Characterization of Humic Substances by Environmental Scanning

Feb 8, 2005 - AND. SERGE STOLL §. School of Geography, Earth and Environmental Sciences, ... CABE, University of Geneva, Switzerland. Environmental ...
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Research Characterization of Humic Substances by Environmental Scanning Electron Microscopy

Environmental scanning electron microscopy (ESEM) is a new technique capable of imaging micron and submicron particles. Here, we have applied it to image and quantify natural aquatic organic matter (standard Suwannee River humic acid, SRHA). Uniquely, we have observed the humic aggregate structures as a function of humidity and pH. Large aggregates of tens of micrometers were observed as the dominant material under all conditions, although much smaller material was also observed. Fractal dimensions (D) were calculated between 1.48 and 1.70, although these values were not statistically different under conditions of low humidity. However, D values calculated at high humidities (85%) during the rehydration phase were significantly lower (1.48 ( 0.01) than in the initial dehydration phase (1.69 ( 0.01). This hysteresis indicated that full rehydration of the HS was either kinetically slow or irreversible after dehydration. Fractal analysis of ESEM images was also performed to probe the change in aggregate structure as a function of pH. Minimum values were calculated at neutral pHs, rising by 0.1-0.2 at both high and low pHs because of a combination of the physical chemistry of HS and the impacts of the drying regime within the ESEM. Thus, ESEM was an important complementary technique to other analytical methods. At present, ESEM cannot be used to image nonperturbed natural samples. However, the method is an ideal method for probing the changes in colloid structure as function of hydration state and has the potential to perform fully quantitative and nonperturbing analysis of colloidal structure.

Mechanistic understanding of the role HS play with regard to pollutant chemistry, transport, and biouptake requires quantitative understanding of HS structure, particularly in their unperturbed state. However, HS are complex, polydisperse, colloidal systems, which are poorly understood (8). Up to the present time, few techniques exist which can quantify their structure in a minimally perturbing manner. In this paper, we have for the first time applied the new technique of environmental scanning electron microscopy (ESEM) to determine the effect of humidity and pH on the size and conformation of standard Suwannee River humic acid (SRHA), a well-characterized humic standard (9-12). ESEM is (through the production of a pressure gradient between electron source and sample) almost unique in that it allows samples to be imaged in their hydrated state (13, 14). In addition, poorly electron dense material such as natural organics do not require staining because of the amplification of signal due to the interactions of the electron beam and vapor within the ESEM sample chamber (13). Together, these properties raise the possibility of imaging unperturbed aquatic organic and colloidal material. The resolution is similar to, but slightly reduced compared with atomic force microscopy (AFM), conventional SEM, and transmission electron microscopy (TEM). Ideally, resolution limits are ca. 5-10 nm but will likely be lower for nonideal environmental materials (15). These properties make it an ideal complementary technique to force and electron microscopy. In addition, ESEM also has the unique capability of allowing the hydration state to be altered between 0 and 100% humidity through the use of controlled pressure and temperature conditions (13), allowing the impacts of dehydration and rehydration processes on organic structure to be probed. Hydration state and cyclical movement between wet and dry states has previously been shown to affect particle size, surface area, and total proton binding sites in synthetic colloids (16). Here, we have quantitatively examined the effect of hydration state on natural organic matter through the first application of ESEM to environmental colloids. In addition, we have investigated the effects of pH on HS aggregate structure and probed the limits of ESEM resolution. Structure has been quantified by analysis of mass fractal dimensions. To the best of our knowledge, this study contains the first data of mass fractal dimensions of HS. In addition, although ESEM has been used to study flocs in wastewater treatment (17), again to our knowledge, this paper reports the first application of ESEM to natural environmental particles and colloids.

Introduction

Methodology

Humic substances (HS) are high molecular weight, heterogeneous organic materials that are the major constituents of soils and aquatic environments (1, 2). They are potentially important in the binding and bioavailability of trace metals and trace organic pollutants (3) because of the large surface area and strength of binding. HS typically fall in the size range 1 nm to several hundred nm (4-7), depending on their source, concentration, solution conditions, and extraction and analysis method.

