Anal. Chem. 1994,66, 3632-3638
Immobilized Cyaaobacteria for On-Line Trace Metal Enrichment by Flow Injection Atomic Absorption Spectrometry Angel Maqulelra,' Hayat A. M. Elmahadl, and Rosa Puchades Department of Chemistry, Polytechnic University of Valencia, Camino de Vera s/n, 4607 1, Valencia, Spain Cyanobacteria(Spirulinaplatensis) immobilizedon controlled pore glass preconcentrate Cu(II), Zn(II), Cd(II), Pb(II), and Fe(II1) from aqueous solution with high efficiency as ascertained using an on-line flow injection atomic absorption spectrometrysystem. The degree of metal binding depends on the pH of the solution. Quantitative retention of copper, zinc, and cadmium occurred at a wide range of pH values, while the retention for lead and iron was pH-dependent. The latter metals were adsorbed strongly only at pH 6 and 7, respectively. The breakthrough capacity was determined from the breakthrough curve, with values of 0.0035,0.0008,0.0011,0.0028, and 0.0017 ng/mL for Cu, Zn, Cd, Pb, and Fe, respectively,being obtained. The analysis of a certified reference sample, sewage sludge of domestic origin (BCR No. 144), for cadmium and copper with a high accuracy ensures the feasibility of this technique for environmental analysis. In spite of the great improvements in sensitivity and selectivity of modern analytical detection systems, separation techniques such as precipitation, solvent extraction, ionexchange, formation of volatile compounds, electrochemical means, chromatography, etc. are still frequently used to overcome matrix interference and/or to enhance sensitivity through preconcentration of the analyte. Manually operated separation procedures are usually tedious, involve high sample and reagent consumption, and are susceptible to contamination. Obviously, improvement in the efficiency of the separation step will greatly contribute to the overall efficiency of such methods. As an interesting concept in solution handling, flow injection (FI) analysis has much to offer in this respect. The advantages of these techniques for different chemical methods' have been summarized. FI provides a novel approach to on-line sample preconcentration: matrix removal with traditional chemical separation procedures being scaled down for the purpose.2 Most studies have utilized microcolumns packed with chemical chelating or ion-exchange materials, and their developments in conjunction with FI-AAS have been pioneered by Olsen et al.3 Luque de Castro4 has reviewed the use of solid-phase reactors coupled on-line to FI which significantly expand the potential of this technique either by enhancing such basic (1) Clark, G. D.; Whitman, D. A.; Christian, G. D.; Ruzicka, J . Crit. Rev. Anal. Chem. 1990, 21, 357. (2) Valchrcel, M.; Luque de Castro, M. D. J. Chromatogr. 1987, 3, 393. (3) Olsen, S.;Pesenda, L. C. R.; Ruzicka, J.; Hansen, E. H. Analyst 1983, 108,
905. (4) Luque de Castro, M. D. Trends Anal. Chem. 1992, 1 1 , 149.
