表面活性剂溶液清洗油污土壤试验研究

选用两种阴离子型表面活性剂十二烷基苯磺酸钠(LAS)和十二烷基硫酸钠(SDS)及两种非离子型表面活性剂Triton X-100和Tween 80,研究了临界胶束浓度(CMC)附近各表面活性剂溶液对柴油的增溶及一次性清洗油污土壤的能力。结果表明:在CMC附近,各表面活性剂对柴油的增溶能力大小顺序为SDSSDS>Triton X-100>Tween 80,两种阴离子型表面活性剂的清洗效果优于两种非离子型表面活性剂。     

塿土对阴-非离子表面活性剂的吸附特征研究

以平衡吸附法研究了塿土对阴离子表面活性剂（SDS）、非离子表面活性剂（TritonX-100、Tween80和Brij35）的吸附特征,考察了pH、阴-非离子表面活性剂混合对塿土吸附表面活性剂的影响。结果表明,非离子表面活性剂在塿土上吸附等温线均呈L型,且均符合Freundlich和Langmuir方程;塿土对SDS的吸附等温线呈LS型,可用Freundlich方程来描述;塿土对4种表面活性剂吸附量的大小顺序为Tween80〉SDS〉Brij35〉TritonX-100。当阴-非离子表面活性剂一起进入土壤中,SDS-Brij35之间的相互影响不大;TritonX-100与SDS相互作用较大,无论二者以何种方式混合都会使TritonX-100在塿土上的吸附量增加,SDS的吸附量下降;SDS与Tween80之间的相互作用最大,混合后吸附量均下降,但Tween80吸附量降低的幅度最大。pH对非离子表面活性剂的吸附影响不大,而随着pH的增加,塿土对SDS的吸附百分率明显下降;在pH为8.0时,塿土对非离子表面活性剂的吸附百分率达到80以上。因此在选择合适的表面活性剂进行有机污染土壤修复和治理时,考虑土壤的特性和表面活性剂的结构是非常重要的。     

Pentachlorophenol interactions with soil

Pentachlorophenol (PCP) adsorption and desorption equilibrium was studied with two Menfro silt loam soils — upper horizon and lower horizon. For the adsorption studies the variables were: temperature (10 and 30 °C) and the amount of organic matter. The variables for the desorption studies were: temperature (10 and 30 °C), pH and the presence of an anionic and a cationic surfactant. The results from these studies confirmed the importance of soil organic matter for adsorption of PCP on the soils. The adsorption data at different temperatures indicated the physical nature of the adsorption process. The desorption data produced non-singularity and some PCP was irreversibly adsorbed onto the soil despite repeated washings. Increased pH increased the desorption of PCP from the soil. The anionic surfactant, sodium dodecylbenzene sulfonate (SDS) was able to desorb significant amounts of PCP from the soil at doses equal to critical micelle concentration (CMC). But, the nonionic surfactant, surfactant, Triton X-405 required a much higher dose (twice the CMC) to cause a significant desorption of PCP from the soil.     

Influence of surfactant charge on antimicrobial efficacy of surfactant-stabilized thyme oil nanoemulsions

Thyme oil-in-water nanoemulsions stabilized by a nonionic surfactant (Tween 80, T80) were prepared as potential antimicrobial delivery systems (pH 4). The nanoemulsions were highly unstable to droplet growth and phase separation, which was attributed to Ostwald ripening due to the relatively high water solubility of thyme oil. Ostwald ripening could be inhibited by incorporating ≥75% of corn oil (a hydrophobic material with a low water solubility) into the nanoemulsion droplets. The electrical characteristics of the droplets in the nanoemulsions were varied by incorporating ionic surfactants with different charges after homogenization: a cationic surfactant (lauric arginate, LAE) or an anionic surfactant (sodium dodecyl sulfate, SDS). The antifungal activity of nanoemulsions containing positive, negative, or neutral thymol droplets was then conducted against four strains of acid-resistant spoilage yeasts: Zygosaccharomyces bailli, Saccharomyces cerevisiae, Brettanomyces bruxellensis, and Brettanomyces naardenensis. The antifungal properties of the three surfactants (T80, LAE, SDS) were also tested in the absence of thymol droplets. Both ionic surfactants showed strong antifungal activity in the absence of thymol droplets, but no antimicrobial activity in their presence. This effect was attributed to partitioning of the antimicrobial surfactant molecules between the oil droplet and microbial surfaces, thereby reducing the effective concentration of active surfactants available to act as antimicrobials. This study shows oil droplets may decrease the efficacy of surfactant-based antimicrobials, which has important consequences for formulating effective antimicrobial agents for utilization in emulsion-based food and beverage products.     