HS Preparation. Suwannee River humic acid (SRHA) was purchased from the International Humic Substances Society (IHSS) and used without further pretreatment. Stock solutions of 100 mg L-1 were freshly prepared 24 h prior to sample preparation. Samples were prepared at 10 mg L-1 and at a variety of solution conditions. Sample pH was investigated between 3.3 and 9.8 with the addition of dilute NaOH or HNO3. Samples were prepared with 5 mM added NaCl. ESEM Theory. Full description of ESEM theory and operation is given elsewhere (13, 14). However, a brief summary of the major modifications compared with traditional SEM is given here. Conventional SEMs perform imaging in a vacuum, precluding the imaging of wet samples in their native state (13, 14). The ESEM is able to overcome this

P A U L S . R E D W O O D , † J A M I E R . L E A D , * ,† ROY M. HARRISON,† IAN P. JONES,‡ AND SERGE STOLL§ School of Geography, Earth and Environmental Sciences, University of Birmingham, United Kingdom, School of Engineering, University of Birmingham, United Kingdom, and CABE, University of Geneva, Switzerland

* Corresponding author phone: (+121) 414 8147; fax: (+121) 414 5528; e-mail: [email protected]. † School of Geography, University of Birmingham. ‡ School of Engineering, University of Birmingham. § University of Geneva. 1962

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10.1021/es0489543 CCC: $30.25

 2005 American Chemical Society Published on Web 02/08/2005

difficulty by means of a series of apertures (usually three) and pumps, which create a gradient in a vacuum along the length of the ESEM. The majority of the ESEM is under high vacuum (13) as with conventional SEM. However, a short working distance of a few mm at the sample stage is at a pressure of 1-10 Torr. By choosing the correct temperature (ca. 2-10 °C), using a water-cooled Peltier stage, the sample can be maintained under liquid water and the water content varied by altering pressure or temperature. In addition to imaging hydrated samples, there is no requirement for staining poorly electron dense material such as HS as with most SEM and TEM analysis. This is due to the interactions of the secondary electrons and the water vapor around the sample. This interaction produces a cascade of ionized gas atoms, which amplify the signal allowing even natural organic material to be imaged. No samples in this study were stained. Preparation and Imaging of Samples. Several preparation techniques were used to allow a direct comparison with previous AFM measurements of the same samples (18, 19). Small (50 µL) aliquots of sample were pipetted onto either a glass or mica surface and placed into the ESEM immediately for imaging. Alternatively, the glass or mica was inserted into the solution, left for 30 min, and rinsed briefly with clean water and placed into the ESEM. Sample deposition and substrate type had no observable effect on sample structure. Several drops of pure water were placed around the sample (but not imaged) to control the sample humidity. All surfaces were fixed to grooved, stainless steel stubs using silver paint to reduce charge effects around the sample which may reduce image resolution. An FEI XL-30 FEG-ESEM in wet mode was used for all imaging. All images were obtained at 2 °C and the pressure was altered below 10 Torr to maintain humidities between 25 and 100%. At 100% humidity, the sample was fully hydrated. Samples were imaged with a standard gaseous secondary electron detector using a 10 kV electron beam and using H2O for the vapor skirt. Fractal Dimension Methodology. Fractal geometry provides a powerful mathematical tool for the description of the structure and properties of random and heterogeneous systems such as HS. A fractal object can be described quantitatively by a nonintegral (fractal) dimension D, which reflects the actual space, occupied by the system. A fractal object is characterized by a power law between “mass” (N) and radius (r) with a noninteger exponent D:

N(r) ∼ rD where D is the fractal dimension. If the system is composed of filamentary branches (20) such as in HS, the number of branches n(r) at a distance r is given by the gradient of the mass:

n(r) ∼ dN(r)/dr ∼ r(D-1) Therefore, a count of the mass of the object inside the circles of radius, r, provides a measure of D (21). Where the fractal object has a clear growth center, counting begins. However, in cases where this is not apparent, the computer program calculates a center of mass and the counting begins at that point. The fractal dimension is then calculated as the gradient of log cumulative number of pixels (mass) for each radial distance r plotted against the log cumulative radius r for each radial distance as shown in Figure 1. When calculating the gradient of this curve, we do not include the plots at either end of the curve as these represent (a) artifacts due to possible irregularities from the calculation of the apparent center of mass and (b) edge effects in the humic aggregate