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Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
analytical parameters as sensitivity, selectivity, and the implementation of specific reactions (e.g., enzymatic, immunoassay, ion-exchange, chromatographic supports, or redox) or by acting as solvent extractants or reagent releasers. An alternative approach is to immobilize the microorganism cell wall on an insoluble substrate which has multibinding sites in order to achieve both selectivity and sensitivity. Living organisms adsorb trace metals from aqueous solution, and hence they engage in an intricate balancing act in their interactions with heavy metals. Metal ions such as Pb(II), Cu(II), Zn(II), Cd(II), and Fe(II1) may bestrongly adsorbed on the cell surface of algae,s.6 fungi,' bacteria,8 and yeast^.^ The uptake of metals by algae and other organisms has been of interest for a variety of reasons, including concern over potentially toxic trace metal accumulation in the food chain, metal recovery technique from process and industrial streams, and contaminated water treatment.'O," Several different cell wall constituents have been implicated in metal binding, although ionic binding has been often assumed. Christ et a1.I2 have suggested the existence of covalent binding for some metal ions, while the exact nature of the binding sites probably varies with the cell wall composition of the organism. The principle of ion-exchange seems to be involved with any microrganisms for which heavy metals binding has been reported.I3 The identification of the particular cell wall constituents responsible for metal adsorption is under study by several researchers. Some authors14 utilized N M R spectrometry to investigate alga-Cd binding. The chemical shift observed for the bound Cd strongly suggested a carboxylic group attachment. OthersI5 studied five different alga species and monitored the metal adsorption before and after carboxylic esterification. The results suggest that carboxylicgroups were ( 5 ) Elmahadi, H. A. M.; Greenway, G. M. J.Anal. Atom. Specfrom.1991,6,643. (6) Gardea-Torresdey. J.; Darnall, D.; Wang, J. Anal. Chem. 1982, 60, 72. (7) Galun, M. P.; Malki, D. F.; Feldstein, H.; Galun, E., Sicgel, S.M.; Siegel, B. 2. Water. Air, Soil Polluf. 1984, 21, 411. (8) Treen-Seans, M. E.; Volesky, B.; Neufeld, E. J. Biorechnol. Bioeng. 1984.26, 1323. (9) Nakajima, A.; Sakaguchi, T. Appl. Microbiol. Biotechnol. 1986, 24, 59. (IO) Darnall, D. W.; Greene, B.; Henzl, M. T.; Hosea, J. M.; McPherson, R. A.; Sneddon, J.; Alexander, M. D. Enuiron. Sci. Technol. 1986, 20, 206. (11) Mahan, C. A.; Majidi, V.; Holcombe, J. A. Anal. Chem. 1989, 61, 624. (12) Crist, R. H.; Oberholser, K.; Shank, N.; Nguyen, M. Enuiron. Sci. Technol. 1981, 15, 1212. (13) Greene, B.; Henzl, M. T.; Hosea, G. M.; Darnall, D. W. Biotechnol. Bioeng. 1986, 28, 764. (14) Magdi, V.; Laude, D. A.; Holcombe, J. A. Enuiron. Sci. Technol. 1990.24, (3),.337. (1 5) Gardea-Torresdey, J. L.; Beaker-Hepak, M. K.; Hosea, J. M.; Darnall, D. W. Enuiron. Sci. Technol. 1990, 24, 1372.
0003-27OOf94/036&3632$04.50/0
0 1994 Amerlcan Chemical Society
involved in Cu and A1 binding but were not responsible for Au binding. Watkins et a1.16 utilized X-ray absorption nearedge spectroscopy (XANES) and extended X-ray absorption fine structure spectroscopy (EXAFS) to study Au binding to an alga. Their results showed that Au(1) was bound to S and/or N atoms and Au(II1) was bound differently from Au(I). It appears that at least two (and possibly several) different functional groups on the algal cell wall may be responsible for metal adsorption. The number and type of binding sites may also be metal-dependent. Other workers" studied the utility of alga biomass to enhance the analytical sensitivities for trace metal spectroscopic analysis. Parts per trillion (ng/L) and parts per billion (ng/mL)18J9 sensitivities have been achieved for some trace metals using alga cells for preconcentration involving a batch method. A potential disadvantage of this batch procedure is the need for multiple extractions of metals having low partition coefficients. Some authors have entrapped alga cells in a copolymer matrixZoor encapsulated them in a polyacrylamide ge1,21,22while others have immobilized the microorganisms in polysulfone beadseZ3 Darnall et al.24reported their results from using silica gel to immobilize alga cells for biotechnological applications, although the procedure of immobilization was not given. Mahan and H ~ l c o m b ehave ~ ~ reviewed some of the methods of immobilization of microorganisms based on physical adsorption, and they also reported a method of immobilization of algae using silica gel by physical adsorption in a batch mode of preconcentration, which was time-consuming. Another disadvantage is that the use of silica gel, although readily modified by a variety of silanizing agents allowing for a myriad of functional groups to be immobilized, is limited to pH < 9 due to the hydrolysis of the silica framework in basic media. An additional drawback of using silica has been the relatively low exchange capacity of many silica-based chelating agents. Controlled pore glass (CPG) is a special type of support. It is formed by phase separation of homogeneous borosilicate glass followed by dissociation of the boron-rich glass phase by strong acid, leaving a highly porous silica-rich glass. It does not swell or shrink upon change of solvent composition, so it is particularly suitable for this type of application. Recently, covalent immobilization of microorganisms has been reported using CPG as a up port.^ The immobilized algae were packed in a minicolumn, and FI was used for handling the sample. The present work is based on the use of bacteria as a microorganism. It investigates the success of the covalent (16) Watkins, W.; Elder, R. C.; Greene, E.;Darnall, D. 26,
1147.
