JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY DECEMBER 199 1 VOL. 6 643 Immobilized Alga as a Reagent for Preconcentration in Trace Element Atomic Absorption Spectrometry Hayat A. M. Elmahadi and Gillian M. Greenway School of Chemistry University of Hull Hull HU6 7RX UK The alga Sebnestrum capricornutum was covalently immobilized on controlled pore glass and packed in a minicolumn which was incorporated into a flow injection system for the preconcentration of trace elements prior to determination by atomic absorption spectrometry. Samples (5 ml) of Cu2+ Pb2+ Zn2+ Co2+ Hg2+ and Cd2+ were preconcentrated to give detection limits of 0.05 2.5 0.2 8.0 30.0 and 2.0 ng ml-l respectively with a sampling rate of 20 h-l. Keywords Immobilized alga; preconcentration; trace metal; atomic absorption spectrometry The ability of algae to accumulate trace metals by biosorp- tion has been known for some years.' This effect has been put to use in water treatment systems,* however only recently has it been exploited for analytical measurement methods3 Many reagents have been investigated for the preconcentration of trace metals however these usually have only one type of binding site.The possible advantage of algae is that potentially the cell wall has many constitu- ents that can be implicated in metal binding including mine and carboxyl groups from amino acids and polysac- charides sulphydryl groups and unmethylated pectin^.^ This range of binding sites means that by altering the elution conditions different metal ions can be retained preferentially.Another advantage of using these organisms is their small and uniform cell size. Most of the analytical work that has been published using algae has been carried out on material that was not immobilized. This can be a difficult and time-consuming procedure requiring several steps including washing and centrifuging. A biosensor has been reported where the alga was immobilized on a carbon paste electrode for the determination of Cu** species.5 Other workers have immo- bilized algae mainly for biotechnology applications.6 This has been either by invasive adsorption of live algae into polymer matrices or by physical entrapment in polymers. Dagnell et al.' reported the use of polyacrylamide immobil- ized alga. These workers have also developed a method of physically trapping the alga in a silica gel polymeric material.* Immobilization of chemical reagentsg and en- zymesl0 by covalent attachment to a water insoluble substrate provides very stable preparations.Controlled pore glass (CPG) has been shown to be a particularly effective insoluble substrate as it exhibits good mechanical properties in flowing streams. For enzymes,1° the CPG is first silanized and then the bifunctional properties of gluteraldehyde are used to cross-link the enzyme to the silanized glass through the lysine amino groups on the enzyme. This work describes the covalent immobilization of an alga on CPG for the preconcentration of Cu2+ Pb2+ Zn2+ Co2+ Hg2+ and Cd2+. The diversity of active binding sites of the alga means that its ability to preconcentrate is not hindered by immobilization.Experimental Reagents The green alga Selenestrum capricornutum was selected as it is a fast growing and easily cultivated species. It was cultured by the Department of Applied Biology at the University of Hull in an aerated medium and harvested after 1 week. Chu 10 medium (medium No. 10 as described by Chdl) was prepared in the laboratory and used for cultivation of the alga. This consisted of Ca(N03)2.4H20 (57.6 mg l-l) Ca(N03)2 (40 mg l-l) K2HPO4 (5.0 mg l-l) Mg!304.7H20 (25.0 mg l-l) Na2C03 (20.0 mg l-l) Na2SiOj (25.0 mg l-l) FeC13 (0.8 mg l-l) 25 mmol 1-1 HN03 and soil extract. The soil extract was prepared by placing 250 g of soil into 