The preparation of 125I-labelled sodium polystyrene sulphonate

Appl.

Pergamon

Rodiar.

hr. Vol. 45, No. 3, pp. 345-351, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0969-8043/94 $6.00 + 0.00

The Preparation of 1251-Labelled Sodium Polystyrene Sulphonate IAN Fluid Processes

HARRISON Group,

and JENNIFER

J. W. HIGGO

British Geological Survey, Kingsley Dunham Nottingham NG12 SGG, England

Centre,

Keyworth,

(Received I9 July 1993) The anionic polymer, sodium polystyrene p-sulphonate (PSSNa), was labelled with iodine-125. Standard radio-iodination for proteins proceeds via phenol groups. As PSSNa contains no phenolic functions these were introduced by reaction with sodium hydroxide. The optimized conditions gave barely discernible phenolic incorporation so the properties of the bulk PSSNa were not noticeably altered. The modified PSSNa was then radio-iodinated by the standard technique. The yield, activity and stability of the product were determined by size exclusion chromatography.

Introduction The role of colloidal-size (submicron) particles in facilitating contaminant transport is still poorly understood. There is evidence that, under favourable conditions, colloids may be mobile in subsurface environments (McCarthy and Zachara, 1989). Association of contaminants with these particles may, thus, enhance the transport of strongly sorbing pollutants. A research programme aimed at understanding the physico-chemical and hydrogeological factors that control colloid transport was, therefore, embarked upon. An array of boreholes equipped with ranging gamma-ray probes had already been constructed in a glacial sand aquifer (Williams et al., 1991) and radiolabelling of the colloid would, accordingly, provide the most convenient method of detection if its migration behaviour was to be studied under field conditions. Sodium polystyrene p-sulphonate, PSSNa, is an anionic polyelectrolyte (as is the naturally occurring colloid, humic acid) and is a water-soluble colloidal polymeric material. Initial experiments, to assess the mobility of PSSNa in a sandy medium were performed using column tests (Harrison et al., 1993). Glass columns, slurry packed with sand from the aquifer and eluted with natural groundwater had PSSNa solution injected onto them. Elution of the PSSNa, estimated by U.V. absorption of the effluent at 254 nm, showed that polymer was markedly mobile. The degree of mobility indicated that this material might have potential as a tracer of colloidal mobility in the field. However, for such a purpose a gamma-ray emitting label had to be incorporated into the structure. 345

Certain criteria for the choice of gamma-ray emitter needed to be addressed initially, i.e. the nuclide needed to be: (i) Readily available commercially. (ii) Able to bond stably to PSSNa, preferably by a relatively simple reaction. (iii) Short lived so that rapid decay would prevent persistence in the ground. (iv) Safe to manipulate with well established handling protocols. (v) Active enough to be easily detected by the field gamma-ray probes. The radio-iodine isotope, 12’1,satisfied the above specifications, except that no method for chemically bonding it into the polymeric structure was immediately apparent. The labelling of proteins with radio-iodine, by means of the cationic iodinium ions (I+), has been widely practised in various forms for over 30 years (Bolton, 1985). The iodinium ions, generated by mild oxidation of iodide ions, at around pH 7.5 + 0.5, electrophilically attack phenolate ions, generated by the slight ionization (pK, = 10.00) of phenolic groups present in the tyrosine residues of the protein. This leads to mono- and di- substitution of radio-iodine at the ortho- positions on the aromatic rings of the tyrosine functions. As there are no phenolic groups in PSSNa, to use the protein labelling technique, it was necessary, firstly, to introduce them into the structure. One standard method for the preparation of phenols is the reaction of aromatic sulphonates with sodium hydroxide (Streitweiser and Heathcock, 1976). The nucleophilic substitution of the sulphonate group by

346

IAN HARRISON and JENNIFERJ. W. HIGGO

the hydroxyl group normally needs temperatures around 25O’C for a reasonable yield of the desired phenol. Such elevated temperatures would inevitably cause profound degradation of the polymeric structure of PSSNa. But because of the high specific activity of ‘*‘I it became apparent that only a very small number of the aromatic sulphonate groups need be converted into phenohc groups (ca 1 in 250,000) in order to obtain a sample with sufficient activity for use as a field tracer. By altering the reaction conditions it was possible to obtain the required small incorporation of phenohc character without noticeable alteration of the properties of the bulk polymer. A qualitative test specifically for p-phenols was used to assess the degree of incorporation and size exclusion chromatography was used to ascertain the effect upon the bulk polymer of the reaction conditions. After radio-iodination of the modified PSSNa the same size exclusion technique was employed to determine the iodination yield and stability of the product. The ability of the product to act as a stable tracer in the field was also tested in the laboratory.