W. Inorg. Chem. 1987,
(17) Shengjun, M.; Holcombe, J. A. Talunru 1991, 38, 503. (18) Magdi, V.; Holcombe, J. A. J . Anal. Atom. Specrrom. 1989, 4, 439. (19) Shengjun, M.; Holcombe, J. A. Anal. Chem. 1990,62, 1194. (20) Harris, P. 0.;Ramelow, G. J. Enuiron. Sei. Technol. 1990, 24, 220. (21) Nakajima,A.;Horikoshi,T.;Sakagyshi,T. Eur. J.Appl. Microbiol.Biotechnol. 1982, 16, 88.
(22) Zimnik, P. R.; Sneddon, J. Anal. Lerr. 1988, 21, 1383. (23) Jeffers, T. H.; Ferguson, C. R.; Seidel, D. C. Bureau of Mines in House Publication, 1989. (24) Darnall, D. W.;Greene, E.; Hosea, M.; Macpherson, R. A.; Henzl, M.; Alexander, M. D. In Trace Mefal Removal From Aqueous Solutions; Thompson, R., Ed.; Royal Society of Chemistry: London, 1986; Special Publication No. 61, p 1. (25) Mahan, C. A.; Holcombe, J. A. A n d . Chem. 1992, 64, 1933. (26) Waterburg, J. E.TheCyanobacteria. Isolation,Purification,and Identification. In The Prokaryotes, 2nd ed.; Balows, A,,Truper, H. G., Dworkin, M., Harder, W., Schleifer, K. H., Eds.; Springer-Verlag: New York, 1992; Chapter 77, pp 2058-2078.
immobilization of bacteria on CPG and explores the system's binding capabilities for trace (Cd, Cu, Fe, Pb, and Zn) metal enrichment prior to flow injection the system's atomic absorption analysis.
EXPER I MENTAL SECTION Reagents. Doubly distilled deionized water was used throughout, and all the chemicals were analytical grade. Spirulina platensis,26a mixed cyanobacteria species, Sigma reference S9134, CPG (PG 240-200, pore diameter 242 A, mesh size 120-200), aminotriethoxysilane, and glutaraldehyde (50%v/v) were from Sigma. Since the latter reagent is toxic and an irritant to skin, all manipulations were carried out in a fume cupboard. Stock solutions of cadmium, lead, zinc, copper, and iron, 1000 pg/mL, were obtained from Titrisol Merck. Working standards were made by a serial dilution of an appropriate volume of a 100 pg/mL stock solution with a recommended volume of water or buffer to obtain the chosen concentration. The phosphate buffer was prepared from 0.1 M sodium dihydrogen orthophosphate, with pH being adjusted with sodium hydroxide to cover a pH rangeof 5.0-9.5. HCl(36%), HNO3 (68%), and HC104 were from Panreac. All glassware was soaked overnight in 10% H N 0 3 before being washed and used. A reference sample material (sewage sludge of domestic origin), BCR No. 144, was obtained from the Community Bureau of References, Belgium. Preparation of the Reference Sample Material Solution. Duplicate samples of 2 g of the reference sample material were weighed each in a separate beaker (50 mL). To each beaker was added 10 mL of a mixture of concentrated HCl and HNO3 (3:l). Both beakers were covered with clinical test ware film and left overnight. The beakers were then put on a hot plate (1 50 "C) for 2.5 h, after which they were allowed to cool to room temperature. To each beaker was added 25 mL of water. The digested material was filtered through Whatman No. 5 filter paper in a 50-mL volumetric flask and washed with cold HNO3 (2 M). The volume was made up to 50 mL with water. A 25-mL aliquot of each sample was then taken and adjusted to pH 7.0 with NaOH prior to being made up to 50 mL with phosphate (pH 7.0) for the determination of copper. The same procedure was carried out for the determination of cadmium, but the sample volume was made up to 50 mL with a pH 7.5 phosphate buffer. Immobilization of Cyanobacteria on CPG. The bacterial solution was prepared by weighing 0.02 g of a sample of cyanobacteria into a small beaker (25.0 mL). NaOH pellets (0.5 g) were dissolved in 25 mL of water and warmed to 6070 OC. This solution was added with stirring to the small beaker containing the