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Analytica Chimica Acta (v.598, #2)
Synthesis and electrochemical applications of gold nanoparticles by Shaojun Guo; Erkang Wang (pp. 181-192).
This review covers recent advances in synthesis and electrochemical applications of gold nanoparticles (AuNPs). Described approaches include the synthesis of AuNPs via designing and choosing new protecting ligands; and applications in electrochemistry of AuNPs including AuNPs-based bioelectrochemical sensors, such as direct electrochemistry of redox-proteins, genosensors and immunosensors, and AuNPs as enhancing platform for electrocatalysis and electrochemical sensors.
Keywords: Gold nanoparticle; Biosensor; Electrocatalysis; Electrochemical sensor; Nanomaterial
Advances in hyphenated analytical techniques for shotgun proteome and peptidome analysis—A review by Lianghai Hu; Mingliang Ye; Xiaogang Jiang; Shun Feng; Hanfa Zou (pp. 193-204).
Proteomics is defined as the analysis of part or all of the protein components of a complex biological system (a cell, organ or tissue) at a given moment. Due to the huge number of proteins encoded by the genome, novel analytical techniques must be developed to meet the need of large scale analysis. This has led to the hyphenation of multiple techniques to achieve this object. Here current status of the hyphenated analytical techniques of one-dimensional and multidimensional liquid chromatography–mass spectrometry for shotgun proteomic analysis is reviewed, and on-line techniques for automated sample preparation and injection are also covered. In addition, the hyphenated techniques for peptidome analysis are also covered.
Keywords: Proteomics; Peptidomics; Hyphenated techniques; Multidimensional separation; Liquid chromatography–mass spectrometry; Review
Determination of trace α-fetoprotein variant by affinity adsorption solid substrate-room temperature phosphorimetry and its application to the forecast of human diseases by Jia-Ming Liu; Zhen-Bo Liu; Qiao-Mei Lu; Fei-Ming Li; Shi-Rong Hu; Guo-Hui Zhu; Xiao-Mei Huang; Zhi-Ming Li; Xiu-Mei Shi (pp. 205-213).
In the presence of ion perturber LiAc, 4-generation polyamidoamine dendrimers (4G-D) could emit strong and stable room temperature phosphorescence (RTP) signal atλexmax/λemmax=511.8/675.3nm on nitrocellulose membrane (NCM), and Triton X-100 could sharply enhance the RTP signal of 4G-D. Triton X-100-4G-D was used to label concanavalin agglutinin (Con A) to get the labeling product Triton X-100-4G-D-Con A. Quantitative specific affinity adsorption (AA) reaction between Triton X-100-4G-D-Con A and α-fetoprotein variant (AFP-V) could be carried out on the surface of NCM, whose product Triton X-100-4G-D-Con A-AFP-V could emit strong and stable RTP and its Δ Ip was proportional to the content of AFP-V. According to the facts above, a new affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) for the determination of trace AFP-V by Con A labeled with Triton X-100-4G-D was established. Detection limits of this method were 0.23fgspot−1 (direct method, corresponding concentration: 5.8×10−13gmL−1) and 0.13fgspot−1 (sandwich method, corresponding concentration: 3.2×10−13gmL−1). It has been successfully applied to determine the content of AFP-V in human serum and forecast human diseases, for its high sensitivity, long RTP lifetime, good repeatability, high accuracy and little background perturbation withλemmax at the long wavelength area. Meanwhile, the mechanism for the determination of trace AFP-V using AA-SS-RTP was also discussed.
Keywords: α-Fetoprotein variant; 4-Generation dendrimers; Label; Concanavalin agglutinin; Affinity adsorption solid substrate-room temperature phosphorimetry
Volatilization of in situ generated antimony trichloride using aqua regia vapors for the determination of ultra trace impurities in high-purity antimony using inductively coupled plasma-mass spectrometry by A.C. Sahayam; Shiuh-Jen Jiang; Chia-Ching Wan (pp. 214-218).
Volatilization of Sb has been carried out using vapors generated from aqua regia for the determination of non-volatile impurities present in antimony. The antimony trichloride (SbCl3) thus generated was volatilized >99.9% at 250°C in 90min. Recoveries of spiked impurities Al, Mn, Co, Ni, Cu, Sr, Ag, Cd, Ba, Pb were found to be >90% (47% for Pb) after volatilization of the matrix element. The only reagent used (aqua regia vapors) was generated in situ in a closed environment minimizing the external contamination. The procedure offers experimental blanks and method detection limits at lower ngg−1 using ICP-MS. The method has been applied for the determination of impurities in two Sb samples of 99.5 and 99.999% (5N) purity. The developed method was validated using bromide volatilization procedure. The results obtained by the present method were also compared with those of Sector Field ICP-MS (SF-ICP-MS).
