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Algae Columns with Anodic Stripping Voltammetric Detection Wladyslaw W. Kubiak' a n d J o s e p h Wang* Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003 Dennis Darnall* Bio-Recovery Systems, Inc., Las Cruces, New Mexico 88003
The use of sllica-lmmobillzed algal cells for on-line column separatlon In conjunction with continuous monltorlng of trace metals is described. Algae-silica preparations are hlghly sultable for flow analysts as they couple the unique reactlvlty patterns and hlgh blndlng capacity of algal biomass wlth the hydrodynamic and mechankal features of porous sillca. Such advantages are Illustrated by uslng on-line anodic strlpplng vdtammetry and the alga Ch(0reua pyrenldosa Selective and exhaustive removal of Interfering constltuents circumvents common problems such as overlapping peaks and intermetallic effects. Effects of flow rate, pH, operation thne, and other variables are reported. The system is characterized by hlgh durabliity, simplicity, and economy and offers an attractive aiternatlve to prevalent columns used for flow analysis.
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The development of on-line analyzers for monitoring heavy metals has been the subject of considerable environmental, clinical, and industrial interest. Atomic absorption spectroscopy (AAS)and anodic stripping voltammetry (ASV) have proved successful for detection in these flow systems ( I ) . Independent of the detection technique or the sample matrix, separatory and enrichment steps are usually desired for on-line monitoring of trace metals. Efforts toward the incorporation of on-line matrix isolation/preconcentration schemes into trace-metal flow analyzers have grown recently. Olsen et al. (2) used an ion-exchange Chelex-100 column connected to an atomic absorption spectrophotometer. Columns containing immobilized 8-quinolinol were found useful in connection with flow injection/AAS determinations (3, 4 ) . Chelate ion-exchange columns and flow-through coulometric cells were used to alleviate matrix effects in ASV flow analyzers (5, 6). The present paper describes the utility of algal biomass for on-line matrix isolation in flow analysis. The binding of metal ions to microorganisms is a rapidly growing area of interest. Such biological processes for collection of metal ions involve biosorption, intracellular uptake, and chemical transformations. Recent work in these laboratories has focused on the accumulation of metals onto algal biomass (7-11). In particular, biosorptive interactions of several heavy metals onto the cell walls of various algae were investigated (7,8) and their utility for wastewater treatment and metal recovery was illustrated (9, 10). Different algae exhibit different affinities toward different metals, and different metals exhibit different pH-binding profiles for a given alga. The field of analytical chemistry can greatly benefit from the unique metal-algae interactions. For example, these interactions were exploited recently for developing electrochemical sensors based on algae-containing carbon-paste electrodes ( I I ) . We have recently developed a method for immobilizing algal cells in silica (7). Such alga-silica preparations combine the IOn leave from the Academy of Mining and Metallurgy, Krakow,
Poland.
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high binding capacity of algal biomass, low resistance to fluid flow, self-supporting rigidity, and excellent durability. In contrast, when nonimmobilized algae cells are packed into a column, the algae clump together and significant flow rates cannot be achieved even with high pressures. Compared with common ion-exchange resins, the algae-silica matrix possesses the advantage that alkali and alkaline-earth metals do not interfere significantly with the binding of toxic, heavy metals. The advantages of using alga-silica columns for on-line monitoring are illustrated in the present work in connection with on-line ASV and the alga Chlorella pyrenidosa. Common problems associated with practical ASV flow analysis, including unsatisfactory resolution or intermetallic effects, can thus be eliminated. The strategy is to selectively remove the interfering constituent in the algae column prior to the ASV detection. The chemical and physical environments experienced by