Environ. Sci. Techno/. 1995, 29, 2176-2184
Characterization of Aquatic Colloids and Macromdecules. 2. -Key Role of Physical Structures on Analytical Results J . BUFFLE',' AND G. G. LEPPARDt Analytical and Biophysical Environmental Chemistry ( W E ) , Department of Inorganic, Analytical and Applied Chemistry, University of Geneva, Sciences 11, 30 Quai E. Ansermet, 1211 Geneva 4, Switzerland, and National Water Research Institute, Environment Canada-CCW, Burlington, Ontario, Canada L7R 4A6
Parts 1 and 2 of this series are tutorial feature articles exceptionally published as Critical Reviews for editorial reasons. They intend to give to the nonspecialist an overview of the major aspects of the characterization of aquatic colloids in relation to their physical and chemical properties. For this reason, there is a close link between parts 1 and 2, which in reality form one single paper. In these papers, the words colloids and macromolecules refer to any organic or inorganic entity in the size range of 1 nm to 1 p m . In aquatic systems, they form inherently unstable suspensions due to their propensity to undergo conformational changes, aggregate, and then sediment. These factors should be considered carefully in the development of any procedure for the characterization of colloidal material. They are discussed in part 1 of the series. Part 2 describes some important characterization procedures commonly reported in the literature by discussing critically their limitations based on the above-mentioned properties and behavior of colloids. The following analytical steps and methods are covered: (i)sampling and sample storage and handling in relation to problems of microbial activity, colloid aggregation, adsorption, and flotation; (ii)fractionation by filtration, centrifugation, and field-flow fractionation techniques; and (iii) characterization by electron microscopy and light scattering techniques.
Introduction The purpose of parts 1 and 2 of this series is to discuss the problems that may arise when trying to characterize aquatic colloids, macromolecules, and their aggregates. The general goal of such a characterization is to understand the role of colloidal material as carriers of toxic or vital compounds in a particular environmental compartment (lake, river, sediment, soil, groundwater, ocean, etc.). This goal can be achieved only if the chemical characteristics (chemical composition and properties) of the colloidal material are +
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related to its physical characteristics (size, morphology, conformational changes). Indeed, the physical characteristics are directly linked to the circulation of the colloids in the environmental system of interest through processes such as coagulation-sedimentation in water columns or fitration in porous media (Figure 1 in part 1). Determination of both chemical and physical characteristics of the same colloidalfractions of an aquatic sample is a challenging task. Indeed, the fine physical structure of colloids, macromolecules, and their aggregates is very delicate (Figure 3 of part 1)and easily perturbed by chemical reagents (pH change, added electrolyte, etc.). On the other hand, chemical and physical characteristics of colloids cannot be determined without some analytical manipulation such as sampling, storage, or fractionation,which may often introduce drastic perturbations of the colloidal structures. Part 1 of this series has discussed the nature of colloidal systems and the factors which may affect their structures. In the present paper, we shall discuss the most important processing steps and techniques of colloid characterization giving special attention to the possible impact on colloidal structures, in order to assist the interested scientist in selecting the best conditions to minimize structural perturbations along the analytical procedure.
Sampling, Storage, and Sample Handling As discussed in part 1, a major characteristic of a water sample containing colloids is its intrinsic instability due in particular to continuing coagulation processes and microbial activity. As a result, sampling, sample storage, sample processing, and in particular fractionation are key steps that should be shortened and simplified as much as possible and whose conditions must be carefully chosen. Sample collectionitself is not necessarilya trivial procedure. Collecting samples representative of all particles, including the largest and smallest ones, is not easy. The largest particles can be easily excluded during the sampling step due to their rapid sedimentation. On the other hand, the submicrometer particles represent only a very small proportion (a few percent) of the total colloid mass (part 1);consequently, their concentration is often low (typically 5 100 p g drn-"), and losses by adsorption on vessel walls may be important. Preequilibration of the sampling vessel is recommended to minimize such losses. Following collection of the sample, the system continues to undergo many of the same elimination processes as those
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occurring in the water body (part 1, Figure 21, but most of the sources of new colloidal material are absent except those linked to microbial activity. A net transformation of the system thus occurs which should be slowed down as much as possible. Apart from adsorption on vessel walls, which may be minimized by preequilibration, the main other potential modifications include the following (1,2): (i) Microbial growth with possible release andlor degradation of organic compounds; these microbes may originate either from the test sample or from bacterial contamination of the sample handling material. (ii) Aggregation of small colloids into large particles or flocs due to the coagulation processes described in part 1. Visible sedimenting particles may be formed, but in many cases coagulation produces aggregates that remain undetected by simple observation since aggregates in the size range 0.1-1.0 pm may be stable for days (part 1). (iii)Accumulation of small colloids or aggregates at the air-water interface due to flotation. This latter effect is observed in particular when the temperature of the water sample is allowed to increase over the natural temperature; it is due to microbubble formation at the colloid surface, resulting from the lower solubility of oxygen and nitrogen at higher temperature. The buoyancy of colloids is subsequently increased, and flotation may occur for the least dense colloidal material. It has been observed (2) that when an aquatic colloid sample is left to stand quietly for a few hours, the fine colloidal material may accumulate in the top part of the water sample instead of sedimenting. This problem is obviously more dramatic when a gas is deliberately bubbled into the water sample, as is required for some analytical methods (e.g., degassing of 0 2 in voltammetric techniques). Antibiotics [e$., Hg(II),NaN3(2-4)] and anticoagulants [e.& hexametaphosphate (2, 8 1 have been proposed to minimize problems i and ii, but generally for different applications or without detailed tests of the influence of these reagents. Systematic experimental tests (2)show that they cannot be used for presening the structural integrity of the colloid sample as they pose various “secondary” problems, e.g.,hexametaphosphate induces disaggregation of natural aggregates and promotes bacterial growth after metaphosphate hydrolysis. To ensure their antibiotic action, Hg(I1) and NaN3 must be used in concentrations that are large enough to produce colloidal mercuric hydroxide or to interfere with analytical measurements, respectively. Although finding nonperturbing anticoagulant or antibiotic agents remains a possible option, their choice is clearly not obvious. Drying and freeze-drying prevent bacterial growth, but they induce changes in colloidal and aggregate structures (6, 7). In a detailed study (1,2),it has been concluded that the use of any “stabilizing” agent should preferably be avoided and that reliable results can be obtained in their absence, provided that measurements are all carried out within 2-3 days after sampling; significant changes by coagulation or bacterial activity mainly occur after that period provided storage is done at 4 “C in the dark. Storing the sample at a low temperature also minimizes the flotation problem. To sum up, important processing conditions include the following: storage (and handling) of sample at 4 “C in the dark for no more than 2-3 days including fractionation and measurements related to colloid structure studies; minimized physical and chemical changes of the water sample; use of sterilized
equipment and reagents; and avoidance of long stirring steps.