Keywords: Antimony; High-purity materials; Aqua regia vapors; Antimony trichloride; Volatilization; Inductively coupled plasma-mass spectrometry
Using mean field approach independent component analysis to fatty acid characterization with overlapped GC–MS signals by Maryam Vosough (pp. 219-226).
In this paper, mean field independent component analysis (MF-ICA) was applied as a deconvolution method to separate complex gas chromatographic–mass spectrometric (GC–MS) signals obtained from fatty acid analysis of fish oil. The separation which is a blind operation was used as a complementary method in identification of the unknown components of a mixture and in quantification purposes, as well. In MF-ICA, the sources (mass spectra) are recovered from the mean of their posterior distributions and mixing matrix (chromatograms) and noise level are estimated through the maximum a posterior (MAP) solution. The number of independent components (ICs) in the overlapping signals can be estimated by the difference between the reconstructed and original GC–MS data. It was found that the chromatographic profiles and the mass spectra of the components in overlapping multicomponent GC–MS data can be accurately recovered with and without previously background correction. The resolved mass spectral sources satisfactory are identified using mass spectral search system. The recovered chromatographic area and the relative content of each analyte considering selected number of ICs are calculated and the results are compared with the ones obtained previously by using heuristic evolving latent projections (HELP) method.
Keywords: Independent component analysis; Mean field; Deconvolution; Fatty acids; Gas chromatography–mass spectrometry
The performance of ν-support vector regression on determination of soluble solids content of apple by acousto-optic tunable filter near-infrared spectroscopy by Dazhou Zhu; Baoping Ji; Chaoying Meng; Bolin Shi; Zhenhua Tu; Zhaoshen Qing (pp. 227-234).
The ν-support vector regression ( ν-SVR) was used to construct the calibration model between soluble solids content (SSC) of apples and acousto-optic tunable filter near-infrared (AOTF-NIR) spectra. The performance of ν-SVR was compared with the partial least square regression (PLSR) and the back-propagation artificial neural networks (BP-ANN). The influence of SVR parameters on the predictive ability of model was investigated. The results indicated that the parameter ν had a rather wide optimal area (between 0.35 and 1 for the apple data). Therefore, we could determine the value of ν beforehand and focus on the selection of other SVR parameters. For analyzing SSC of apple, ν-SVR was superior to PLSR and BP-ANN, especially in the case of fewer samples and treating the noise polluted spectra. Proper spectra pretreatment methods, such as scaling, mean center, standard normal variate (SNV) and the wavelength selection methods (stepwise multiple linear regression and genetic algorithm with PLS as its objective function), could improve the quality of ν-SVR model greatly.
Keywords: Support vector machine; ν; -Support vector regression; Apple; Soluble solids content; Near-infrared spectroscopy
Dissolved iron analysis in estuarine and coastal waters by using a modified adsorptive stripping chronopotentiometric (SCP) method by Ricardo D. Riso; Benoît Pernet-Coudrier; Matthieu Waeles; Pierre Le Corre (pp. 235-241).
An electrochemical method based on adsorptive stripping chronopotentiometry (SCP) with a rotating mercury film electrode has been developed for the determination of dissolved iron (III) at subnanomolar concentrations in estuarine and coastal waters. The detection limit was 0.11nM after adsorption time of 60s. Compared to the other chronopotentiometric methods available for dissolved iron measurement in natural and estuarine waters, the procedure described here exhibits a 15-fold better sensitivity. Therefore, it allows one to accurately quantify concentrations commonly found in estuarine and coastal waters. Moreover, by using the speciation scheme proposed by Aldrich and van den Berg (Electroanalysis 10 (1998) 369), several forms could be measured, i.e. reactive iron (Fe R) and reactive iron (III) (FeIII R), or estimated, i.e. complexed iron (Fe C) and reactive iron (II) (FeII R). The method described here is reliable, fast, inexpensive and compact. It was applied successfully to the study of the chemical speciation of dissolved iron along the salinity gradient of the Aulne estuary (Brittany-France).