metals incorporated onto algae lead to new reactivity patterns, not found in conventional ion-exchange columns. Coupled with their stability and simplicity, algae columns seem well-suited for continuous monitoring of heavy metals in aqueous media. EXPERIMENTAL SECTION Apparatus. The flow system consisted of a 0.25-L sample reservoir, connected to the detector by Tygon tubing (1.6 mm id., 4.8 mm 0.d.). A three-way valve allowed passage of the sample through the algae-silica column or its bypass straight to the detector. The column was a stainless steel cartridge (4.6 mm i.d., 30 mm long),filled with the dry algamilica powder (50-100 mesh particles), and supported on a guard column holder. The algae-silica (Chlorella pyrenidosa immobilized in silica gel) was obtained from Bio-Recovery Systems. The solution flow was maintained with a variable speed Masterflex pump (Cole Palmer No. 7553-10) together with the solid-state Masterflex controller. The flow cell was a glassy carbon thin-layer detector (Model TL-5, Bioanalytical Systems). The Ag/AgCl (3 M NaC1) reference electrode (Bioanalytical Systems) and stainless steel auxiliary electrode were located in the downstream compartment. An IBM ECL22.5 voltammetric analyzer, combined with a Houston Omniscribe strip-chart recorder, served to obtain the current-potential data. Reagents. Double-distilled water was used to prepare all solutions. Metal ion stock solutions,lo4 M, prepared by dissolving the corresponding nitrate salts in nitric acid and diluting as required, were stored in polyethylene containers. The supporting electrolyte was prepared by dissolving 8.2 g of sodium acetate in 1 L and adjusting the pH with acetic acid. A 5 X M mercury(I1) nitrate solution (in 0.1 M acetate buffer) was used for the mercury plating. Procedure. The new column was conditioned by passing a 0.1 M nitric acid solution through at 0.3 mL/min for 2 h. The mercury film was deposited at the beginning of each day and yielded reproducible results during the course of a day. The mercury film was deposited by holding the potential of the glassy carbon electrode at -0.9 V and passing the deaerated plating solution through at 1.0 mL/min for 15 min. (The mercury solution bypassed the column.) The potential was then switched to 0.0 V, for 3 min, to strip any contaminants that may have codeposited with the film. The ASV procedure involved passage of the 0 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 5, MARCH 1, 1989
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Flgure 2. Dependence of metal binding efficiency of the algae column on flow rate for a 2 X lo-’ M lead solution. Other conditions are given in Figure 1.
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Flgure 1. Stripping voltammograms recorded with (b-h) and without (a, i) passing the solution through the algae column. Curves b-h are representative voltammograms recorded at 30-min intervals over a 180-min period of continuous operation. Conditions were as follows: 1 X lo-’ M lead in acetate buffer (pH 6.0); deposition, 3 min at -0.9 V; Row rate, 0.40 ml/min; differential pulse ramp with 20 mV a m p l i and 10 mV/s scan rate. deaerated sample solution, usually at 0.26 mL/min, while applying the deposition potential. At the end of the 3-min deposition period, the solution flow was stopped and, after a 20-9 rest period, a positive-going differential pulse scan was initiated with a simultaneous recording of the voltammogram. After 60 s at +0.1 V the system was ready for the next ASV cycle. After each day, the column was regenerated by passage of a 0.1 M nitric acid solution, for 30 min, at 0.26 mL/min; the column was stored in the acid overnight. The mercury film was removed at the end of the day by wiping the surface with a wet tissue. RESULTS AND DISCUSSION Metal Ion Uptake. Figure 1demonstrates the high degree of metal binding efficiency obtained with the algae-silica column. It shows stripping voltammograms for a 1 X lo-’ M lead solution obtained with (b-h) and without (a, i) passage through the column. Curves b-h were recorded over an uninterrupted l8O-min period, with no column regeneration. The absence of a lead peak indicates that the algae-silica matrix maintains its lead-binding characteristics under continuous operation conditions. Following this series, the algae column was bypassed; the corresponding voltammogram (curve i) shows a lead peak identical with that recorded at the beginning of the experiment (curve a). Analogous metal removal and response characteristics were obtained in another prolonged (150-min) experiment involving a 1 X lo4 M copper solution (pH 4.5; conditions as in Figure 1, except of a deposition