bacteria. A clear bacterial solution was obtained. This solution was carefully adjusted to pH 7.0 with 1 M HCl (as in acidic media, turbidity is observed). A 10-mL aliquot of this solution was diluted up to 25 mL with the phosphate buffer (pH 7.0). The CPG was activated by boiling 0.5 g of it with 5.0-mL aliquot of 5% v/v nitric acid for 30 min. After filtration through porous sintered glass (GC3), the filtrate was washed with water and dried in an oven at 95 "C for 2 h. The CPG was then silanized with aminotriethoxysilane (ATEOS) solution and treated with glutaraldehyde to prepare the CPG Analytical Chemistry. Vol. 66,No. 21, November 1, 1994
3033
~ ~ ~ ~ r o 'Fl d
H + EtO-{i
glass
-
OEt -(CH,),NH,
OEt CPG-o-Si-(CH,),NH, I
I
OEt
(CPG)
A + OHC - ( C H 2 ) , -CHO
OEt
-A-
-
OEt
H
I
I
CPG-O-Si+CH,),-N=C-(CHZ),4HO
I
OEt
'
B
-
OEt bacteria -CPG-O-Si-(CH,),-N=:-(CH
I
B + l>N+
K
I
I
) -CH=N 2 3
OEt
I bacteria
Figure 1. Reaction scheme for the bacteria immobilization process.
glutaraldehyde derivative as described previ~usly.~The treated CPG was washed with water and transferred to a 25-mL beaker, to which 25-mL of cyanobacterial solution was added. The beaker was then flushed with nitrogen for 10 min. The mixture was kept for 2 days at 4 OC, filtered, and air-dried. The immobilized cyanobacteria were packed in a minicolumn. The immobilizationof cyanobacteria, which is illustrated in Figure 1, was a modified from the processes used for the immobilization of SeZenestrum capricornutum on CPG5 and for enzyme imm~bilization.~'The differences lie mainly in the method of dissolution of the chelating agent and the time of coupling of bacteria with the activated CPG. Scanning Electron Photomicrograph of Immobilized Bacteria. The immobilized cyanobacteria were observed with a scanning electron microscope to investigate the completeness of immobilization (Figures 2A-E). Figure 2A shows a general view of the spreading of the bacteria on the surface of CPG particles. The high-density coverage in some areas but incomplete or empty surface in other areas revealed that it may be possible to immobilize a greater quantity of bacteria cells by improving the method of immobilizationin the future. This could result in an increase in the adsorbable surface area of the material and hence in increased adsorption capacity. Figure 2B shows a morphological comparison between an empty or incomplete CPG with other completely covered particles. Figure 2C shows a closer view of the immobilized bacteria, and in some areas the bacteria tend to cluster together. Figure 2D shows an even closer view which reveals that there is a great density in bacteria cells, with some of the cell wall material appearing to overlap. Figure 2E shows the large surface contact area existing between the CPG and the bacteria. As reported by some authors,25the overlapping of the cell wall and the large surface contact evident in these photomicrographs suggest that either some of the metal ion adsorption sites on the bacteria have been lost by the immobilization process or there may be failures in the process, thereby reducing the adsorption capacity in comparison with the same mass of the free microorganism. A comparison of the binding parameters (such as formation constant, density of adsorption sites, etc.) of unimmobilized bacteria with those of immobilized bacteria should provide some information on the binding sites suspected to be lost by the immobilization process. Research is currently in progress in this direction. Capacity Study. For the determination of the column capacity, 2-mL aliquots of a series of concentrations (2-25 (27) Massoom, M.; Townshend, A. Anal. Chim.Acta 1984, 160, 11 1.