Keywords: Stripping chronopotentiometry; Dissolved iron; Estuary; Coastal seawater
Detection of thrombin using electrogenerated chemiluminescence based on Ru(bpy)32+-doped silica nanoparticle aptasensor via target protein-induced strand displacement by Xiaoying Wang; Jingming Zhou; Wen Yun; Shasha Xiao; Zhu Chang; Pingang He; Yuzhi Fang (pp. 242-248).
A sensitive and selective aptasensor using tri(2,2′-bipyridyl)ruthenium(II)-doped silica nanoparticles (Ru(bpy)32+-doped SNPs) as DNA tags for detection of thrombin is developed based on the target protein-induced strand displacement of the DNA probe. For the proposed aptasensor, the aptamer was assembled on the surface of the Au electrode through Au-S binding. The hybridization event between the DNA probe labeled by the Ru(bpy)32+-doped SNPs and the aptamer was evaluated by electrogenerated chemiluminescence (ECL) measurements. Then, the DNA probe was displaced by thrombin and the binding event between the thrombin and the aptamer was monitored by ECL measurements again. The difference of ECL intensity (Δ IECL) of the two events could be used to quantify the thrombin. Other proteins, such as bovine serum albumin and bovine hemoglobin, had almost negligible Δ IECL. Under the optimal conditions, the Δ IECL was linearly related to the concentration of the thrombin in the range of 10fM to 10pM and the detection limit was down to 1.0fM since SNPs containing a large number of Ru(bpy)32+ molecules were labeled on the DNA probe.
Keywords: Thrombin; Electrogenerated chemiluminescence; Ru(bpy); 3; 2+; -doped silica nanoparticles; Aptasensor; Target protein-induced strand displacement
A simple and rapid method for measuring dissolved oxygen in waters with gold microelectrode by You-Peng Chen; Shao-Yang Liu; Han-Qing Yu (pp. 249-253).
Dissolved oxygen (DO) is an essential indicator in chemical, biological and biochemical processes and needs to be rapidly measured in many cases. In this work, a rapid, sensitive and simple electrochemical method, first derivative linear sweep voltammetry (FD-LSV), was established for the determination of DO. The peak height of FD-LSV was found to be influenced by scan rate and microelectrode surface area. An empirical formula was proposed to describe the relationship between the FD-LSV peak height and the influencing factors. Compared with other electrochemical methods for DO determination, the FD-LSV method needed lesser time and was more accurate. This method was successfully applied for the determination of the DO levels of various environmental water samples.
Keywords: Dissolved oxygen; First derivative linear sweep voltammetry; Microelectrode; Scan rate; Surface area; Water
Application of NiTi alloy coated with ZrO2 as a new fiber for solid-phase microextraction for determination of halophenols in water samples by Dilma Budziak; Edmar Martendal; Eduardo Carasek (pp. 254-260).
A new fiber for solid-phase microextraction (SPME) employing a metallic support coated with an inorganic material is proposed. A nitinol alloy (NiTi) was used as the support material due to its super elasticity and shape memory properties. Zirconium oxide (ZrO2) was electrodeposited onto NiTi using chronoamperometry. The surface characteristics and morphology of the coated and uncoated support were evaluated through scanning electronic microscopy and dispersive energy microanalysis. This assembly was applied in the extraction of three halophenols from aqueous samples. A multivariate approach was used for optimization of the variables involved in the system. The Doehlert matrix was used for evaluation of the best derivatization conditions and a Box-Behnken design to obtain the best extraction conditions. In order to investigate the repeatability, one fiber was used for six extraction tests under similar conditions and the relative standard deviations (R.S.D.) were lower than 12.5%. Detection limits were lower than 0.30ngmL−1. Correlation coefficients were higher than 0.997. Extraction efficiency of the NiTi–ZrO2 fiber was similar to a PDMS 7μm commercial fiber, even though it had a lower coating thickness of 1.35μm. Considering the amount extracted per unit volume, the NiTi–ZrO2 fiber had a better extraction profile when compared to commercial fibers. The new SPME fiber has a lifetime of over 500 extractions. Thus, it is a promising alternative for low-cost analysis, as the proposed fiber is robust, and easily and inexpensively prepared.