500 ml of water and boiling in a steamer for 2-3 h. This was filtered through a Whatman No.1 filter paper autoclaved and stored in a refrigerator for 2-3 d to allow the sediment to settle. A 10 ml volume of this solution was added to the medium before making it up to 1 1 with water and autoclaving. The alga was centrifuged and washed three times with 50 ml of distilled de-ionized water. After washing it was heat treated and then freeze dried. The CPG (CPG-240,22.6 nm pore diameter 80- 1 20 mesh) and 8-aminopropyltriethyox- ysilane were obtained from Sigma. Cadmium chloride and cobalt(@ sulphate and all of the other reagents were of analytical-reagent grade from Merck. Distilled de-ionized water was used throughout. Immobilization of the Alga A 0.1 g amount of the ground freeze-dried material was weighed into a small beaker (20 ml) then 2 ml of 0.01 mol dm-3 hydrochloric acid followed by two 5 ml aliquots of ethanol were added whilst stirring.This mixture was heated on a steam-bath for 1-2 min until all of the material appeared to go into solution. A 2 ml volume of water was then added and 10 ml of the solution were transferred into a clean beaker. The volume was made up to approximately 25 ml using phosphate buffer (0.1 mol dm-3). The solution was adjusted to pH 6 using sodium hydroxide solution. The immobilization procedure which is presented in Fig. 1 was a modified form of that used for the immobilization of enzymes on CPG.l0 The CPG was activated by taking 1 g and boiling it in 10 ml of 5% v/v nitric acid for 30 min. It was then filtered through a porous sintered glass filter washed with water and dried in an oven at 95 "C for 1 h.The CPG was then silanized with 8-aminotriethy- oxysilane solution by taking 2.5 ml of the reagent and diluting it with water to 25 ml. This solution was adjusted to pH 3.45-3.50 with 6 mol dm-3 hydrochloric acid and 5 ml of the solution were then added to the dried CPG and heated on a water-bath at 75 "C for 150 min with stirring. The resulting product was washed with water and dried at 95 "C for 2 h. This procedure was repeated twice. The aldehyde derivative was then prepared by taking 2.5 ml of an aqueous gluteraldehyde solution (50% v/v Merck) and making it up to 50 ml with phosphate buffer (pH 7). A 5 ml volume of this solution was added to the treated glass in a firmly stoppered round-bottomed flask flushed with nitrogen. The reaction was allowed to continue for at least 1 h at room temperature during which time a brown644 solution De-ionized ’ water JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY DECEMBER 199 1 VOL.6 3 To waste 1 W I 3 4 i 5 OEt OEt Controlled I I I glass I pore ?OH + EtO-Si-(CH&NH - C P a k ( C H z ) a N H OEt OEt (4 OEt H I I I OEt (B) A + OHC-(CH2)rCHO - CPai-(CH&-N=C-(CH2),-CHo OEt H I I C PG-o-sii( CH,)s-N = C -. (CH&- CH =N I alga B + H\N$ H / alga - OE I t Fig. 1 Reaction scheme for the covalent immobilization of the alga on controlled pore glass where ci is the initial concentration of metal ions and cf is the concentration after equilibration (in mol dm-3); m is the mass of controlled pore glass (in g) and v is the volume of solution (in cm3).Fig. 2 Electron micrograph of Selenestrum capricornutum immo- bilized on controlled pore glass coloration was observed. Then the activated glass was washed with water. A 0.5 g amount of the activated CPG was added to the 25 ml of alga in phosphate buffer (pH 6). Nitrogen was bubbled through the solution at 10 min intervals for the first hour of the immobilization process the reaction was left under nitrogen for a further hour at room temperature (with stirring) and then the solution was stored at 4” C for 2 d. The resulting immobilized algal cells were then filtered and air dried. The immobilized alga was observed