Isothermal titration calorimetry study of pectin-ionic surfactant interactions

Isothermal titration calorimetry (ITC) was used to measure enthalpy changes resulting from injection of anionic (sodium dodecyl sulfate, SDS) or cationic (dodecyl trimethylammonium bromide, DTAB) surfactants into aqueous 1 wt % pectin solutions (30, 60, or 90% methoxylated). In the absence of pectin, the critical micelle concentrations (cmc) determined by ITC were 14.7 mM for DTAB and 7.7 mM for SDS. Binding of DTAB to pectin was endothermic and was attributed to electrostatic attraction between the cationic surfactant and anionic biopolymer. Binding of SDS to pectin was exothermic and was attributed to hydrophobic interactions. Pectin reduced the cmc of SDS, probably because of long-range electrostatic repulsion between the molecules. Above a particular concentration, which depended on pectin and surfactant type, both ionic surfactants promoted pectin aggregation (monitored by turbidity increase). This study demonstrates the potential of ITC for providing valuable information about interactions between polysaccharides and amphiphiles.     

Effectiveness of the Iodide Ligand Along with two Surfactants on Desorbing Heavy Metals from Soils

Used with one of two surfactants (SDS, an anionic surfactant, and Triton X-100, a nonionic surfactant), the ligand, I^? was evaluated as a washing agent for the desorption of Cd from naturally and artificially contaminated soils. Increasing amounts of the ligand, I^?, with a surfactant, specifically removes higher levels of Cd but not Cu, Zn and Pb. After seven washings, the ligand, I^? with the nonionic surfactant, Triton X-100, removed 65 and 90% of the Cd from soils I and II, containing respectively, to 15 and 1275 mg of Cd/kg. The ligand, I^?, and the anionic surfactant, SDS, removed 35 and 70% of the Cd from soils I and II, respectively. Before washing, the carbonate fraction of soil I contained the most Cd (48%) while the exchangeable and carbonate fractions of soil II contained 29 and 33% of the total Cd, respectively. For soil I, SDS with/ without the ligand desorbed Cd mainly from the carbonate and oxide fractions, while only Triton X-100 with ligand I^? could remove Cd from the exchangeable fraction. For soil II, Cd was desorbed from the exchangeable fraction only when either surfactant was used in combination with the ligand. Thus, a surfactant with ligand can extract specific heavy metals from soils and selective sequential extraction is useful in identifying which fraction can be targeted by the surfactant – ligand agent.     

Influence of rhamnolipids and triton X-100 on the biodegradation of three pesticides in aqueous phase and soil slurries.

The effect of surfactants on the biodegradation of trifluralin and atrazine (by Streptomyces PS1/5) and coumaphos (by degrading consortia from a contaminated cattle dip) in liquid cultures and soil slurries was tested at different concentrations of a rhamnolipid mixture (Rh-mix) and Triton X-100 (TX-100). The extent of trifluralin biodegradation in liquid culture was improved at high concentrations of both surfactants. The extent of atrazine degradation dropped in the presence of either surfactant. Coumaphos biodegradation improved slightly at Rh-mix dosages >3000 microM; however, it was readily inhibited by TX-100 at amounts above the critical micelle concentration. In soil slurries, the extent of both trifluralin and atrazine biodegradation was higher in Hagerstown A (HTA) soil than in Hagerstown B (HTB) soil and was not significantly affected by the presence of either surfactant. The onset of trifluralin biodegradation was retarded at higher concentrations of surfactants. In the absence of surfactant, up to 98% of coumaphos in both soil slurries was transformed. At increasing dosages of Rh-mix, the onset of coumaphos biodegradation was retarded, but the removal efficiency of the pesticide increased. Rh-mix and TX-100 depletion was observed during Streptomyces PS1/5 growth in liquid cultures. Rh-mix concentration also decreased during coumaphos biodegradation, whereas TX-100 concentration was not affected. These results suggest that surfactants, added for the purpose of increasing the apparent water solubility of hydrophobic organic compounds, may have unintended effects on both the rate and extent of biodegradation of the target compounds if the surfactants can also be degraded by the microorganisms in the system.     

