, e.g., AP+, Al(OH)2+, Al(OH)2+, Al(OH)3, A12(OH)24+, A13( OH)45-, Al( OH)4- and even A113(OH)327+.17

Above ca. pH 4, the insoluble Al(OH)3 species (or Al2o3.3H20) begins to precipitate and redissolves only if the pH becomes high enough to form the soluble Al(OH)4- species. The isoelectric point, with a minimum solubility lies around pH 7. From our experience, a pH of about 7 provides the best results, which means that the isoelectric point, favouring the formation of Al(OH)3, offers the best condition to retain the metal in the mini-column.

A second experimental fact is that MES and PIPES behaved similarly and were far superior to the rest of the buffers tested. Both reagents have a structure featuring a polar sulphonic group and an organic "tail." Hence they could be adsorbed on the colloidal surface of A1(OH)3 particles (lake formation of these particles with dyes is a well known phenomenon) giving a negatively charged (through the -SO3- group of the organic buffers) soluble species of aluminium able to be fixed or changed by the anion exchanger.

Chloride ions not only increased the signal observed (Fig. 3) but also proved to be the best reactivating anion for the resin (1 ml of 1 M solutions of KI, KF, H2S04, H N 0 3 or HCl were tested for that purpose). After repeated elutions with 1 M NaOH the height of the peak observed tended to decrease, but washing by injection of 1 M HC1 restored the normal signal. This demonstrates that when the resin is loaded with C1- ions the ionic exchange with the A1111 species formed is at an optimum. Considering the affinity order of a basic anion- exchange resin (F- < OH- < C1- < NO3- < I- < S042-),18 a possible mechanism accounting for the deposition and elution observed could include a typical ion-exchange reaction between the C1-, as the exchanger ion, and the negatively charged AIII1 species formed with the buffer having a sulphonic group, Al(OH)3.RS03-. In FIA, the contact time between the solution and resin is very short and thus equilibrium is unlikely to be attained. Therefore the displace- ment of C1- ions by the A1(OH)3.RS03- species must also be kinetically favoured. Other anions of the sequence with a lower affinity for the resin such as fluoride or hydroxyl would form complexes with A1111 and as the exchange of these complexes is probably slower, the deposition of the metal is adversely affected.

In conclusion, the results show that the mechanism of anion-exchange in the anionic resin under the conditions of the proposed procedure is different from the classical mechan- ism. The different steps could be formulated as follows:

Activation: R,N+OH- + Cl- 2 R4N+C1- + OH-

A1111 deposition: R4N+Cl- + Al(OH)3.RS03- (1) 6) (r) 6)

(r) (4 R,N+Al(OH)3.RSO3- + C1-

(r) (4 A1111 elution: R4N+Al(OH)3.RS03- + 20H-

(r) (s) R,N+OH- + Al(OH)4- + RS03-

(r) 6) (9 Determination of Aluminium in Biological Samples Serum samples without previous treatment were directly injected into the FIA - AAS system described for the determination of Al. No signals for the metal were observed even when aluminium (up to 1 p.p.m.) was added to the serum. Dilution and the addition of mineral acids, bromine water, strong chelating agents and metal ions such as Cu2+ (with a high affinity for proteins) to serum were tested without any success. Physical treatment of the serum samples by irradiation with UV light or sonication in acid media did not

Table 2. Results of determination of aluminium in tap waters and haemodialysis fluids

Aluminium concentrationhg ml- 1

Flow injection pre-concentration

ICP-AES GFAAS Samples Tap water:

Ref. 1 . . . . . . 100.4k2.4 88.6 2 4 . 1 315.2 k 7.1 Ref.2 . . . . . . 316.8k0.7 471.0 k 1.1 Ref. 3 . . . . . . 441.2 k 0.7

Dialysis fluids: 61.7 k 6.0 Ref. 1 . . . . . . 64.5k0.7 84.9 k 0.2 Ref.2 . . . . . . 71.1 k 3.0

Ref. 3 . . . . . . 109.9 k 1.5 109.7 k 2.5

improve the situation: even the aluminium added to the serum could not be determined by our detection system. This clearly indicates that aluminium binds so effectively to serum proteins, and even to protein fragments formed by the chemical or physical treatments investigated, that free alu- minium does not seem to exist in human serum. Serum aluminium should be complexed, probably in the protein transferrin719J0 forming a species which cannot be collected by the ion exchanger. Our findings agree with a recent report20 showing a bond between A1111 and transferrin which cannot be dissociated by prolonged dialysis or desferrioxamine treat- ment. Other workers have reported, however, that dialysis of aluminium-saturated transferrin does release aluminium by lowering of the pH 4.5 or by adding the drug desferrioxamine to the dialysate.21 It is possible that the kinetic behaviour of the complex rather than the thermodynamic stability con- stants of AP+ for the protein and the drug would explain the different and controversial reports on the forms in which aluminium is found in serum.19-21

