Patricia A. Maurice

Considerations in the use of high-pressure size exclusion chromatography (HPSEC) for determining molecular weights of aquatic humic substances

Abstract High-pressure size exclusion chromatography (HPSEC) is a powerful technique for determining molecular weight (MW) distributions of aquatic humic substances (HS). Previous researchers have shown that HPSEC can provide values of weight average molecular weight ( M w ), number average molecular weight ( M n ), and polydispersity ( ρ ) that are comparable to values determined by other techniques such as vapor pressure osmometry and field flow fractionation. We have observed that HPSEC does not always provide reproducible results for HS—variability by 10–20% is common. Since certain applications require better precision for MW of HS, this research focuses on improving precision and reproducibility of HPSEC measurements. While the method of baseline correction or of high MW (HMW) cutoff of the HPSEC chromatogram is not critical, the choice of low MW (LMW) cutoff can greatly affect M n and ρ . For HS, we recommend either 2% of the maximum chromatogram height or MW=50 as the LMW cutoff, whichever is the higher value, and 1% of the maximum chromatogram height as the HMW cutoff. Polystyrene sulfonate (PSS) standards are commonly used in combination with acetone; we further recommend the inclusion of salicylic acid as a charged low MW standard. Analysis of UV detection wavelengths shows that wavelengths between 230 and 280xa0nm give reasonable results, but that higher wavelengths can bias measurements to higher MWs. We recommend 254xa0nm except for low carbon concentration samples, where 230 will provide better sensitivity. Our results show that “standardization” of HPSEC procedures according to these suggestions can lead to excellent reproducibility of M n and M w measurements for HS (2–3% RSD).

FRACTIONATION OF AQUATIC NATURAL ORGANIC MATTER UPON SORPTION TO GOETHITE AND KAOLINITE

Natural organic matter (NOM) consists of a complex mixture of organic molecules; previous studies have suggested that preferential sorption of higher molecular weight, more hydrophobic, and more aromatic components may lead to fractionation of the NOM pool upon passage through porous media. Our work expands upon previous studies by quantifying the change in solution-phase weight average molecular weight (Mw) upon sorption of bulk (rather than isolated) surface water NOM from the Suwannee River (SR) and the Great Dismal Swamp (GDS) to goethite and kaolinite at different sorption densities and at pH 4, 22°C. High pressure size exclusion chromatography (HPSEC) was used to quantify changes in Mw upon sorption, and molar absorptivities at λ=280 nm were used to approximate changes in solution NOM aromaticity. Two SR water samples were used, with Mw=2320 and 2200 Da; a single GDS sample was used, with Mw=1890 Da. The SR NOM was slightly more hydrophobic and aromatic. These differences were reflected in greater sorption of SR NOM than GDS NOM. Both surface water NOMs showed a much greater affinity for goethite than for kaolinite. HPSEC analysis of the NOM remaining in solution after 24 h reaction time with goethite revealed that the largest changes in solution phase Mws (decreases by 900–1700 Da) occurred at relatively low equilibrium sorbate concentrations (approximately 5–20 mg C l−1); the decrease in solution Mw suggested that reactive surface sites were occupied disproportionately by large and intermediate size NOM moieties. At higher equilibrium NOM concentrations (>20 mg C l−1), as percent adsorption decreased, Mw in solution was similar to original samples. A smaller decrease in solution NOM Mw (300–500 Da at 10–20 mg C l−1 ∼100 Da at >20 mg) also occurred upon sorption to kaolinite. Overall, our results showed that factors (as related to NOM composition, clay mineral surface properties, and position along the sorption isotherm) which promote a higher percent sorption lead to the most pronounced decreases in solution Mw.

Dissolution of well and poorly crystallized kaolinites; Al speciation and effects of surface characteristics