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Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
Flgure 2. Electron photomicrographs of S. platensis immobilized on CPG at different magnifications. (A) a general view of the spreading of bacteriaon CPG; (B) comparison betweencompletely covered CPGbacteriawith uncoveredone; (C) a closer view of immobilizedbacteria: (D) an even closer view which revealsthe density in bacteria cells; and (E) detailed comparison between the surfaces of CPG and bacteria.
~
-
~~~~
Table 1. Estlmatlon of the Dispersion Coefficient for Different Metal Ion Concentratlons with FI/AAS
element
concentration,fig/mL
dispersion coefficient
Cu(I1) Zn(I1) Cd(I1) Pb(1I) Fe(II1)
4.0 6.0 5.0 14.0 10.0
2.90 2.80 2.70 2.68 2.70
cu
/
zn
0 -
u e
m
f! 0
0,21-
v)
n
a pg/mL) made at the appropriate pH were preconcentrated and eluted. The amount of metal adsorbed at each concentration level was determined from the following equation: C,cV
4
5
7
6
8
9
10
W
PH Flgure 3. Effect of buffer pH on the absorbance signal. Metal
Where C is the capacity, c is the concentration (in pg/mL) of metal eluted, u is the volume (in mL) of metal used, and w is the weight (in g) of the immobilized material. Evaluation of breakthrough capacity was made from a breakthrough curve by plotting the total metal concentration (mg/L) vs the millimoles of metal adsorbed per gram, as will be discussed later. Instrumentation. A Perkin-Elmer Model 2380 atomic absorption spectrophotometer was used. A Perkin-Elmer recorder 56 was used to record the signal. The air:acetylene ratio was 229. No background correction was used. Details of the flow injection manifold have been described elsewhere.28 The immobilized bacteria were packed in a home-made minicolumn (5 cm long and 2.5 mm i.d.).
RESULTS AND DISCUSSION Flow Injection Atomic Absorption Spectrometry. The manifold and results obtained for the direct injection of 100 r L of the standard metal ion solutions studied here, including the equation of the regression line, the correlation coefficient, the relative standard deviation, and the detection limit (pg/ mL), have previously been shown.28 The dispersion coefficient was determined by comparing the absorbance of a known concentration of metal ions with direct aspiration with that after injection of 100 pL of the same concentration of metal ions in a carrier stream of water. The results have been presented in Table 1. From these results, it is evident that there is a little dispersion in the direct injection system, which has no influence in this work, as FI is used only as a means to transport the sample to the detector via a column reactor. Optimization Study. Among the parameters that affect the preconcentration process, pH and the concentration of eluent were the most important. Optimization for the effect of flow rate on the preconcentration procedure has been carried only to copper solution. It was found that flow rate has a slight effect on the preconcentration process. For 0.7, 1.4, and 2.1 mL/min, theabsorbances were0.255,0.258, and0.260, respectively. On the basis of this result, a flow rate of 2 mL/ min was used in this work. Figure 3 shows the effect of pH on the ability of S.platensis to bind metal ions, which was assessed by the change in the absorbance signal in the pH range 5.0-9.5. All the metals tested, with the exception of (28) Maquieira,A.; E1mahadi.A. M. H.; hchades, R. AndChem. 1994,66,1462.
concentrations in ng/mL, Cu(I1) Pb(I1) = 1000; Fe(II1) = 400.