Beta-lactoglobulin structure and retinol binding changes in presence of anionic and neutral detergents

Bovine beta-lactoglobulin (beta-LG) in vivo (in milks) has been found in complexes with lipids such as butyric and oleic acids. To elucidate the still unknown structure-function relationship in this protein, the structural changes of beta-lactoglobulin variant A (beta-LG A) in the presence of anionic surfactant such as sodium n-dodecyl sulfate (SDS) and in the presence of nonionic surfactant such as Triton X-100 have been investigated. Subsequently, the retinol binding by beta-LG has been investigated in the presence of various amounts of these surfactants as its binding indicator. The results of UV-vis and fluorescence studies show a higher denaturating effect of SDS at acid pH that can be due to greater positive charges of beta-LG at this pH indicating also the nonspecific hydrophobic interactions of Triton X-100 with beta-LG at all studied pHs. Isothermal titration calorimetry (ITC) measurements indicate the endothermic nature of beta-LG/SDS interactions and the exothermic nature of Triton X-100/beta-LG interactions. The analysis of the binding data demonstrates the absence of considerable changes in retinol binding properties of beta-LG in the presence of various amounts of these surfactants. This implies that surfactant binding does not change the conformation of beta-LG in the regions defining the retinol-binding site.     

离子型表面活性剂对菠菜生长与土壤酶活性的影响

为评价离子型表面活性剂对土壤生态的安全性,利用盆栽试验,研究了十六烷基三甲基溴化铵（Cetyltrimethyl ammonium bromide,CTAB）和十二烷基硫酸钠（Sodium dodecyl sulfate,SDS）对菠菜生物量、叶片抗氧化酶和土壤酶活性的影响。结果表明：CTAB和SDS都对菠菜的生长表现出低浓度促进高浓度抑制,其对菠菜生长造成抑制的临界浓度分别是500、1000 mg·kg-1。各CTAB和SDS浓度处理都显著增加了叶片超氧化物歧化酶（SOD）的活性。随着CTAB浓度增加,SOD活性呈现先增加后下降的趋势;SDS处理下,在50~750 mg·kg-1浓度范围内SOD活性无显著变化,浓度>750 mg·kg-1 SOD活性显著降低。CTAB处理下,叶片过氧化氢酶（CAT）活性主要受到促进作用,SDS处理下CAT活性先受到促进后受到抑制。除个别浓度外,CTAB和SDS都使叶片过氧化物酶（POD）的活性降低,不同浓度之间POD活性变化规律性不强。各CTAB浓度处理下,土壤脱氢酶、脲酶、蔗糖酶和中性磷酸酶的活性几乎都受到抑制;各SDS浓度处理下,土壤脱氢酶和蔗糖酶的活性都不同程度的增加,土壤脲酶和中性磷酸酶的活性,除个别情况外都受到抑制。CTAB和SDS都在一定程度上表现出土壤生态毒性,CTAB的毒性要大于SDS。     

Maltodextrin-anionic surfactant interactions: isothermal titration calorimetry and surface tension study

Interactions between maltodextrin (DE = 10) and an anionic surfactant (sodium dodecyl sulfate, SDS) were studied in a buffer solution (pH 7.0, 10 mM NaCl, 20 mM Trizma, 30.0 degrees C) using isothermal titration calorimetry (ITC), surface tension, differential scanning calorimetry (DSC), and turbidity techniques. ITC measurements indicated that the binding of SDS to maltodextrin was exothermic and that, on average, one SDS monomer bound per 24 glucose units of maltodextrin at saturation. Surface tension measurements indicated that there was a critical surfactant concentration ( approximately 0.05 mM SDS) below which surfactant and maltodextrin did not interact and that the amount of surfactant bound to the maltodextrin above this concentration increased with increasing maltodextrin concentration. Turbidity measurements indicated that the solutions remained transparent at all maltodextrin (0-1 wt %) and SDS (0-20 mM) concentrations studied, which suggested that phase separation did not occur. DSC measurements indicated that no phase transitions occurred between 10 and 110 degrees C for maltodextrin solutions (0.5 wt %) in the presence or absence of surfactant. A phase diagram was developed to describe the interactions between SDS and maltodextrin.     