Abstract This study compared surface characteristics and dissolution behavior of well-crystallized (KGa-1b) and poorly crystallized (KGa-2) kaolinite standards. Atomic force microscopy (AFM) revealed that particles of KGa-1b generally have nicely hexagonal micromorphology and crystallographically controlled microtopographic features. Particles of KGa-2 are also hexagonal, but their micromorphology tends to be more rounded. Basal-plane surfaces tend to be more irregular with fewer clearly crystallographically controlled features. KGa-1b particles tend to be larger in diameter and thicker than KGa-2 particles. Micromorphologic measurements showed that both KGa-1b and KGa-2 have modal edge- to total surface-area ratios of approximately 0.1 (mean ~ 0.2), although these measurements did not include the potentially large contribution of basal-plane step edges (additional 20% or more). Dissolution experiments were conducted in oxalic acid and inorganic acids at pH 3, 22 °C, I = 0.01 M, under batch dissolution conditions. Dissolution rates (measured as Si release) in 1 mM oxalic acid were approximately twice as fast for KGa-2 as for KGa-1b (2.27 vs. 0.96 nmol/m2·h). Rates for KGa- 2 and KGa-1b were similar in HNO3 (0.86 and 1.16 nmol/m2·h, respectively). The comparable rates for these two sedimentary kaolinites and for a hydrothermal kaolinite studied by Wieland and Stumm (1992) suggests that the fundamental structure of kaolinite, rather than specific surface details, exerts the greatest influence on dissolution kinetics. High-performance cation exchange chromatography (HP-CEC) was used to determine the distributions of monomeric Al species over the course of kaolinite dissolution. For dissolution in 1 mM oxalate, Al-oxalates were observed almost exclusively in agreement with results of equilibrium speciation calculations. For dissolution in HNO3, the peak representing uncomplexed Al species, Alf, was predominant but not exclusive, as predicted by calculations. A peak having a retention time characteristic of species with +2 charge may be evidence for an AlOSi(OH)32+ species, and warrant further investigation.

Simultaneous dissolution of hydroxylapatite and precipitation of hydroxypyromorphite: Direct evidence of homogeneous nucleation

Abstract Results of in situ atomic force microscopy (AFM), ex situ AFM, optical microscopy, scanning electron microscopy, energy dispersive spectroscopy, and x-ray diffraction were combined with previous macroscopic investigations to characterize aqueous Pb(NO 3 ) 2 reaction with hydroxylapatite (Ca 5 (PO 4 ) 3 OH) (HAP). Experiments were conducted by immersing particulate HAP crystals in the AFM fluid cell in solutions with initial Pb concentrations = 100 mg L −1 , at pH 6 and 22°C under static conditions. Experimental results showed that transport-controlled HAP dissolution provided phosphate for precipitation of hydroxypyromorphite (Pb 5 (PO 4 ) 3 OH) (HPY), which in turn sequestered aqueous Pb. The combination of in situ and ex situ techniques provided direct evidence that HPY formed primarily by homogeneous nucleation in solution. Nevertheless, HPY needles were found in close association with HAP surfaces, most probably due to diffusional controls on phosphate concentrations. Diffusion of phosphate away from dissolving HAP appeared to be the rate-limiting step in the overall reaction sequence.

Iron acquisition from hydrous Fe(III)-oxides by an aerobic Pseudomonas sp.

Iron is a metabolic requirement of living systems, yet iron is very insoluble in aerobic, neutral environments. Therefore, the amount of iron in solution under these conditions is not sufficient to meet the nutrient requirements of microorganisms. It has been assumed that microorganisms acquire iron in these environments through the use of specific iron chelating compounds called siderophores. Interestingly, there is little quantitative data in the literature to support this hypothesis. Our studies were initiated to investigate the mechanism(s) used by an aerobic microorganism to acquire iron from a relatively insoluble iron oxide. When iron was supplied in the form of hematite, a Pseudomonas sp. was able to achieve moderate growth, as compared to growth on FeEDTA. Analysis using high-performance liquid chromatography (HPLC) of the metabolic products of this species showed significant differences between growth on hematite as compared to FeEDTA. The results of these experiments will be discussed in terms of our current knowledge of microbial enhanced iron dissolution, and compared to abiotic dissolution rates.

Applications of atomic-force microscopy in environmental colloid and surface chemistry

Abstract Atomic-force microscopy (AFM) is a powerful technique for imaging mineral surfaces in air or immersed in solution and at sub-nanometer-scale resolution. As the technique continues to develop, AFM is emerging as an important means not only for imaging the surface structure and microtopography of environmental particles, but also for determining changes in microtopography over the course of dissolution, growth, sorption, heterogeneous nucleation, and redox reactions. Additionally, AFM can provide a direct means of probing the structure of the double layer. As AFM use continues to proliferate, there is an increasing need for critical evaluation of image acquisition and processing techniques. This manuscript reviews the “state of the art” in force microscopy with regard to theoretical considerations, technological advances, and current and potential applications. Some common artifacts are discussed, with the purpose both of preventing new users from re-making old mistakes and of enabling non-users to evaluate AFM results. Finally, examples of applications in colloid and surface chemistry are used to illustrate the potential of AFM for providing new insight into a wide range of environmental processes.