=
100; Zn(I1) = 80; Cd(I1)
= 200;
Pb(I1) and Fe(III), were weakly bound below pH 7.0 and strongly bound above it, while Pb(I1) and Fe(II1) were bound strongly at pH 6.0 and 7.0, respectively, and weakly above these values. The maximum uptake for Cu(II), Zn(II), and Cd(I1) occurred at pH 7.0,8.0, and 7.5, respectively, and above these specified values the absorbance signals remain unchanged, unlike their uptake with the immobilized Sel. cupricornutum under similar conditions,5 in which a sharp absorbance maximum occurred only at a specified pH value. This suggests that different binding sites may be involved in the process of binding.’ The decrease of the absorbance signal for lead and iron above a certain pH value may reveal that the biosorption of these cations occurs via an ion-exchange process, with metal cations competing with protons for negatively charged binding sites of the cell wall. Although the binding is pH-dependent, there are significant differences in behavior. For example, at pH 5.0, nearly 30% of Pb was bound under the conditions of the experiment, while lower amounts of Cd (1 1%) and Zn (1 7%) were bound. This variation should permit a selective elution of bound metal ions with this immobilized reagent by a judicious choice of pH, which will be investigated in the future. Note that the five metal ions tested here essentially bind to the cell wall of microorganisms at pH > 5.0, do not bind at pH < 2.0, and are, in fact, eluted by nitric acid below pH 2.0. This was found to be consistent with the metal cation being the species which is bound to the ligand on the cell wa11.24 The elution of the adsorbed metal ions could be achieved by using an appropriate eluent solution capable of effectively stripping them from the biomass and bringing it into the solution. In this work, nitric acid was used. Figure 4 shows the effect of eluent concentration on the metal recovery from the column. For each concentration level, a 1OO-pL aliquot of acid was used. It was observed that 0.5 M HN03 was enough for the complete elution of Cd(I1) and Pb(I1). Zn(I1) needs only 0.1 M acid, while Cu(1I) required at least a 1.5 M concentration to achieve a complete elution. Fe(II1) was more problematic and was not completely eluted until a mixture of 1.0 M HN03/0.1 M HCl was incorporated into Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
3835
Q
0
c m n ~
z
’1
I
0.2
v)
n
a
0
3
2
1
0
-1
Nitric acid concentration/moi L
(1OOpL)
Flgure 4. Effectof eluent ( 100pL)acid concentration on the absorbance signal. Concentrations in ng/mL, Zn(I1) = 80; Cu(I1) = 100;Cd(I1) = 200;Fe(II1) = 400;Pb(I1) = 500.
0
200
400
600
800
Concentration of metal ion/ng mLFlgure 5. Calibration graphs for a series of standard metal ion solutions in ng/mL; x-axis, X50 for Pb, X0.5 for Cd; y-axis, 1 mm = 0.0042 for both Cu(I1) and Zn(I1).
100 pL to recover it from the column. This suggests a much stronger binding of Fe(II1) with the cell wall of bacteria. Preconcentration and Analytical Measurements. Aliquots (5 mL) of a series of buffered standard metal ion solutions were passed through the column, preconcentrated, and eluted at the above established conditions.28 The calibration graphs, which are shown in Figure 5 , are linear, the correlation coefficients being 0.9998,0.9994,0.9998,0.9999, and 0.9996 for copper, zinc, iron, cadmium, and lead, respectively. The relative standard deviation (RSD) was less than 3% at concentrations of 200, 100, 400, 200, and 1000 ng/mL for copper, zinc, iron, cadmium, and lead, respectively. In a recent some authors described Donnan dialysis preconcentration in terms of signal enhancement factors (SEFs) as opposed to enrichment factors (EFs). They stated that “the E F is the ratio of the concentration of the ionic species of interest after dialysis in the receiver to the initial concentration in the sample and thus describes the exact level of preconcentration. The SEF is the ratio of the peak signal (29) Kasthurikrishnan, N.; Koropchak, J. A. Anal. Chem. 1993, 65, 1857.