Keywords: NiTinol; Zirconium oxide; Electrodeposition; Solid-phase microextraction
Evaluation of carbohydrate antigen 50 in human serum using magnetic particle-based chemiluminescence enzyme immunoassay by Xu Wang; Jin-Ming Lin; Xitang Ying (pp. 261-267).
A magnetic particles (MPs)-based chemiluminescence enzyme immunoassay (CLEIA) with high sensitivity, specificity, rapidity, and reproducibility was proposed for the evaluation of tumor marker, carbohydrate antigen 50 (CA50) in human serum. The immunomagnetic particles coated with anti-fluorescein isothiocyanate (FITC) antibody was used as dispersed solid phase for the immunoassay, which was based on a sandwich immunoreaction of FITC-labeled anti-CA50 antibody, CA50 antigen, and alkaline phosphatase (ALP)-labeled anti-CA50 antibody, and was based on a subsequent chemiluminescence reaction of ALP with 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2′-adamantane) (AMPPD) solution. The CL emission intensity was directly proportional to the amount of analyte present in a sample solution. The effects of several physicochemical parameters, including the concentration of FITC-labeled anti-CA50 antibody, the dilution ratio of ALP-labeled anti-CA50 antibody, the volume of MPs and substrate, the immunoreaction time and other relevant variables upon the immunoassay were studied and optimized. The proposed method exhibited advantages in a lower minimum detectable concentration of 1.0UmL−1 with comparison to the commercially available immunoradiometric assay (IRMA), and showed a larger linear range of 0 to 300UmL−1, as well as less total assay time of only 50min with comparison to both IRMA and microplate CLEIA. The coefficient of variation was less than 7 and 11% for intra- and inter-assay precision, respectively. This method has been successfully applied to the evaluation of CA50 in human serum with recoveries from 82 to 112%, and showed a good correlation with the commercially available CA50 IRMA.
Keywords: Magnetic particles; Chemiluminescence enzyme immunoassay; Tumor marker; Carbohydrate antigen 50
Differentiation of isomeric allylic alkenyl methyl ethers by Raman spectroscopy by Paul Clifford; Richard D. Bowen; Howell G.M. Edwards; Dennis W. Farwell (pp. 268-279).
The Raman spectra of several pairs of alkenyl methyl ethers of general structure R1R2CCR5C(R3R4)OCH3 and R1R2C(OCH3)C(R5)CR3R4 (R1, R2, R3, R4, R5=H or C nH2 n+1, n=1–3) are reported and discussed, with a view to establishing whether Raman spectroscopy offers a viable means of distinguishing between these isomeric unsaturated species. Key bands associated with the ν(sp2CH) and ν(CC) stretching modes are found to be particularly useful in this connection: R1R2CCHCH2OCH3 and R1R2C(OCH3)CHCH2 ethers (R1, R2=CH3, C2H5) are easily distinguished on this basis. Differentiation of their lower homologues, R1CHCHCH2OCH3 and R1CH(OCH3)CHCH2 (R1=CH3, C2H5, C3H7), by similar means is also quite straightforward, even in cases where cis and trans isomers are possible. Pairs of isomeric ethers, such as CH3CHC(CH3)CH2OCH3 and CH3CH(OCH3)C(CH3)CH2, in which the structural differences are more subtle, may also be distinguished with care. Deductions based on bands ascribed to the stretching vibrations are usually confirmed by consideration of the signals associated with the corresponding δ(sp2CH) deformation vibrations. Even C2H5CHCHCH(C3H7)OCH3 and C3H7CHCHCH(C2H5)OCH3 are found to have distinctive Raman spectra, but differentiation of these closely related isomers requires additional consideration of the low wavenumber region.
Keywords: Raman spectroscopy; Allylic; Ethers; Vibrational spectroscopy; Isomeric
Direct, non-destructive quantitative measurement of an active pharmaceutical ingredient in an intact capsule formulation using Raman spectroscopy by Jaejin Kim; Jaegeun Noh; Hoeil Chung; Young-Ah Woo; Mark S. Kemper; Youngil Lee (pp. 280-285).