potential of -0.7V). These data indicate that algae-silica preparations are highly durable under continuous flow conditions. The excellent stability of the ASV response is also obvious. The algae-silica column was regenerated only at the end of a day’s work, after more than 100 ASV cycles. The pH dependence of the metal-ion binding to Chlorella allows convenient column regeneration by passage of diluted acid. In this manner, the same column was able to be used for a period of 3 months, without any noticeable deterioration in performance. Such behavior is of great promise for analytical flow systems. Figure 2 shows the dependence of the metal binding efficiency of the algae column upon the volume flow rate for a 2 X lo-’ M lead solution. About 99% removal of the lead ion is observed between 0.07 and 0.40 mL/min. A sharp decrease in the metal binding efficiency is observed above 0.40 mL/min, as expected from the decreased residence time of an element of solution in the column. Longer columns would allow use of higher flow rates, while maintaining the high metal removal efficiency. Other aspects of the metal-ion uptake a t algaesilica columns, and particularly the effect of the pH, are discussed in the following sections. Analytical Applications. Algae columns can greatly benefit the performance of analytical flow systems. In the following sections we will demonstrate the analytical advantages accrue from the utility of an algae column as a matrix isolation tool. In particular, a selective and exhaustive removal of interfering constituents will be shown to circumvent major problems common to on-line stripping measurements. Even though the concept is presented in the context of ASV flow systems, the approach could be easily extended to other on-line trace metal analyzers. Overlapping peaks, caused by similarity in oxidation potentials, represent a common problem in ASV. Algae columns can eliminate various resolution problems. Such improvements are based on the different affinities that various metals possess toward the alga surface and on the strong dependence of these affinities upon the solution pH. Figure 3 demonstrates the pH dependence of two pairs of metals, that commonly exhibit an overlapping ASV response (copper and bismuth (A) and lead and thallium (B)). Under the conditions shown, the binding of bismuth and copper ions increases upon increasing the pH. While copper is completely (>99%) bound by the algae and consequently removed at pH values higher than 3.5, bismuth is significantly removed only when the pH is raised above 5. Increasing binding of lead accompanies the increase of p H from 3 to 5 ; exhaustive removal is observed as the pH is raised further. Thallium(I), in contrast, is only slightly removed in the pH range 3-4, with no removal a t higher values.
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PH Figure 3. Effect of pH on the removal of (A) copper (a)and bismuth (b) and (B) lead (a)and thalllum (b) by the algae column. Deposition potential: -0.7 V (A) and -0.9 V (B); deposition time, 3 min; flow rate, 0.26 mL/min; metal concentration, 5 X lo-’ M ((A), a, b; (6)a) and 8 X M (B(b)). Other condltions are given In Figure 1.
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Figure 5. Effect of pH on the removal of copper (a) and zinc (b) by the algae column: deposition potentiel, -0.7 V (a) and -1.4 V (b); flow M. rate, 0.26 mL/min; metal concentration, 5 X
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Stripping vottammograms recorded with (A) and without (B) passing the solutlon through the algae column: (a) 1.5 X 10-’M zinc; (b) same as (a) but after adding 7 X lo-’ M copper. Solution pH, 4.6; deposition potential, -1.4 V; flow rate, 0.26 mL/min. Flgure 6.
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Stripping voltammograms recorded with (A) and without (B) passing the solution through the algae column: (a) 5 X lo-’ M thallium; (b) same as (a) but after adding 2 X lo-’ M lead. Solution pH, 6.0. Other conditions are given in Figure 3B. Flgure 4.