FIGURE 1. Variation of number of pixels for each radial distance with cumulative radius at each radial distance. Fractal dimensions are calculated from the gradient. Sections a and b are excluded from calculations because of errors in estimating the growth center and because of edge effects. itself whereby it ceases to be fractal around its edges. For a perfectly isotropic object, that is, a perfectly spherical object with an evenly distributed density throughout, we would find a D of 2.0. This figure would decrease as we move away from this perfect condition reaching a minimum value of 1.0 under perfectly anisotropic conditions, that is, a completely random distribution of particles and particle sizes with no association to one another (22). Therefore, D values allow a quantitative measurement on the level of anisotropy of a humic aggregate between the values of 1.0 and 2.0. All image analyses were performed with Sigma Scan Pro 4.0, whereas a code written in Turbo basic Plus was used to extract, calculate, and analyze the mass distributions

Results and Discussion Hydration and Dehydration. Effects of dehydration and subsequent rehydration of an initially fully hydrated HS sample are shown in Figure 2a-d (dehydration) and 3a-c (rehydration). The variation of fractal dimension with humidity is shown in Figure 4. On initial inspection of Figures 2 and 3, it is clear that we are viewing a large aggregated structure. While there was also smaller material found, this is the dominant structure observed and was consistent under all conditions studied. These structures are generally somewhat inconsistent with images produced by AFM (18, 19) and TEM. AFM images for the same sample under identical conditions produced in our laboratories and elsewhere indicate that small macromolecules of 1-5 nm are dominant. However, aggregates of natural organic matter (NOM) with similar dimensions have also been observed by AFM (8). Most likely the differences sometimes observed are due to the different modes of operation, including different resolution levels of the two types of microscopy, rather than artifacts in one or both methods. For instance, the ESEM operates at much poorer resolution with limits of ca. 50 nm for this sample (although ultimate resolution on ideal samples is likely to be better), while AFM operates at much finer resolution (ca. 0.1 nm). In addition, ESEM is well resolved in lateral dimension, while AFM accurately measures heights above a substrate surface (8). The two techniques are therefore strongly complementary and reveal different aspects of the HS colloidal structure. It is possible that the larger aggregate observed by ESEM is composed of a large number of much smaller macromolecules which are observed by AFM, although this idea needs further testing. The need for a range of methodologies is well established (4, 23, 24), VOL. 39, NO. 7, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. ESEM images at 45% humidity (a), 65% (b), and 100% (c). All samples prepared at pH 9.8 and 5 mM NaCl.

FIGURE 2. ESEM images at 85% humidity (a), 65% (b), 45% (c), and 25% (d). All samples prepared at pH 9.8 and 5 mM NaCl. because of both the complexity of these organic colloids and the analytical bias and “window” inherent in different 1964

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FIGURE 4. Variation of fractal dimensions (D) with humidity. All samples prepared at pH 9.8 and 5 mM NaCl. methods. Development of ESEM therefore provides another powerful technique to probe these organic colloidal structures.