3638 Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
for on-line Donnan dialysis to the steady-state signal resulting from direct aspiration at the optimized flow rate of that of that particular intrument (ICP-AES)”. In an ideal case, the two factors are similar, but with ICP-AES and a flow injection Donnan dialysis combination, matrix effects, dispersion, and spectral interferences may cause the SEF to differ from the EF.29 In this work, approximately similar values were obtained for the tested metal ions, and in our view the reason could be that AAS has the minimum spectral interferences compared to the emission methods (AES). Note that in this work the FI system was used only to transport the sample into the detector via a column reactor, and a minimized dispersion was obtained by injecting the sample into a manifold having the shortest possible distance between the injection port and the detector. As confirmed by Tyson30and in another similar study,31the contribution of the column to the dispersion was negligible compared to the dominant effect of the nebulizer in this respect.30 Two factors were used to assess the improvement obtained with preconcentration. The first was the improvement in detection limit, Le., the enhancement factor (EF), by which the detection limit was decreased using preconcentration as opposed to direct injection. The other measurement was the improvement in the sensitivity, which was calculated by comparing the slope of the calibration graph of preconcentration with that obtained with direct injection. Table 2 shows the analytical data which present the detection limit before and after preconcentration, the improvement in sensitivity, and the enhancement factor. As can be seen from the table, cyanobacteria were found to be very effective for the preconcentration of all metals, and lower detection limits were obtained. The improvement in the sensitivity was maximum for Cu and Zn, based on quantitative adsorption, elution, and a concentration factor of 50 ( 5 mL of sample concentrated in 100 pL of acid), probably due to their strong affinity for the coordinating group on the cell wall of the bacteria. However, the sensitivity of atomic absorption spectrometry for all tested metal ions was improved very much, and a lower detection limit was obtained. Moreover, in this investigation, higher enhancement factors were obtained for all tested metal ions compared to those obtained with other chemical chelating reagents in a similar study.31 Also, sub-parts per billion levels could be obtained by increasing the sample volume, and hence higher enrichment factors could be achieved. Capacity and Recovery Studies. The ion-exchange capacity for the column depends on the quantity of the resin in the column and the nature of the ion-exchange material used. The breakthrough capacity of the resin is defined as the amount of metal ions that can be extracted per unit mass under the operating conditions prevailing prior to being detected in the column effluent.32 This breakthrough capacity was used in this work to evaluate the amount of metal adsorbed onto the coordination sites of the immobilized cyanobacteria, since there is more useful than the apparent capacity when lower levels of metal ions were used. As described in the Experimental Section, the breakthrough capacity was evaluated from a (30) Tyson, J. F.; Appleton, J. M. H.; Idris, A. B. Anal. Chim. Acta 1983, 145, 159. (31) Devi, S.; Habib, K. A. J.; Townshend. A. Quim. Analltica 1989, 8 , 159. (32) Marhol, M. Ion-exchangers In Analytical Chemistry. Their Properties and Use in Inorganic Chemistry; Elsevier: New York, 1982.
Table 2. Analytlcal Data for Dlred Injectlon and On-Llne Preconcentrrtlon'
DLb
slope
element
based on direct injection (pg/mL)
based on preconcentration (ng/mL)
cu Zn Cd Pb Fe
0.20 0.20 0.05 1.oo 0.45
0.3 0.2 0.4 10.0 0.5
EP
direct injection ("/re)
preconcentration ("/ne)
improvementd in sensitivity
667 1000 125 100 900
15.50 17.23 29.05 4.69 12.15
0.32 0.90 0.91 0.12 0.33
53 52 31 26 27
Sample volume, 5 mL; sampling rate, 24 h-1. DL (detection limit), 2u,,4 for 10 replicate injectionsof the blank. EF is a comparison of DL obtained with direct injection as opposed to preconcentration (DL direct injection/DL preconcentration). Improvement in sensitivity is obtained by comparison of slope obtained with preconcentration to that obtained with direct injection. Table 4. Effect of Dlfferent Interfering Ions'
A
CI)
2 0
5
Y
Pb
ionsb
Cu(I1)
Cu(I1) Zn(I1) Cd(I1) Mg(W Pb(I1)
0.0 0.0 0.0 -8.0
Zn(I1)
Cd(I1)
Pb(I1)
Fe(II1)
-13.0
-07.0 -10.0
0.0 0.0 0.0
-15.0 0.0
0.0
0.0 0.0 0.0 0.0 0.0
-15.0 -20.0 -20.0
'Percent change in peak height for 1 pg/mL metal ion solutions. Interfering ions are in 10 pg/mL concentration. Table 5. Rewtls of the Analysk, of Reference Sample Materlal calculated value' certified value' recovery (%) 0 1
0
I
I
10
20
I 30
Total concentration (pglmL)
Cu(I1) Cd(I1)
0.058 0.035
0.056 0.037
103.57 94.60
In mg/kg.