Solubilization of Nitrogen Heterocyclic Compounds Using Different Surfactants

In order to develop surfactant-enhanced remediation for nitrogen heterocyclic compounds (NHCs) (aniline, indole, and quinolone), the solubilization properties of micellar solutions of five surfactants, namely sodium dodecyl sulfate (SDS), rhamnolipid (RL), polysorbate (Tween 80), sorbitan monolaurate (Span 20), and iso-octyl phenoxy polyethoxy ethanol (TX-100) were investigated in this work. The solubilization capacities were quantified using critical micelle concentration (CMC) as well as thermodynamic and kinetic experiments. Besides, nuclear magnetic resonance (¹H NMR) spectra were used to infer the locus of NHCs solubilized by SDS and TX-100. The results from the properties of five surfactants indicated that CMC was affected by temperature, while the micellization was spontaneous and could be both endothermic and exothermic based on the type of surfactant and temperature. Furthermore, the difference in compensation temperature was caused by different solubilization mechanism for various surfactants. The solubilization results showed that the solubilization of NHCs in the surfactant solutions followed a pseudo-first-order kinetic model. Meanwhile, the change in proton’s chemical shift depended on the structure of NHCs and the solubilization ability of surfactants. Finally, the orthogonal experiment (L16(4³)) was elementarily designed to optimize the solubilization conditions of indole and the results showed that RL could be a better choice for solubilizing NHCs.

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Graphical Abstract ?

     

复合表面活性剂（SDS/Tw-80）对长链烷烃的增溶和柴油污染土壤的洗脱作用

表面活性剂能够快速高效处理石油污染土壤,但由于所选用表面活性剂的类型和配比、土壤性质、污染物种类的差异,洗脱效果和作用原理不尽相同。通过研究复合表面活性剂SDS/Tw-80对石油烷烃的表观增溶和从受试土壤中的洗脱过程,探讨和阐释影响洗脱过程的各种因素。结果表明,复合表面活性剂对污染土壤中的石油烷烃洗脱效果较单一表面活性剂更好,且随复合表面活性剂SDS/Tw-80中的组分配合比例的增大而显著提高,同时能够降低胶束的成束浓度要求,扩大目标物在单位胶束内的容纳量,促使各目标长链烷烃进入胶束内部的趋势加大。复合表面活性剂的适用不仅能够改善胶束构成,且组分间表现出显著的协同作用,从而使在保证较高洗脱效率的同时能够大幅降低试剂用量,有效地克服土壤的吸附作用,把对土壤性状的影响降低,对土壤质量的恢复具有积极意义。     

Arsenic Contamination in Rice, Wheat, Pulses, and Vegetables: A Study in an Arsenic Affected Area of West Bengal, India

Water retention and transport in soils is dependent upon the surface tension of the aqueous phase. Surfactants present in aqueous solution reduce the surface tension of aqueous phase. In soil–water systems, this can result in water drainage and reductions in field capacity and hydraulic conductivity. In this investigation, the surface tension of surfactant solutions mixed with soil—in a constant fixed ratio—was measured as a function of surfactant concentration. Two anionic surfactants were used: sodium dodecyl sulphate and sodium bis (2-ethylhexyl) sulfosuccinate. Two soils were also used—a clay soil and a sandy soil. The key observation made by this investigation was that the addition of soil to the surfactant solution provided a further component of surface tension reduction. Neither soil sample reduced the surface tension of water when surfactant was absent from the aqueous phase, though both soils released soil organic matter at low surfactant concentrations as shown by measurement of the chemical oxygen demand of the supernatant solutions. Furthermore, both surfactants were shown to be weakly adsorbed by soil as shown by the use of a methylene blue assay. It is therefore proposed that the additional reduction in surface tension arises from synergistic interactions between the surfactants and dissolved soil organic matter.     