by scanning electron microscopy to investigate theeffect ofthe chemical processon the integrity of the cells. As can be seen from Fig. 2 the alga was successfully immobilized and in general the cell walls were still intact showing the characteristic half-moon shape.Instrumentation and Preconcentration Procedure The flame atomic absorption spectrometer was a Varian Model AA75 and was used with an air-acetylene flame. Table 1 shows the wavelengths used for the different elements. Results were recorded on a chart recorder (Chessell Model BD4040). The flow injection (FI) manifold is shown in Fig. 3. It consisted of a peristaltic pump (Ismatec Minipuls SA 8031) and a rotary polytetrafluro- ethylene (PTFE) valve (Rheodyne 5020). All connections were made with 0.8 i.d. PTFE tubing. Two 3-way valves (Omnifit Anachem) were utilized the second being in- cluded in an effort to minimize the amount of buffer passing into the flame.The immobilized alga was packed into a glass tube 5 cm long x 2.5 mm i.d. In the procedure volumes of up to 5 ml of metal ions in buffer solution were passed through the minicolumn which was then washed with water. The accumulated ions were then released by injection of acid and transported to the flame for detection by atomic absorption spectrometry (AAS). Table 1 Conditions for preconcentration-AAS Wavelength/ Metal ion nm PH cu* + 324.8 7.5 Zn2+ 21 3.9 7.5 coz+ 240.7 8.0 Hg2+ 253.7 6.5 Cd2+ 228.8 8.5 PbZ+ 2 17.0 5.5 Evaluation of Exchange Capacity In order to evaluate the exchange capacity of the immobil- ized alga for different metals a 25 ml aliquot of a 0.025 mol dm-3 metal standard (either Cu2+ Pb2+ Zn2+ Coz+ Hg2+ or Cd2+) in the appropriate buffer was added to 0.1 g of the immobilized alga.The mixture was then allowed to equilibriate for 16 h at room temperature while stirring after which the solid was filtered off and the metal ion concentration of the supernatent liquid was determined. Fig. 3 FI manifold for the preconcentration of metal ions by the immobilized alga 1 3-way valve; 2 peristaltic pump; 3 injectionJOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY DECEMBER 199 1 VOL. 6 645 Results and Discussion The effect of the pH of the buffer solution on the ability of the column to preconcentrate was investigated for the different metal ions (Fig. 4). Solutions of 0.1 mol dm-3 phosphate buffer and TRIS [ 2-amino-2-(hydroxymethyl)- propane-1,Idioll buffer were used to cover a pH range of 4-10. As can be seen in Fig.4 for Cu2+ Hgz+ and Pb2+ the highest sensitivity was obtained in phosphate buffer at pH values of 7.5 6.5 and 5.5 respectively. The highest sensitivities for Zn Co and Cd were obtained for TRIS buffer at pH values of 7.5 8 and 8.5 respectively. The other main factor affecting the preconcentration technique is the eluent acid that releases complexed ions from the algal surface. The concentration of acid must be limited to the lowest possible level in order to prevent degradation of the biomass. Fig. 5 shows the effect of acid concentration on the absorbance signal for hydro- chloric acid identical results were obtained for nitric acid. A 100 pl volume of 0.5 mol dm-3 acid was required to elute Cu2+ Zn2+ and Pb2+ completely. The Co2+ was not fully eluted until 100 p1 of 1 mol dm-3 acid were used whereas Cd2+ only required 100 pl of 0.1 mol dm-3 acid (sulphuric acid was also found to be suitable in this instance). Mercury was more problematic in that lower sensitivity and peak broadening with tailing was observed when it was eluted with acid (0.1-3 mol dm-3 nitric or hydrochloric acid).In order to overcome this problem thiourea was dissolved in the 100 pl of 0.1 mol dm-3 hydrochloric acid eluent so that the solution was 0.1 mol dm-3 in thiourea; the thiourea released the Hg from the