Flgure 6. Breakthrough curve for the different metal ions investigated. Table 3. Breakthrough Capaclty and Recovery of Dlfferent Metal Ions
metal ion
capacity (mmol/g)
recovery (7%)
Cu(I1) Zn(I1) Cd(I1) Pb(I1) Fe(II1)
0.0035 0.0008 0.001 1 0.0028 0.0017
102.0 100.2 95.00 85.0 98.0
breakthrough curve plot, which is presented in Figure 6. These values were shown in Table 3. From this table, it was found that Cu(I1) has the highest breakthrough capacity, followed byPb(I1) and Fe(II1). Zn(I1) has a lower uptake. However, the breakthrough capacity for this material is higher for Pb(11) than that obtained for immobilized yeast.28 The recovery of the tested metal ions was evaluated by injecting a known concentration of standard metals and comparing it with the results obtained after preconcentration of a more diluted solution containing the same amount of metal ions. Quantitative recoveries were obtained for all metals except Pb, for which the recovery was only 85% (Table 3). Interference. Table 4 shows the effect of various interfering ions (10 mg/L) on the preconcentration process. Up to a concentration of 8 pg/mL of interfering ions, there was no change in the absorbance signal for all metals tested. Interference was encountered only when the concentration of interfering metal ions was up to 10 pg/mL. From the table it can be concluded that there is no interference in the determination of lead, perhaps because this metal ion is
selectively extracted in acidic media, unlike the other competing ions. There is no interference in the determination of Fe(III), which may reveal that its formation constant with this biomass should be higher and thus not easily displaced by other competing ions. This conclusion could also be drawn from its elution behavior, since a mixture of strong acids was required to elute it. The interference in the determination of Cu and Cd is not severeconsidering the high level of interfering ions. The interference could be explained by the difference in the strength of binding with the microorganism cell wall or by thedifference in the formation constant33with the binding sites. Again, the phenomenon is more complex because there is more than one binding site. Analysis of Reference Sample Material. Buffered sample (5-mL aliquots) was preconcentrated as reported in the Experimental Section, using the standard addition method. The results are shown in Table 5 . From this table it is clear that the calculated values for Cu and Cd agreed very well with the certified values.
CONCLUSIONS Bacterial cell walls were successfully immobilized on CPG and used in a minicolumn for trace metal enrichment. The results revealed excellent reproducibility for standard metal ion solutions. The determination of copper and cadmium in sewage reference sample material agreed very well with the certified values. Covalent immobilization on CPG gave a very stable preparation. Concentration of trace metals from 5 mL of sample provided enhancement factors of 100-1000. The relatively low capacity of this material is not a disadAnalytical Chemistty, Vol. 66,No. 21, November 1, 1994
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vantage, provided that its use is restricted to very low metal ion concentrations. Furthermore, the use of S . platensis provides more than two binding sites, so both selectivity and sensitivity could be achieved; additionally, the bacterial solution is cheaper compared to chemical chelating agent. Thecolumn retains its activity for 3 months (6 h of continuous use per day).
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ACKNOWLEDGMENT The authors are grateful to the Spanish Ministry of Education for financing this research (CICYT, project ALI 90/0633). Recelved for review February 14, 1994. Accepted July 7, 1994.a e
Abstract published in Aduance ACS Abstracts, September 1, 1994.