Bioremoval of Surfactant from Laundry Wastewater in Optimized Condition by Anoxic Reactors

Effects of ethanol and nitrate on linear alkyl benzene sulfonate (LAS) degradation were investigated using central composite design. At experimental design, removal of 99.9% was observed in batch reactors (1 L) with 9.8 to 41.2 mg L^?1 of LAS. The batch reactors were kept under agitation at 120 rpm and 30 °C. Ethanol (co-substrate) and nitrate (electron acceptor) were statistically significant factors (p?<?0.05) in surfactant removal. Optimal values were 97.5 and 88 mg L^?1 for ethanol and nitrate, respectively. LAS removal was kinetically investigated by varying surfactant concentration while using optimal values. Batch I (27 mg L^?1 LAS) exhibited greater degradation rate (K^LAS) (0.054 h^?1) in the presence of ethanol and nitrate. Nonetheless, in Batch II (60 mg L^?1 LAS), the K^LAS values decreased in those reactors probably due to inhibition by excess substrate for same concentrations of nitrate and ethanol added in reactors. As LAS concentration increased, the dominance of bacterial populations also increased, whereas diversity index decreased from 2.8 (inoculum) to 2.4 and 2.5 for reactors with both added nitrate and ethanol and those with only added ethanol, respectively. Probably, a selection of microbial populations occurred in relation to LAS concentration. The nitrate and ethanol, at able concentration, made it possible the induction of denitrifying microrganisms foward to LAS removal.     

Kinetic,Equilibrium, and Thermodynamic Analyses of Ni(II) Biosorption from Aqueous Solution by Acorn Shell of <Emphasis Type="Italic">Quercus crassipes</Emphasis>

Exposure to divalent nickel [Ni(II)] poses a significant risk to human health. The present study was conducted to evaluate the biosorption capacity of acorn shell of Quercus crassipes Humb. & Bonpl. (QCS) for removal of Ni(II) ions from aqueous solutions in terms of kinetics, equilibrium, and thermodynamics. Batch biosorption studies showed that the Ni(II) biosorption behavior of QCS is strongly dependent on solution pH, shaking contact time, initial Ni(II) concentration, and temperature. Specifically, Ni(II) biosorption was found to increase with increasing solution pH, contact time, initial Ni(II) concentration, and temperature. Modeling of the Ni(II) biosorption kinetic and equilibrium data showed that the best agreement of experimental data was achieved with the pseudo-second-order kinetics model and the Freundlich isotherm model, respectively. The calculated thermodynamic parameters indicated that the Ni(II) biosorption process was endothermic, non-spontaneous, and chemical in nature. Fourier-transform infrared (FTIR) spectroscopy analysis showed that acidic functional groups, namely hydroxyl, carbonyl, and carboxyl functional groups, present on the QCS surface are likely to be involved in the biosorption of Ni(II) ions. The performance of QCS was compared with those of other reported biosorbents in terms of the efficiency of Ni(II) removal from aqueous solutions, revealing that QCS is highly effective in terms of its biosorption capacity. These findings indicate that QCS can be used as a low-cost, highly effective, and environmentally friendly alternative biosorbent for the detoxification of Ni(II)-contaminated water and wastewater.     

Rhamnolipid Foam Enhanced Remediation of Cadmium and Nickel Contaminated Soil

Column experiments were conducted to evaluate the feasibility of using a rhamnolipid foam to remove heavy metals (Cd and Ni) from a sandy soil contaminated with Cd (1706 ppm) and Ni (2010 ppm). Best results were obtained from the foam generated by a 0.5% rhamnolipid solution with an initial pH value of 10.0 after flushing with 20-pore-volume of solution. These conditions removed 73.2% of the Cd and 68.1% of the Ni. Removal efficiencies by foam generated by a chemical surfactant, Triton X-100, were investigated as a comparison. It removed 65.5% of the Cd and 57.3% of the Ni under the same conditions. After a 20-pore-volume liquid solution flushing, 0.5% rhamnolipid (initial pH 10.0) without foam generation removed 61.7% of the Cd and 51.0% of the Ni, whereas 0.5% Triton X-100 (initial pH 10.0) removed 52.8% of the Cd and 45.2% of the Ni. Distilled water with adjusted pH only was also used to flush through the contaminated soil column as a control. It removed 17.8% of the Cd and 18.7% of the Ni. This study shows that rhamnolipid foam technology can be an effective means for the remediation of cadmium and nickel contaminated soil.     