The active pharmaceutical ingredient (ambroxol) in an intact capsule formulation has been non-destructively quantified using Raman spectroscopy. To improve the problem of insufficient representive sampling inherent in Raman measurements, we have employed a wide area illumination (WAI) scheme that enables much improved sample coverage through a circular excitation laser spot with a 6mm diameter. One of the anticipated sources of variation for this measurement was variation in the capsule shells. However, the WAI scheme significantly decreased the spectral variation among empty capsules compared to a measurement with a traditional small-spot excitation. Therefore, measurement variations emanating from the capsule shell did not significantly influence the accuracy of the determination of ambroxol concentrations. The resulting standard error of prediction (SEP) using the WAI scheme was comparable to that from previous Raman measurements which used a conventional small-spot excitation and employed a sampling scheme that involved rotation of an ambroxol pellet. It is further noteworthy that the SEP was also similar to that obtained from the use of transmission NIR spectroscopy, which was achieved by collection of spectra of the powdered capsule contents removed from the shell. The proposed Raman measurement using the WAI scheme in this case was sufficient to achieve the quantitative measurement of the active pharmaceutical ingredient (API) content of capsules non-destructively.
Keywords: Pharmaceutical capsule; Ambroxol; Non-destructive analysis; Raman spectroscopy; Wide area illumination
An integrated portable hand-held analyser for real-time isothermal nucleic acid amplification by Matthew C. Smith; George Steimle; Stan Ivanov; Mark Holly; David P. Fries (pp. 286-294).
A compact hand-held heated fluorometric instrument for performing real-time isothermal nucleic acid amplification and detection is described. The optoelectronic instrument combines a Printed Circuit Board/Micro Electro Mechanical Systems (PCB/MEMS) reaction detection/chamber containing an integrated resistive heater with attached miniature LED light source and photo-detector and a disposable glass waveguide capillary to enable a mini-fluorometer. The fluorometer is fabricated and assembled in planar geometry, rolled into a tubular format and packaged with custom control electronics to form the hand-held reactor. Positive or negative results for each reaction are displayed to the user using an LED interface. Reaction data is stored in FLASH memory for retrieval via an in-built USB connection. Operating on one disposable 3V lithium battery >12, 60min reactions can be performed. Maximum dimensions of the system are 150mm ( h)×48mm ( d)×40mm ( w), the total instrument weight (with battery) is 140g. The system produces comparable results to laboratory instrumentation when performing a real-time nucleic acid sequence-based amplification (NASBA) reaction, and also displayed comparable precision, accuracy and resolution to laboratory-based real-time nucleic acid amplification instrumentation. A good linear response ( R2=0.948) to fluorescein gradients ranging from 0.5 to 10μM was also obtained from the instrument indicating that it may be utilized for other fluorometric assays. This instrument enables an inexpensive, compact approach to in-field genetic screening, providing results comparable to laboratory equipment with rapid user feedback as to the status of the reaction.
Keywords: Field detection; Real-time nucleic acid sequence-based amplification; System in Package; Fluorometer
Optical fluoride sensor based on monomer–dimer equilibrium of scandium(III)-octaethylporphyrin in a plasticized polymeric film by Youngjea Kang; Jeff W. Kampf; Mark E. Meyerhoff (pp. 295-303).
A fluoride-selective optical sensor based on scandium(III)-octaethylporphyrin (Sc(III)OEP) as an ionophore within a plasticized PVC film is described. The presence of fluoride ion in the aqueous sample phase increases the formation of a difluoro-bridged Sc(III)OEP dimer species in the polymer film. The ability of the Sc(III) porphyrin to form the dimeric structure in the presence of fluoride is confirmed by UV–vis spectroscopy and X-ray crystallography. For more practical sensing applications, a pH chromoionophore (ETH 7075) is added to the plasticized PVC film along with Sc(III)OEP and the observed optical response is based on coextraction of protons with sample phase fluoride to create the dimeric porphyrin and a protonated chromoionophore species. The selectivity pattern observed is F−≫ClO4−, SCN−, NO3−>Br−, Cl−. Only organic salicylate is a significant interferent. Fast and reversible fluoride response is observed over the range of 10−4 to 10−2M fluoride, allowing use of the sensing film in a waveguide configuration for flow-injection measurements.
Keywords: Fluoride sensors; Scandium(III) porphyrin; Waveguide optical sensors; Dimer–monomer equilibrium
Resonance Rayleigh scattering spectral method for the determination of raloxifene using gold nanoparticle as a probe by Shao Pu Liu; You Qiu He; Zhong Fang Liu; Ling Kong; Qun Min Lu (pp. 304-311).