Fractionation Filtration and Centrifugation. Because of the extreme complexity of an aquatic colloid mixture, fractionation is often required before applying characterization techniques. Filtration and ultrafiltration are largely used to fractionate colloidal material in the range 1 nm to 100 pm and to separate the so-called “particulate” material from the “dissolved”compounds (with a cutoff limit often arbitrarily set at 0.2 or 0.45pm). As discussed in ref 8, filtration should not be regarded as a sievingprocess except when very large pore filters are used (’20prn). Different secondary factors may playa predominant role in the various pore size ranges (8) and make the cutoff limit very much operationally defined (9, 10). For instance, ultrafiltration of small ions and molecules (0.5-5.0 nm) is influenced by their hydration layer and electric charge. Ultrafiltration of organic macromolecules (1-50 nm) depends on their conformation and interactions with the membrane. Drastic transformations of the colloidal material in the size range 50 nm to 5.0 pm may be induced by two important processes: (i) the interactions between the membrane and aquatic macromolecules and colloids, leading to conformation changes and/or irreversible adsorption and (ii) the self-coagulation of colloids at the membrane surface. This latter effect results from the increased concentration of colloids (the so-called concentration polarization) in the diffusion layer at the membrane surface because of the slow back-diffusion into solution of the larger retained colloids (Figure 1). As a result, colloid concentrations at the membrane surface may be orders of magnitude larger than in the bulk solution (8),leading to an almost instantaneous coagulation. In this case, colloids smaller than the pore size may be entrapped into large aggregates and retained on membranes through which they should have passed. This process largely controls the retention of colloids or macromolecules larger than 20-50 nm because of their very low diffusion coefficient. Theoretical calculations and experimental results [Figure 2 (8)l demonstrate that colloid retention due to surface coagulation strongly increases with flow rate. Note that SEM pictures (Figure 2) may suggest that larger particles are retained at high flow rates than at low flow rates. This, however, is an example which demonstrates that SEM observations alone may be misleading. Indeed, TEM observations of sections of the membrane and particles seen in Figure 2 indicate (8) that these particles are composed of much smaller colloids aggregated at the membrane surface. Surface coagulation may be minimized (i) by cascade filtration (Le.,filteringthe sample through successive filters with decreasing pore sizes), taking care to minimize the number of filtration steps; (ii) by decreasing the thickness ofthe diffusion layer above the membrane (Figure11,either by stirring the bulk solution or by using tangential flow filtration; and/or (iii) by employing low flow rates (8). The use of condition ii however does not allow complete elimination of the diffusion layer and consequently of the concentration polarization effect; strong stirring above the membrane cannot be used for long periods of time since it favors coagulation in the bulk solution (part 1);tangential flow filtration is a clear improvement over classical filtration, but calculations suggest that it is not sufficientto completely VOL. 29. NO. 9. 1995 I ENVIRONMENTAL SCIENCE & TECHNOLOGY 12177
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FIGURE 1. Schematic represontation of colloid fluxes in classical filtration and in tangential flow filtration. The colloids that am too large to pass through the membrane accumulate in the ditlusion laver whew they may relf-coagulate or coagulate with smallsr colloids which otherwise would have passed through tho membrane. To minimizs this pmblsm, the dfiusion layer thickness must be decreased aithar by stining the bulk solution (classical filtration) or using a higher tangential flow rate.
avoid surface coagulation (81, although experimental systematic tests are required. Surface coagulation can be avoided by usingvery low flow rates (conditioniii) (Figures 2 and 3); however, fitration of large volumes requires long periods of time for which coagulation or microbial activity in the bulk solution may become important disturbing factors(Figure 3). Combination of the various aforementioned effects leaves only an often small window of acceptable flow rates that must thus be carefully chosen to minimize colloid perturbation, both at the membrane surface and in the bulk solution (Figure 3). Additional problems of filtration a n related to contaminations, which may be particularly important for the fractionation of suspensions of aquatic colloids