Materials Introduction

of phenolic

character

The sodium polystyrene p-sulphonate, PSSNa (completely sulphonated) of average mol. wt 500,000 Da, that was used in the preparation, together with some narrow mol. wt range sodium polystyrene sulphonate standards, used to calibrate the size exclusion technique, were obtained from Park Scientific Ltd, Northampton, U.K. Chemicals for the test reagent were obtained from the Aldrich Chemical Co. Ltd, Gillingham, U.K. Dialysis was performed with Spectra/Par” tubing (MWCO 100,000 Da) supplied by Pierce & Warriner, Chester, U.K. Size exclusion

chromatography

(SEC)

The reagents employed for the SEC were of HPLC grade and were from B.D.H. Ltd, Atherstone, U.K. Sephacryl” S300HR (stationary phase), Blue Dextran, the LKB Uvicord S U.V. detector and the Redifrac fraction collector were all from Pharmacia LKB (U.K.), Milton Keynes, U.K. The sample injection valve, glass column and fittings were from Omnifit, Cambridge, U.K. The pump was a Lichrograph L6000 supplied by E. Merck, Darmstadt, Germany. The chart integrator was an HP3396B from Hewlett Packard, Avondale, PA, U.S.A. Radio -iodination

[iZSI]Sodium iodide in NaOH solution was obtained from Amersham International plc, Amersham, U.K. The Iodobeads’” were from Pierce, Rockford, 111, U.S.A. The chloramine-T hydrate and sodium metabisulphite were from the Aldrich Chemical Co. Ltd, Gillingham, U.K. Visking dialysis tubing

(MWCO 12,000-14,000 Da) was from Medicell International Ltd, London, U.K. Gamma counting was performed on a Minaxi Auto GammaH 5000 series gamma counter supplied by Canberra Packard, Pangbourne, U.K.

Methods, Results and Discussion Introduction

of phenolic

character

A set of experiments, to investigate the effect of reaction conditions on the substitution of hydroxyl groups for the sulphonate groups, was conducted. In each, PSSNa was stirred into sodium hydroxide that had been dissolved in a small amount of water. The resulting paste, in a covered nickel crucible, was placed in an oven for a set period of time. After neutralization of the product, the effect of temperature and duration upon the production of p-phenol groups was assessed. A qualitative test (Pesez and Barbos, 1974) was employed, in which a yellow coloration was produced by reagents containing I-nitroso-2-naphthol and sodium nitrite. The intensity of the colour was indicative of the amount of phenohc incorporation. It was found that a discernible positive result could be obtained with a temperature as low as 125 C provided the crucible was maintained at this temperature for approx. 1 week. At higher temperatures, the test showed that, phenohc incorporation was greater and faster. Though this, it transpired, was at the expense of more damage to the polymeric structure of the PSSNa. Size exclusion

chromatography

(SEC)

SEC was used to indicate the extent of the damage caused during the reactions employed to introduce phenolic character into PSSNa. Because the technique provides a separation based broadly on molecular size, evidence of rupture of the polymer backbone is reflected by changes in the chromatograms for the reacted materials when compared with those of the original, unreacted material. The system had the following characteristics: Eluant-80% 0.1 M sodium sulphate AR (aqueous) : 20% acetonitrile Column-Sephacryl ’ S300HR (20 cm x I cm i.d.) Slurry packed Flowrate-0.5 mL/min Sample loop volume---0.2 mL Detector-Fixed wavelength 254 nm. Absorbance range 0.5 aufs Chart-HP3396B Integrator The system was calibrated using Blue Dextran and PSSNa standards with narrow range average molecular weights (M,/M, < I. 10) of 400,000 Da, 220,000 Da, 100,000 Da and 70,000 Da. Chromatograms of these made it apparent that the system had