Toxicity of Synthetic Surfactants to the Marine Macroalga, Ulva lactuca

The toxicities of three synthetic surfactants to the marine macroalga, Ulva lactuca, have been examined by monitoring chlorophyll a fluorescence quenching. The anionic surfactant, sodium dodecyl sulphate (SDS), exerted no measurable toxicity over the concentration range 0?C10 mg L^?1, while presence of the non-ionic surfactant, Triton X-100 (TX), elicited a small reduction in photochemical efficiency that was independent of concentration. The cationic surfactant, hexadecyltrimethylammonium bromide (HDTMA), incurred a dose-dependent response to ??3 mg L^?1 (EC₅₀?=?2.4 mg L^?1), but a reduction in toxicity thereafter. Presence of TX had little effect on the toxicity of HDTMA but an equimolar concentration of SDS directly offset the impact of HDTMA on photochemical efficiency. Relative toxicities of the surfactants are attributed to differences in affinity for the algal surface and tendencies to disrupt cell membranes and interact with intracellular macromolecules. Non-linear dose responses and antagonistic effects are attributed to non-specific interactions between molecules of the same surfactant and electrostatic interactions between molecules of different amphiphilic character.     

Determination of Ni (II) in beverages without any sample pretreatment by adsorptive stripping chronopotentiometry (AdSCP)

The purpose of this paper was to use adsorptive stripping chronopotentiometry for the determination of Ni (II) in worldwide consumed beverages without any sample pretreatment, using dimethilglyoxime (DMG) as complexing agent and a glassy carbon mercury film electrode as the working electrode. Ni (DMG)2 complex is adsorbed onto the mercury film at an electrolysis potential of -500 mV for 60 s and then reduced by a -5 microA constant cathodic current. The sensitivity of the method was studied for certified reference water and black tea in the pH range 6.5-11. At pH 9.5 in ammonia buffer, a detection limit of 0.2 microg L(-1) was achieved; the instrumental precision (expressed as rsd %) was 1.5%, and the accuracy, expressed as obtained recoveries both from certified and not certified matrixes, ranged from 93.0 to 95.5 %. The chronopotentiometric analysis executed on commercial beverages provided evidence that black tea samples were the richest source of Ni (II) (1500-3700 microg L(-1)), followed by coffee (100.0-300.5 microg L(-1)); bottled mineral water showed a Ni (II) concentration lower than 4.6 microg L(-1). Among alcoholic beverages, red wines presented the highest content of Ni (II) (55.5-105.0 microg L(-1)). Significant differences were noticed between Ni (II) levels of fermented and distillated alcoholic beverages; moreover, canned cola and beer did not show higher Ni (II) levels with respect to the glass-bottled products.     

Sorption of Anionic Surfactants SDS,AOT and Cationic Surfactant Hyamine 1622 on Natural Soils

Sorption of anionic surfactants Aerosol-OT (AOT) and Sodium Dodecyl Sulfate (SDS), as well as cationic surfactant Hyamine1622 on two soil surfaces were investigated. Batch equilibriumtechniques were used under laboratory conditions to observe the amount of surfactant adsorbed and desorbed to and from thesoil surfaces. Data obtained were fitted to two sorption isotherms in order to characterise the sorption process. The nature of the sorbate (i.e. the surfactant) was found to be important in the sorption process. Anionics adsorbed via hydrophobic bonding, whereas `cation-exchange' mechanism was dominant in the sorption of cationic Hyamine 1622. Anionic AOT,with a double chain, and hence a higher critical packing parameter, adsorbed more strongly onto the surface compared tothe singly-chained SDS.     

Adsorption of anionic surfactant (sodium dodecyl sulfate) on silica

The adsorption of an anionic surfactant sodium dodecyl sulfate (SDS) on a negatively charged silica was studied to provide a better understanding of surfactant adsorption phenomena in an electrostatic repulsion environment between surfactant and soil. The adsorption experiment was conducted under different electrolyte concentration and pH. Results indicated that adsorption happened with hydrophobic interaction, although electrostatic repulsion was generated between SDS and silica surface. The adsorption amount decreased with decreasing electrolyte concentration and increasing pH due to the increase of electrostatic repulsion. The influence of electric potential near the silica surface on the adsorption was confirmed with the modified Langmuir adsorption equation, 1-pK basic Stern model and zeta potential. Because silica is ubiquitous in soils and the water environment, the adsorption characteristics of an anionic surfactant is important when we consider the fate of an anionic surfactant in the environment. The result is also useful when considering the fate of agricultural chemicals which contain negative charge and hydrophobic sites.