Analysis of Water and Dialysis Fluids

The proposed analytical method was applied to the determina- tion of aluminium in tap waters and haemodialysis fluids. Tap water samples were adjusted to pH 7 with 1 M MES - NH3 buffer solution, and to a final 3500 pg ml-1 chloride matrix with 1.7 M NaCl solution, giving a final dilution of 5.5%. For haemodialysis fluids only the MES - NH3 buffer solution was added and the final dilution of the sample was only 2.5%. The samples were then analysed using the flow injection ion- exchange pre-concentration ICP-AES method described. The results obtained were compared with those obtained by the GFAAS method4 for the same samples.

Samples from different urban areas were analysed in duplicate and a direct calibration against simple aluminium standards in a 3500 pg ml-1 NaCl matrix was used. The results (Table 2) show that the two methods gave equivalent results.

In conclusion, a flow injection ion-exchange pre-concentra- tion technique such as that used in this work, improves the sensitivity and detection limits for the determination of aluminium by AAS and/or ICP-AES by more than one order of magnitude and offers a great potential, still unexploited, to study the binding, stability and reaction kinetics of the complex formed between A1111 and serum proteins.

Financial support form the Comision Asesora para la Investi- gacion Cientifica y Tkcnica (CAICYT) for this work (project no. 2837/83) is gratefully acknowledged. We thank J. L. Fernandez Martin and J. Pkrez Parajon for the GFAAS measurements in this work.

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1. 2. 3. 4.

5.

6.

7.

8.

9. 10. 3 1. 12.

References Galle, P. , Recherche, 1986, 61, 857. Florence, T. M., Trends Anal. Chem., 1983, 2, 162. Robinson, J. W., and Deano, P. M., Znt. Lab., 1986, 16, 14. Sanz Medel, A., Rodriguez Roza, R., Gonzalez Alonso, R., Noval Vallina, A., Cannata, J . B., ZCP Znf. Newsl., 1986, 11, 708. Cannata, J. B., Suirez, S. C., Cuesta, V., Rodriguez, R. R., Allende, M. T., Herrera, J . , Pkrez, L1. J . , Proc. Eur. Dral. Transplant. Assoc., Eur. Renal Assoc., 1984, 21, 354. Rodriguez Roza, R. , Sanz Medel, A . , and Cannata, J . B., Quim. Anal., 1986, 5 , 150. Sanz Medel, A., Rodriguez Roza, R. M., Gonzalez Alonso, R., Noval Vallina, A , , and Cannata, J . B., J. Anal. At. Spectrom., 1987, 2, 177. Rodriguez, R. R., Gonzalez, A. R., Cannata, J . B., and Sanz Medel, A. , paper presented at “XX Reuni6n Bienal de la Real Sdad. Espanola de Quimica, Castellh, 24-28 September, 1984. ‘’ Tyson, J. F., Anal. Proc., 1983, 20, 488. Greenfield, S . , Spectrochim. Acta, Part B , 1983, 38, 93. RfiiiEka, J., Fresenius 2. Anal. Chem., 1986,324, 745. Nord, L., and Kalberg, B., Anal. Chim. Acta, 1983, 145, 151.

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Olsen, S . , Pessenda, L. C. R., RfiiiEka, J . , and Hansen, E. H. , Analyst, 1983, 108, 905. Fang, Z . , Rfiiitka, J., and Hansen, E. H . , Anal. Chim. Acta, 1984, 164,23. Marshall, M. A., and Mottola, H. A., Anal. Chem., 1985, 57, 729. Hartenstein, S. D., Rfiiieka, J . , and Christian, G. D., Anal. Chem., 1985, 57, 21. Mesmer, R. E., and Baes, C. F., “The Hydrolysis of Ctions,” Wiley, NewYork, 1976. Samuelson, O., “Ion-exchangers in Analytical Chemistry,” Wiley, New York, 1952. Rahman, H . , Channon, S. M., Skillen, A . W., Ward, M. K., and Kerr, D. N. S., Proc. Eur. Dial. Transplant. Assoc., Eur. Renal Assoc.. 1984, 21, 360. Rahman, H., Skillen, A. W., M. K., Channon, S . M., and Kerr, D. N. S., Znt. J. Artif. Organs., 1986, 9, 93. Moshtagnie, A. A., and Skillen.. A. W . , Biochem. Soc. Trans., 1986, 14, 916.

Paper 57/51 Received April 16th, 1987 Accepted April 30th, I987

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