347

‘251-labelledsodium polystyrene sulphonate a small fractionation range between 70,000 and 100,000 Da. The total exclusion volume was 7mL and the total permeation volume was 17mL. The chromatogram (Fig. 1) for PSSNa (1) showed a large early peak because most of the polymer eluted at the total exclusion volume i.e. most of the molecules were above 100,000 Da. The peak tail was shallow as it passed through the fractionation range showing that very few of the molecules were between 100,000 and 70,000 Da. The small peak at the total permeation volume represented the small number of polymer molecules with molecular weights < 70,000 Da. The other chromatograms (2,3,4) showed varying degrees of thermal polymeric degradation, indicated by the diminution of the high molecular weight peak and the appearance of material at lower molecular weight i.e. in the fractionation range and at the total permeation volume. However, for the PSSNa that had been heated at 125°C for 168 h (2) only very slight disruption was apparent. Radio-iodination In the 250” and 160°C cases, where the polymer backbone had suffered gross rupture, the value as a field tracer was dubious because the average molecular weight of the bulk material would no longer be known. Nevertheless, radio-iodination of the 160°C product was undertaken for comparison with the potentially more useful 125°C product. For the initial small scale investigations Iodobeads’” were employed as oxidant, instead of the more usual chloramine-T, because of their greater ease of use (Hearn, 1982). Thus, 0.1 g of a solution of carrier-free Na’*‘I (ca 5 MBq/mL) was mixed with 1 mL of water and four Iodobeads’” were added. The mixture was allowed to stand for 5 min to permit

generation of iodinium ions. One mL of the modified PSSNa solution (ca 25,000 ppm) was added and the reaction allowed to proceed with occasional agitation for ca 2 h. The reaction was terminated by pipetting the liquid phase from the beads into a Visking dialysis tube (MWCO 12,000-14,000 Da) surrounded by water. The dialysis, which was continued for 5-6 days with daily changes of the water, removed unreacted Na’*‘I, chloride ion and low molecular weight degradation products. During dialysis and afterwards samples of the radio-iodinated products were taken and analysed using the SEC system. Fractions of the effluent leaving the U.V. detector were collected and counted on a gamma-ray counter (lower threshold 15 KeV and upper threshold 80 KeV-‘2SI counting efficiency 71%). All the radio-iodinated products showed similar initial levels of radio-iodine associated with the high molecular weight polymer (Fig. 2). However, activity loss for the unmodified PSSNa was much more rapid over the ensuing 15 days than for the 160°C and 125°C modified PSSNa. Thereafter, the loss from the 160°C and 125°C products was imperceptible but seemed to continue slowly from the unmodified PSSNa. These observations were rationalized in terms of weak and strong radio-iodine bonding. Thus, the PSSNa in the samples, regardless of their phenolic content, underwent a weak interaction with a species or radio-iodine present in the iodination mixture. This weakly associated PSSNa/iodine combination broke down quickly compared with the radio-iodine that had been covalently bound by substitution of phenolic groups via the iodinium ions. Accordingly, all samples showed an initial rapid loss of high molecular weight activity. Once the weakly bound radio-iodine had been lost, it was the materials

1

-r

0.6

0.5 8 9 s$

0.4

2 9

0.3

w 2

0.2

$ 5

0.1

0

5

10

Elution

15

Volume

Fig. 1. Size exclusion chromatograms

20

1 .................

pSSNa

2-

125°C for 168 h

3-

160°C for 20 h

4-

250 “C for 1.5 h

25

in mls.

showing the effect on PSSNa of heating with NaOH

IAN HARRISONand JENNIFER J. W. HIGGO

0

l.

a

0 .

a

X

X

0 i 0 Time

OO

0

Aa .

X

I

I

I

20

30

elapsed

since preparation

0

12YC Product

X

Unmodified PSSNa

X

X

10

160°C Product

1

1

40

50

in days

Fig. 2. Loss of ‘25Iactivity from the PSSNa associated with the SEC high M.W. Peak.