Atomic force microscopy of soil and stream fulvic acids

Abstract Atomic force microscopy (AFM) was used to image fulvic acid (FA) deposited from aqueous solution on to the basal-plane surfaces of freshly cleaved muscovite, and allowed to air dry. Two fulvic acid samples were used: a soil fulvic acid (SFA) prepared by NaOH extraction from a muck soil underlying a freshwater fen in the New Jersey Pinelands and the IHSS standard Suwannee River fulvic acid (SRFA). The use of tapping-mode AFM (TMAFM), a relatively new technique which reduces the lateral frictional forces generally associated with contact-mode AFM, allowed excellent images of delicate FA structures to be obtained with minimal sample disturbance. Four main structures were observed on SFA. At low concentrations, sponge-like structures consisting of rings (∼ 15 nm in diameter) appeared, along with small spheres (10–50 nm). At higher concentrations, aggregates of spheres formed branches and chain-like assemblies. At very high surface coverage, perforated sheets were observed. On some samples, all of these structures were apparent, perhaps owing to concentration gradients on drying. SRFA samples were only imagined at higher concentrations. Spheres, aggregated branches, and perforated sheets were apparent. The results agree with previous work by Stevenson and Schnitzer [Soil Sci., 133(1992) 179], who applied TEM to soil FAs freeze-dried on muscovite. However, the TEM images did not detect the smaller spheres and sponge-like structures observed by AFM at low concentrations. The relevance of imaging dried samples remains questionable; hence, it is hoped in the future to use new in situ TMAFM to image FAs sorbed to surfaces in solution. Although TMAFM provided excellent images, a variety of artifacts and potential problems were encountered, as discussed.

Attachment of a pseudomonas sp. to Fe(III)‐(hydr)oxide surfaces

As a first step towards understanding microbial dissolution processes, our research focuses on characterizing attachment features that form between a Pseudomonas sp. bacteria and the Fe(III)‐(hydr)oxide minerals hematite and goethite. Microbial growth curves in Fe‐limited growth media indicated that the bacteria were able to obtain Fe from the Fe(III)‐(hydr)oxidesfor use in metabolic processes. A combination of scanning electron microscopy, epifluorescence, and Tapping Mode™ atomic‐force microscopy showed that the bacteria colonized some fraction of mineralogical aggregates. These aggregates were covered by bacteria and were linked together by relatively open biofilms consisting of networks of fiber‐like attachment features intertwined through thin films of amorphous‐looking organic material. The biofilm material encompassed numerous individual bacteria, as well as mineralogie particles. We hypothesize that the bacteria first attached to mineral aggregates, perhaps via their flagella, forming colonies. Fo...

APPLICATION OF ATOMIC-FORCE MICROSCOPY TO STUDIES OF MICROBIAL INTERACTIONS WITH HYDROUS FE(III)-OXIDES

Abstract We are using atomic-force microscopy (AFM), to address the interactions of a Pseudomonas sp. aerobic soil microbe with Fe(III)-(hydr)oxides. Research results to date show that AFM is a viable technique for in-situ and ex-situ imaging of bacteria attached to rnineral surfaces. Delicate microbial structures and mineral surface microtopographies are readily accessible. However, sample preparation has proven to be crucial. For example, we found that an organic extractant used to separate bacteria from particle surfaces left a residue that could be mistaken for dissolution features in AFM images. Additionally, care must be taken to image samples in numerous locations and under a wide variety of conditions. The need for extensive imaging was reinforced by our observations that most reacted particles had no obvious dissolution features, but a small fraction of particles were extensively eroded. Hence, microbe-Fe(III)-(hydr)oxide interactions lead to enhanced dissolution, but the dissolution process is heterogeneous in nature. Thus, although AFM imaging of such complex systems is difficult and time-consuming, a painstaking approach is needed.

Using Atomic Force Microscopy to Study Soil Mineral Reactions

Publisher Summary The chapter discusses the application of AFM (atomic force microscopy) to researchon the structure, chemical composition, and chemical reactivity of soil particle surfaces. First, basic operating principles are reviewed. Second, tip-sample interactions are discussed, including forces between the tip and the sample, special considerations in the new tapping-mode AFM (TMAFM), and tip shape considerations. A variety of common artifacts are discussed, with the dual purpose of alerting new users to potential pitfalls and of enabling nonusers to evaluate more fully AFM results. Third, examples of applications to studies of soil chemistry—particularly soil particle chemistry is presented. Finally, several “new frontiers” in AFM research are discussed. The aim is to provide the reader with a sense of the ever-increasing capabilities of AFM and to point out some of the potential problems and limitations that need to be recognized, addressed, and eventually overcome.