column by forming a strong complex with it. The parameters affecting the FI system were also investi- gated such as flow rate and the length of the preconcentra- tion column but these factors were found to be negligible in terms of dispersion compared with the effect of the nebulizer.12 \ F k 1 1 1 1 1 1 0 1 2 3 4 5 6 7 8 9 1 0 PH Fig.4 Effect of pH on metal ion uptake A 500 ng ml-1 PbZ+; B 100 ng ml-1 Zn2+; C 80 ngml-1 Cd2+; D 50 ngml-1 Cu2+; E 250 ng ml-1 Co2+; and F 5000 ng m1-I Hg7+ Calibration Fig. 6 shows a typical calibration graph and the series of calibration traces obtained for each individual element using the procedure described with the conditions as established above. The figures of merit are given in Table 2. The limits of detection were compared with the limits of detection obtained without preconcentration by calculating the en- hancement factor. As can be seen from the Table 2 the alga was effective at preconcentrating all the elements investi- gated but was particularly good for Cu2+ ZnZ+ and Cd2+.Capacity and Recovery The effectiveness of the alga in preconcentrating metal ions was assessed further by measuring its capacity by the batch method described under Evaluation of Exchange Capacity. The results given in Table 3 show that the alga has a high uptake capacity for Cu2+ Zn2+ Cd2+ and Pb2+. The uptake for Co2+ and Hgz+ was lower. Some of these values are high compared with those obtained for chemical preconcentra- tion reagents such as silica-immobilized 8-hydroxyquinoline (230 pmol g-l for Cu2+) and 8-hydroxyquinoline-5-sul- phonic acid (6.0 mmol g-l for Cd2+).9 The recovery of the metal ions was found by comparing the signal obtained with direct injection with that obtained after preconcentration and elution with the appropriate volume and concentration of acid (Table 3).The recoveries obtained were good with only Coz+ showing a low recovery. The lifetime of the immobilized alga was 3 months if stored below 4 "C whilst not in use. Interferences Table 4 shows the interference effects of high concentrations 200 160 a 6 120 .- Q) Y m 80 2 40 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 Acid conce nt rat io n/m ol d m-3 Fig. 5 Effect of hydrochloric acid concentration on elution of metal ions A 1 10 ng ml-1 Zn2+; B 60 ng ml-l CuZ+; C 250 ngml-1 Co2+; D 5000 ng ml-l Hg2+; E 60 ng ml-I Cd2+; and F 250 ng ml Pb2+ 160 140 E 120 E 2 100 m .- 2 80 60 40 20 Y L I 1 1 L I I . 1 0 10 20 30 40 50 60 70 80 90 100 Concentration of metal ion/ng ml-' [~n'+l/ng mi ' Fig. 6 Calibration graph for a series of metal ion standards and the calibration trace for Zn ions A Co2+ ( x 10); B Pbz+ ( x 10); C Cu2+; D Hg2+ (x 100); E,CdZ+; and F Zn2+646 JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY DECEMBER 199 1 VOL.6 Table 2 Analytical performance of the on-line preconcentration FI-AAS system for 5 ml of sample (sampling rate 20 h-l) Parameter cuz+ Zn2+ co2+ Hg2+ CdZ+ Pb2+ Linear rangel ng ml-a 5-45 10-100 55-300 650-5000 15-80 60-450 Correlation coefficient 0.9992 0.9998 0.9997 0.9988 0.9995 0.9999 RSD* (%) ( n = 5 ) 1 .5( 40) 1.3( 100) 1.4(250) 1.2(4000) 1 .5( 30) 1.1(300) Detection limit?/ Enhancement ng ml-1 0.05 0.2 factor+ 4000 lo00 8 30 2 2.5 50 167 500 80 *Values in parentheses are the concentrations of the metal ions in ng ml-I. t2a value. $Factor by which the limits of detection decrease using preconcentration as opposed to direct injection.Table 3 Exchange capacity and recovery for the immobilized alga Metal uptake Recovery Metal ion capacity/mol g-' (%I effect is also seen for Pb2+ interference with Hg2+ preconcen- tration. This behaviour needs further study but might be due to the release of ions that have not been completely recovered from previous determinations. cu2+ Zn2+ co2+ HgZ+ Cd2 + Pbz+ 9.70 