with appreciable phenolic character, containing covalently bound radio-iodine, that showed the higher and more stable levels of activity. The qualitative phenol test had indicated that the 160°C product, not surprisingly, contained more phenolic character than the 125°C product. Yet more activity was present in the high molecular weight material of the 125°C product (Fig. 2). The reason for this can be seen by comparing the U.V. and gamma-ray traces obtained during the SEC of the two products (Figs 3 and 4). The SEC of the 160°C product revealed that most of the activity (and most of the phenolic character) was associated with the degraded polymer responsible for the distortion of the high molecular weight peak and not with the high molecular weight material

itself. However, when the same comparison was applied to the 125°C product it was apparent that the U.V. and gamma-ray elution were similar, with coincident maxima, but with somewhat more pronounced tailing for the elution of high molecular weight activity, indicating a small degree of degradation attending phenol introduction. A significant reduction of the activity tailing (Fig. 5) was achieved by dialysis of the 125°C product withe Spectra/par” tubing (MWCO 100,000 Da). Radio-iodination yield experiments were conducted on the 125°C product. The procedure was essentially that used for the labelling except that no dialysis was undertaken. Thus, when the SEC analysis was performed the ratio of the radio-iodine associated with the high molecular weight material to

0.60

- 3000 Gamma Detection

s

0.50

2

e

,o

0.40

2

T

0.30

5

0.20

j 5

U.V. Detection

0.10

Elutlon Fig. 3. Comparison

of U.V. and gamma

Volume detection

in mls. for the SEC of the 160°C product

349

‘251-labelledsodium polystyrene sulphonate 0.6

U.V. Detection

Elution

Volume

in mls

Fig. 4. Comparison of u.v. and gamma detection for the SEC of the 125°C product.

the total radio-iodine in the system could be determined directly. In order to determine the total radioiodine it was necessary to collect about 60mL of column effluent because some of the radio-iodine activity was found to elute well after the total permeation volume (with a distinct peak at ca 40mL elution). Much the same yield (Table 1) was obtained when chloramine-T was used instead of Iodobeads’“. When no oxidant was present no iodination occurred showing that iodide will not react with modified PSSNa and that the agent responsible for radio-

iodination was radio-iodine in a positive oxidation state. Reaction time and number of beads did not appear to greatly influence the yield. For use as a field tracer a sample of modified PSSNa with an activity of ca 74 MBq was needed. Our radiochemical facilities were not able to cope with the radio-iodine activities that this would involve. Consequently, a 5 mL sample of the dialysed (MWCO 100,000) 125°C modified PSSNa (ca 33,000 ppm) was sent to Amersham International plc, together with the experimental procedures. Following these, they were able to return 4mL of radio-

6000

‘5000

i

,P .I

-4000

With dialysis

a

- 3000 Without dialysis

0

10 Elution

20 Volume

30

- 2000

40

in mls.

Fig. 5. Effect of dialysis on the SEC of the 125°C product. Dialysis performed with Spectra/Par” (MWCO 100,000 Da) prior to radio-iodination.

350

IAN HARRISON and JENNIFER J. W. HIGGO Table I. Radio-iodination Mass PSSNa (mg) 26.0 26.0 26.0 25.5 25.5 75.2 75.2 25.9 25.1 *Chloramme-T

yield experiments.

No. of Iodobeads

Amount of “‘I used 0%)

RGXtiOIl time (h)

4 4 4 IO IO 5 5 0 IO.1 I mg*

35,062 35,062 35.062 34.61 I 34.61 I 33.698 33,69X 33,950 100.394

2.50 4.33 22.50 2.25 28.50 2.00 24.00 2.00 0.50

(reaction

terminated

by 20 mg Na,S,O,

iodinated sample containing 37 MBq/mL. This was quoted to cyntain I. 1% radio-iodide determined by TLC. Eight days after preparation it was found to contain 8.3% radio-iodide by SEC. This accords with the loss of weakly bound radio-iodine that had been observed during the initial radio-iodination experiments. The sample was stored, at 4°C and periodically monitored by SEC for its radio-iodide content. After 2 months this had slowed to 0.087% per day which was deemed acceptable for the tracer. The 3.9 mL that now remained was mixed with 6 mL of unmodified PSSNa solution (32,OOOppm) and the mixture dialysed in Visking tubing. (Note-the osmotic pressure caused the level in the tubing to rise considerably. Overflow was prevented by exchanging the water dialysate for one of unmodified PSSNa solution ca 32,000 ppm). Thus freed of radio-iodide the labelled material was mixed with more unmodified PSSNa solution, to act as its carrier. The final constitution of the field tracer before dispatch was: Total tracer mass Concentration PSSNa Mass PSSNa Total “‘1 activity in tracer Radio-iodide content Stability