Such variations should enable one to improve the selectivity via p H control. For example, on the basis of the profiles of Figure 3 the optimum pH values for selective measurements of thallium and bismuth (in the presence of lead and copper, respectively) are 6 and 4. Figure 4 illustrates the ASV response for a mixture of thallium(1) and lead, as obtained with (A) and without (B) passage through the algae column. The thallium peak is not affected by the addition of lead when the solution is pumped through the column. In contrast, partially merged peaks are observed in the absence of passage through the algae column. Note also that (in accordance with Figure 3B) the sensitive thallium response is not degraded by passage through the column. A series of seven successive increments of the thallium concentration (over the (3-21) X M range), in the presence of 2 X lo4 M lead, yielded a linear calibration plot with a slope of 5.7 nA/kM and correlation coefficient of 0.999. The use of an algae column yielded analogous improvements in thallium measurements in the presence of indium (not shown). The latter had a pH-binding profile M similar to that of lead. In another experiment, 8 X bismuth was measured selectively in the presence of 8 X M copper after passage through the column (not shown; deposition at -0.7 V; pH 4). A severe copper interference-with identical peak potentials-was observed without passage through the column. Other resolution problems may be circumvented with a judicious choice of the operating pH or via
the use of other algae (with different reactivity patterns). The formation of intermetallic compounds between metals deposited in the mercury electrode is another inherent difficulty in ASV (12). The most frequently noted example is the interaction of copper and zinc that can result in erroneous analytical data. The different pH dependences for the removal of these metals by the algae column (Figure 5) can be exploited to circumvent intermetallic effects. For example, by a proper adjustment of the pH to a value of 4.6 the copper is exhaustively removed and thus the zinc can be measured without interference. Figure 6A illustrates such determination of zinc in the presence of copper. The zinc peak (ca. -1.1 V) is not affected by the copper addition (compare a and b). In contrast, if the same solution is not pumped through the column, the presence of copper results with a 30% suppression of the zinc peak (Figure 6B). Similar advantages can be obtained for other binary systems with intermetallic interactions (via a judicious choice of the operating pH). In conclusion, the above results confirm the feasibility of exploiting algae-silica columns as effective matrix isolation tools in flow analysis. The algawilica system is unique among columns that have been employed in on-line metal analysis due to the coupling of unique reactivity patterns and the high binding capacity of algal biomass with the structural features of porous silica. Under the experimental conditions employed, exhaustive removal can be achieved over the 105-10-9 M concentration range (of interest to stripping measurements). Competition of some metal ions with one another for binding sites on the algae is not expected at this concentration range; some competition may occur at significantly higher concentrations (7). Alkali and alkaline-earth metals can be present at large concentration excess (e.g., 0.1 M sodium, as of the
Anal. Chem. 198% 61, 471-482
supporting electrolyte) without affecting the binding of heavy metals. Although these advantages are presented within the framework of continuous-flow ASV, silica-immobilizedalgae columns could greatly benefit other on-line analytical schemes, e.g., flow-injection atomic absorption spectroscopy. While on-line separation is the focus of the present study, the utility of algae columns as preconcentration tools can be easily envisioned. Because of their foundation on bioaccumulation processes, algae columns should be extremely useful for on-line metal speciation measurements. LITERATURE CITED Ruzlcka. J.: Hansen. E. A. Flow Inlectbn Analvsis: Wilev: New York. 1988. Olsen, S.; Pessenda, L. C. R.; Ruzlcka, J. Hansen, E. A. Analyst (London) 1983, 108, 905. Marshal, M. A.; Mottola, H. A. Anal. Chem. 1985, 57, 729. Maiamas, F.; Bengtsson, M.; Johansson, G. And. Ch/fn. Acta 1984, 160, 1.
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(5) Wang, J.; Dewald. H. D. Anal. Chem. 1983, 55, 933. (8) Yang, X.; Rislnger, L.; Johansson, G. Anal. Chim. Acta 1987, 792, 1. (7) Darnall. D. W.; Qreene. B.; Hosea, M.; McPherson, R. A.; Henzi, M.; Alexander, D. In Trace Metal Removal Irom Aqueous Solutions; Thompson, R., Ed.; Royal Society of Chemistry: London, 1986; Special Publication No. 81, p 1. (8) Watklns. J. W., Elder, R. C.; Oreene, B.: Darnall. D. W. Inorg. Chem. 1987, 26. 1147. (9) Greene, B.; Henzl, M.; Hosea, M.; Darnall, D. W. Bbtechnol. Bioeng. 1988, 26, 764. (10) Darnall, D. W.; Greene, B.; Henzl, M.; Hosea, M.; McPherson, R. A,; Sneddon, J.; Alexander, D. Envlron. Scl. Techno/. 1988, 20, 206. (11) Gardea-Torresdey, J.; Darnall, D.; Wang, J. Anal. Chem. 1988. 60, 72.