When gold nanoparticles were being prepared by sodium citrate reduction method, citrate anions self-assembled on the surface of gold nanoparticles to form supermolecular complex anions with negative charges, and protonated raloxifene (Ralo) was positively charged and could bind with the complex anions to form larger aggregates through electrostatic force and hydrophobic effects, which could result in the remarkable enhancement of the resonance Rayleigh scattering intensity (RRS), and the appearance of new RRS spectra. At the same time, the second-order scattering (SOS) and frequency-doubling scattering (FDS) intensities were also enhanced. The maximum wavelengths were located near 370nm for RRS, 520nm for SOS, and 350nm for FDS, respectively. Among them, the RRS method had the highest sensitivity and the detection limit was 5.60ngmL−1 for Ralo, and its linear range was 0.05–2.37μgmL−1. A new RRS method for the determination of trace Ralo using gold nanoparticles probe was developed. The optimum conditions of the reaction and influencing factors were investigated. In addition, the reaction mechanism and the reasons for the enhancement of RRS were discussed.
Keywords: Gold nanoparticles; Resonance Rayleigh scattering; Raloxifene
Stability-indicating high performance thin layer chromatography determination of Paroxetine hydrochloride in bulk drug and pharmaceutical formulations by A. Venkatachalam; Vidya S. Chatterjee (pp. 312-317).
A simple selective precise and stability-indicating high performance thin layer chromatographic method of analysis of Paroxetine hydrochloride both as a bulk drug and in formulations was developed and validated. The method employed TLC (Thin Layer Chromatography) aluminum precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of butanol:acetic acid:water (8:2:0.5, v/v/v). This system was found to give compact spots for Paroxetine HCl (Rf, retardation factor, value–0.48±0.02). Paroxteine HCl was subjected to acid and alkali hydrolysis, oxidation and photodegradation, where the degraded product was well separated from the pure drug. Densitometric analysis of Paroxetine hydrochloride was carried out in the absorbance mode at 295nm. The linear regression analysis data for the calibration spots showed good relationship with (regression) r2=0.9903 in the amount range of 300–1500ng (nanogram) per spot. The mean value of co-relation co-efficient, slope and intercept were 0.9903±0.001, 5.38±0.058 and 182.5±2.16 respectively. The method was validated for precision, recovery and robustness. The limits of detection and quantitation were 50 and 150ng, respectively. The drug doesnot undergo degradation with oxidation, but gets affected in acidic and alkaline conditions. The acid and alkali degradation showed extra peaks at 0.4 and 0.08 Rf, respectively. This indicates that the drug is susceptible to acidic and alkaline medium. As the method could effectively separate the drug from its degradation products, it can be employed as a stability-indicating one.
Keywords: Pharmaceutical formulations; Paroxetine hydrochloride; High performance thin layer chromatography (HPTLC); Densitometry
Competing ligand exchange-solid phase extraction method for the determination of the complexation of dissolved inorganic mercury (II) in natural waters by Frank J. Black; Kenneth W. Bruland; A. Russell Flegal (pp. 318-333).
A method employing dual competitive ligand exchange followed by solid phase extraction (CLE-SPE) for characterizing the complexation of inorganic Hg(II) in natural waters is described. This method employs parallel use of two competing ligands: diethyldithiolcarbamate (DEDC), which forms hydrophobic complexes with Hg(II), and thiosalicylic acid (TSA), which forms hydrophilic complexes with Hg(II). Inorganic mercury complexed by natural and competing ligands are separated based on hydrophobicity using C18 solid phase extraction columns.Data modeling allows for the calculation of the concentration and conditional stability constants of natural ligands capable of complexing Hg(II) in both the operationally defined hydrophilic and hydrophobic fractions. The use of multiple ligand concentrations, and thus multiple analytical windows, to characterize different ligand classes within both of these two fractions is described. Studies of the kinetics of the ligand exchange involved, potential for changes in the stability of natural ligands during freezing and thawing, potential breakthrough during solid phase extraction, as well as the method's precision and estimation of error, are presented and discussed.Results from the application of the method to natural freshwaters demonstrated that in the limited samples collected over 99.99% of the ambient inorganic mercury is strongly complexed by ligands with conditional stability constants (KHgLcond, Hg2+) on the order of 1030, values similar to that of reduced sulfur ligands. At ambient conditions 85–90% of the mercury exists in hydrophobic complexes in these freshwaters, but strong Hg-binding ligands exist in both the hydrophobic and hydrophilic fractions.
Keywords: Mercury; Complexation; Speciation; Ligands; Freshwater