= = = = =

100.0 g 30.900 ppm (SEC) 3.09 g 63.1254 MBq 2.22% max. (SEC)

in the aquifer

Having successfully produced a stable tracer, consisting of the radio-iodinated PSSNa in a carrier of unmodified PSSNa, it was necessary to resolve two issues: (i) Would the radio-iodinated PSSNa behave in the same manner as the bulk PSSNa (acting as its carrier) under field conditions, given the processes of modification and labelling it had undergone? (ii) Would the geological medium promote decomposition of the labelled PSSNa? A sample of the tracer was therefore passed through a column (ca 45 cm x 1.5cm i.d.) packed with sand from the aquifer, in which the field trial was to take place. The column was eluted with groundwater from the aquifer and the effluent was monitored by U.V. absorption at 254 nm and gamma

Determination

by SEC

High MW SEC lz51 activity @pm)

Total SEC ‘211act1wty (cpm)

10,429

50,068 5 I.220 55, I72 47,908 54,664 48,590 50.003 57,825 166,779

I 1,936 I I ,03x 8992 l2,83 I 12,375 12.623 90 50,412

Radio-mdination yield (%) 20.8 23.3 20.0 18.8 23 5 25.5 25.2 0.16 30.2

in I mL water).

counting. The elution profiles for the U.V. and gamma were found to coincide remarkably well, indicating that the labelled material faithfully mirrored the behaviour of the unmodified PSSNa carrier. The total recovery of PSSNa as determined by U.V. was found to be 40% and by gamma-ray counting was found to be 43.2%. To examine the possibility of dissociation, a sample of tracer was passed through a shorter column of the sand (6.7 cm x 1 cm i.d.) and the radio-iodide content of the effluent determined by SEC and compared with that of the same solution that had not been through the column. Both had the same radio-iodide content. The radio-iodinated PSSNa in unmodified PSSNa carrier was accordingly considered a suitable field tracer for investigations into the transport of polymeric colloidal materials.

Summary and Conclusions Sodium polystyrene p-sulphonate was successfully labelled with “‘1 without perceptibly altering its bulk characteristics. This was accomplished by the introduction, under carefully controlled conditions, of phenolic groups into the polymer followed by radio-iodination using either lodobeads’” or chloramine-T as oxidant. The final product was stable when passed through columns of natural aquifer material and was subsequently used in a field experiment (Higgo et al., 1993). Acknowledgemenrs-Funding for this work was provided by the United Kingdom Department of the Environment. The results will be used in the formulation of Government Policy but at this stage do not necessarily represent that policy. This paper is published with the permission of the Director of the British Geological Survey (NERC).

References Bolton A. E. (1985) Radioiodination technirrues. Reoiew 18. 2nd edn. Amersham International plc., Bucks., U.K. Harrison I., Higao J. J. W. and Williams G. M. (1993) The preparation of lz51 labelled sodium polystyrene sulphonate. British Geological Survey Technical Report WE/92/8 British Geological Survey, Nottingham. U.K. DOE Report No. DOE/HMIP/RR/93/009.

1251-labelled sodium Hearn M. T. W. (1982) Radio-iodination of proteins with pre-loaded lodobeads’“. Pierce Previews November, 3. Higgo J. J. W., Williams G. M., Harrison I., Warwick P., Gardiner M. and Longworth G. (1993) Colloid Transport in a Glacial Sand Aquifer-Laboratory and Field Studies. Colloids and Surfaces 73, 179. McCarthy J. F. and Zachara J. M. (1989) Subsurface transport of contaminants. Environ. Sci. Technol. 23,496. Pesez M. and Barbos J. (1974) Calorimetric and Fluorimetric

polystyrene

sulphonate

351

Analysis of Organic Compounds and Drugs. Marcel Dekker, New York, U.S.A. Streitweiser A. and Heathcock C. H. (1976) Introduction fo Organic Chemistry. Macmillan, New York, U.S.A. Williams G. M., Higgo J. J. W., Sen M. A., Falck W. E., Nov D. J.. Wealthall G. P. and Warwick P. (1991) The influence of organics in field migration experiments; Part 1. In sifu tracer tests and preliminary modelling. Rodiochim. Acta 52153, 457.