8.36 2.52 1.60 11.70 11.45 1 00 98 85 96 102 96 ~~ (20 pg ml-*) of different metal ions on the preconcentration process for particular metal ions. From the table it can be seen that the interference effects for Cu2+ Pb2+ and Zn2+ are generally insignificant considering the high level of interfer- ing ions present with Co2+ and Hg2+ being affected to a greater extent.The interference effect can be explained by the differences in strength of the different ion complexes formed with the algal cell wall. Once all of the binding sites of a chemically immobilized chelating reagent are occupied then those ions that are less strongly bound can be displaced by interfering ions present in excess thus depressing the final absorbance rnea~urement.'~ This process is more complex for the alga because there is more than one type of binding site present and more selective binding occurs. From the results obtained for the interferences the relative affinities of the different metal ions for Selenestrum capricornutum in decreasing order are Hg>Cu>Pb>Zn>Co>Mg. Mercury was bound most strongly and could only be removed by introduction of the thiourea ligand to the eluent.The behaviour of Cd was anomolous. The Co2+ Hg2+ and Mg2+ ions do not interfere in the preconcentration of Cd2+ but an enhancement of the absorbance is seen as a result of the interference of Cu2+ Pb2+ and Zn2+. This enhancement Table 4 Interference effects of high concentrations of different interfering ions (20 pg ml-l) on the preconcentration process for particular metal ions Metal ion& ml-I cu2+ 100 Interfering ion cu2+ - Zn2+ -2.8 co2+ -0.0 Hg2+ -0.0 Cd2+ -0.0 Pb2+ -0.1 Mg2+ -0.0 Zn2+ Co2+ HgZ+ Cd2+ 200 200 5000 80 Change in peak height (%) -11.1 -50.0 -18.0 +lo7 - - 55.0 -27.0 + 100 -3.8 - -5.5 -0.0 -25.0 -0.0 - -0.0 -2.3 -36.0 -27.0 - -3.8 -22.7 +45.5 +lo7 -0.0 -0.0 -0.0 -0.0 Pb2 + 500 - 0.0 -0.0 -0.0 - 57.9 -2.8 - 0.0 -0.0 Conclusions The immobilized alga is shown to be an effective reagent for the quantitative preconcentration of a number of trace metals.The covalent immobilization procedure was success- ful with the column retaining activity for 3 months if stored at below 4°C when not in use. In addition to being a useful analytical reagent the immobilized alga can be used for general studies of the accumulation of metals on algal cell walls allowing rapid flow through studies. Further work will investigate the use of the method for real samples. Dr. R. Goulder and S. Lythe of the Department of Applied Biology University of Hull are thanked for cultivating the alga used in this work. 1 2 3 4 5 6 7 8 9 10 1 1 12 13 References Shumate S. E. 11 Strandberg G. W. McWhirter D. A. Parrott J. R. Bogacki G. M. and Locke B. R. Biotechnology and Bioengineeringsymposium No. 10 Wiley New York 1980 p. 27. Greene B. Hosea M. McPherson R. Henzel M. Alexander M. A. and Darnell D. W. Environ. Sci. Technol. 1986,20,627. Mahan C. A. Majidi V. and Holcombe J. A. Anal. Chem. 1989,61 624. Crist R. H. Oberholser K. Shank N. and Nguyen M. Environ. Sci. Technol. 1981 15 1212. Gardea-Torresdey J. Darnell D. and Wang J. Anal. Chern. 1988,60 72. Trevan M. D. and Mak A. L. Trenh Biotechnol. 1988,6,68. Dagnell D. W. Greene B. Henzel M. Hosea M. McPherson R. A. Sneddon J. and Alexander M. D. Environ. Sci. Technol. 1986 20 206. Darnall D. W. Gabel A. US EPA Res. Dev. [Rep.] EPA EPA/600/9-89-072 Int. Conf New Front. Hazard. Waste Manage. 3rd 1989 pp. 217-225. Devi S. Habib K. J. andTownshend A. Quim. Anal. 1989,8 159. Masoom M. and Townshend A. Anal. Chzm. Acta 1984,166 111. Chu S. P. J. Ecol. 1942 30 284. Tyson J. F. Appleton J. M. H. and Idris A. B. Anal. Chim. Acta 1983 145 159. Bysouth S. R. and Tyson J. F. Anal. Chim. Acta 1988,214 329. Paper I /02492B Received May 28th I991 Accepted July 22nd 1991