(12) Wang, J. Stripping Analysis : Principles , InstrumntaMOn and Applica fbns ; VCH Publishers: Deerfield BeechlWeinheim, 1985.
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RECEIVED for review September 12,1988. Accepted December 1,1988. This work was supported by the National Institutes of Health (Grant No. GM 30913-05) and the National Science Foundation (Grant No. CBT-8610461).
Theory of Homopolymer and Oligomer Separation and Its Application to Gradient Elution Liquid Chromatography Richard E. Boehm* and Daniel E. Martire Department of Chemistry, Georgetown University, Washington, D.C. 20057
A slmpler, more general derlvatlon is developed for a prevlously Introduced theory of homopolymer fractionation by gradient elutlon high-performance liquld chromatography (HPLC). Improved treatments are given for the determlnatlon of the solvent-stationary phase adsorption Isotherm and the soivent-entralnedsolute expansion factors. Retentlon tlmes are evaluated for (hypothetical) homologous serles of flexible, chalntlke, chemlcalty homogeneous solutes In gradlent elutlon HPLC. The feasiblllty of HPLC fractionation of flexible oilgomers and homopolymers is confirmed. The theory also Investigates the signHicant ellects that sample concentratlon has on the retentlon behavior of hlgh molecular welght homopolymers. For example, the molecular weight dependence of the variation of the capacity factor with mobile phase composition Is predicted to depend strongly on the sample concentration in the elution band. The theory Is compared wlth the empirical linear solublllty strength theory proposed by Snyder and co-workers.
INTRODUCTION Successful fractionation of high molecular weight homopolymers using gradient elution high-performance liquid chromatography (HPLC) has bees reported (1-4,5-11). For example, polystyrene homopolymers and/or oligomers in a molecular weight range 105 I molecular weight I lo7 have been separated efficiently and rapidly by means of gradient elution high-performance liquid chromatography (HPLC) utilizing either a C-18 or C-8 chemically bonded stationary phase (CBSP) and a methylene chloride-methanol mixed mobile phase (1-4). Other mixed mobile phases such as H,O-THF have also been employed (5,11). Separation of styrene oligomers also has been achieved by supercritical fluid chromatography with a density-programmed supercritical n-pentane mobile phase (12,13). A considerable variety of other homopolymers (2-4) and biopolymers (6-10,14-16)also 0003-2700/89/0361-047 1$0 1.50/0
have been fractionated effectively through gradient elution HPLC in various mixed mobile-stationary phase combinations. A statistical thermodynamic theory of homopolymer fractionation and its application to gradient elution HPLC has been developed by Boehm, Martire, Armstrong, and Bui (hereafter designated BMAB-1) to describe the retention behavior of an isolated flexible homopolymer molecule distributed between a binary mixed mobile phase and an idealized stationary phase consisting of sorbed solvent (s) on a homogeneous planar surface (17). In this analysis the Flory-Huggins lattice model approach is applied to an isolated polymer molecule and entrained solvent molecules in each chromatographic phase and nearest neighbor interactions are included in the Bragg-Williams random-mixingapproximation (18,19). The BMAB-1 theory successfully accounts for the observed experimental trends of an increasingly abrupt transition from very high to very low retention as the degree of polymerization, M , of the homopolymer increases and as the mobile phase becomes sufficiently enriched to a critical composition of the better solvent for the polymer. The critical composition represents that mobile phase composition where the capacity factor becomes unity. The critical composition is predicted to be a monotonically increasing function of M for flexible homopolymers of sufficiently large M (M L 15) and this dependence emanates primarily from the molecular flexibility of the polymeric solute which allows size and shape alterations by solvent uptake or expulsion in response to its solvent and/or surface environment (18,19). The dependence of the critical composition on M also generates the opportunity for polymer fractionation by gradient HPLC. The BMAB-1 theory was subsequently extended to include intermolecular polymer segment-polymer segment interactions and thus be applicable to semidilute polymer solutions. This extension is hereafter referred to as the BMAB-2 theory (20). The onset of the semidilute regime where different flexible polymer molecules begin to interpenetrate appreciably occurs in good solvents for a volume fraction of chain monomers given 0 1989 American Chemical Society