In addition to the size of the aggregate, it is clear that large crystals of the added NaCl are observable, even at high humidities. At 100% humidity, liquid water is present and the HS cannot be seen. However, as the humidity drops to 85%, the liquid water disappears and relatively dry humic aggregates become visible, with a concomitant precipitation of salt. Despite a low concentration of salt in the initial samples (5 mM), the loss of all free liquid water likely causes a large increase in salt concentration leading to crystal formation. These images undercut our initial thoughts about the ability of the ESEM to image unperturbed organic material from natural waters, because of this precipitation of salt crystals and concomitant aggregation of the HS. At present, analysis of unperturbed samples is not completely possible. From the images in Figures 2 and 3, it is clear that the HS and salt are intimately connected when partially dry. Further investigation of the mechanism of interaction during aggregation is underway. However, it is clear presently that these images cannot directly be used to infer the structure of the solution-phase HS. Rather, the ESEM allows quantification of the effects of wetting and drying and analysis of HS structure when sorbed to the surface of a second (solid) phase. Closer investigation of Figures 2 and 3 indicates an increasingly less bulky and clearer image as dehydration occurs, which could be due to removal of water from the HS, as individual parts of the aggregate shrink and separate. As rehydration occurs, there is an indication that the salt begins to dissolve at 85% humidity before fully hydrating at 100% humidity. Although the HS cannot be seen under water, the water droplets form over the HS, picking out its shape. The change in D values partly support this picture on the basis of ESEM images, although the D values calculated are not significantly different at low humidities (Figure 4). Interestingly, there appears to be a degree of hysteresis in rehydration, which becomes significant at high humidities. As humidity decreased and then increased during dehydration and rehydration, the D values decrease and then increased but the two curves are not significantly different. However, a D of 1.48 ( 0.01 (85% humidity, rehydration) is significantly lower than the D of 1.69 (0.01 ( 85% humidity dehydration), indicating a less compact shape on rehydration, presumably because of a kinetically hindered uptake of water or an irreversible loss of water by the HS during dehydration. Because of the difference between aggregates, the data represents only a single aggregate during a single hydration/ dehydration cycle. Nevertheless, the hysteresis was observed consistently for all aggregates imaged with slightly different absolute D values of different aggregates. The method of ESEM is therefore ideal for the quantitative determination of structural changes to organic and colloidal matter during rehydration and dehydration, shown as an important process in the chemical behavior of a synthetic manganese oxide (16). A few possible applications of ESEM include (1) the impact on HS structure because of the extraction and purification procedures, for instance, the SRHA in this study was previously freeze-dried to allow transport and storage, and in the absence of an initial dehydration stage, it is likely that the hysteresis observed would be altered; (2) the impact on the structure of relatively dry atmospheric particles after deposition in wet environments (e.g., surface waters or the human lung) and how this affects pollutant sorption/desorption; and (3) the impact on the structure of fully hydrated aquatic colloids as a result of dehydration, for example, because of drought or water abstraction. In all of these processes, the hysteresis in Figure 4 prompts the question are any structural changes reversible and if so, what are the rates of change? Changes observed are likely to have subsequent effects on pollutant sorption or desorption. Ascertaining the answers is not possible in this initial study,

FIGURE 5. Variation of fractal dimensions with pH. All samples prepared at 5 mM NaCl and imaged at 65% humidity. in part because the HS have previously been dried. Further studies on natural nonperturbed organic matter using ESEM and wet chemical methods are being pursued on samples which have not undergone preliminary drying. Effects of pH. The effects on fractal dimensions of the HS aggregates of pH is shown in Figure 5. Observation of the images shows no substantive differences with the images already presented in Figures 2 and 3 and are therefore not shown. Figure 5 shows different absolute values (ca. 0.1) of two separate aggregates, which have been obtained under identical conditions. The difference suggests there is significant interaggregate variability because of the unique growth properties of each individual aggregate. However, both aggregates show the same trend with a minimum D value at neutral pH values, which increases by small but significant amounts at higher and lower pH values. The reasons for these trends are not completely clear although may be related to interactions such as electrostatic or hydrophobic processes at high and low pH values. Fuller interpretation awaits a greater understanding of the physical chemistry of HS and their interaction with the ESEM drying process. In conclusion, ESEM is an important new technique for the analysis and quantification of organic and colloid structure and ideally should be used in conjunction with other methods to provide a true picture of HS structure and behavior. The method can image and quantify the structure of these colloids, potentially with minimal perturbation. In addition, ESEM can probe important processes such as the effect of wetting and dehydration on colloid structure, with a realistic resolution limit of ca. 30 nm.

Acknowledgments Paul Redwood was supported by a studentship grant from the Natural Environmental Research Council (NER/S/A/ 2001/05983). The authors would like to acknowledge the advice and help with ESEM from Professor Athene Donald.

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Received for review July 8, 2004. Revised manuscript received December 20, 2004. Accepted December 21, 2004. ES0489543