On-line coupling of size exclusion chromatography with electrospray ionization-tandem mass spectrometry for the analysis of aquatic fulvic and humic acids

A method was developed for the analysis of humic and fulvic acids by size-exclusion chromatography-electrospray ionization-tandem mass spectrometry using a completely volatile eluent. Humic and fulvic acids were separated into three peaks. These fractions occupied different mass ranges and showed differences in the fine structure of their mass spectra. The low-molecular-weight (LMW) fraction of fulvic acids is most sensitively determined by ESI-MS, and it appears that previous results obtained by infusion-ESI-MS were primarily determined by this fulvic acid fraction. The average molecular weight of this fractions turned out to be lower than that reported from infusion-ESI-MS measurements. Its scan spectra and the product ion spectra of some of its molecular anions perfectly match those previously obtained from whole fulvic acid mixtures. Obviously, a class of well-defined polycarboxylated molecules exist that occurs in all fulvic acid fractions thus far investigated. With decreasing elution time and increasing molecular weight, detection by ESI-MS loses sensitivity as compared to the parallel UV recording, and the fine structure of the scan spectra becomes increasingly uniform for both fulvic and humic acids. The average molecular weight of the HMW fraction exceeds those values calculated from infusion experiments. Scan spectra and product ion spectra of the high-molecular-weight (HMW) fraction of both the humic and the fulvic acids suggest that the HMW fraction consists of several subunits that originate from the LMW fraction.     

Impulse-driven heated-droplet deposition interface for capillary and microbore LC-MALDI MS and MS/MS

An automated off-line liquid chromatography-matrix-assisted laser desorption ionization (LC-MALDI) interface capable of coupling both capillary and microbore LC separations with MALDI mass spectrometry (MS) and tandem mass spectrometry (MS/MS) has been developed. The interface is a combination of two concepts: analyte concentration from heated hanging droplets and impulse-driven droplet deposition of LC fractions onto a MALDI sample plate. At room temperature the interface allows the coupling of capillary LC separations (i.e., flow rate of <5 microL/min) with MALDI MS. With heating, it can be used to combine microbore LC operated at a relatively high flow rate of up to 50 microL/min with MALDI MS. The collected fractions can be analyzed by MALDI MS and MS/MS instruments, such as time-of-flight (TOF) and quadrupole-TOF MS. Performance of the interface was examined using several peptide and protein standards. It was shown that, using MALDI-TOF MS, [GLU1]-fibrinopeptide B could be detected with a total injection amount of 5 fmol to microbore LC. Chromatographic performance was also monitored. A peak width of 12 s at half-height for [GLU1]-fibrinopeptide B showed no evidence of band broadening due to the interface. The ability of the interface to mitigate ion suppression was studied using a mixture of 100 fmol of [GLU1]-fibrinopeptide B and 10 pmol of cytochrome c tryptic digest. Although fully suppressed under direct MALDI conditions, LC-MALDI analysis was able to detect the 100 fmol peptide with 10 s fraction collection. Finally, the ability to inject relatively large sample amounts to improve detectability of low-abundance peptides was illustrated in the analysis of phosphopeptides from alpha-casein tryptic digests. A digest loaded on column to 2.4 microg and analyzed by LC-MALDI MS/MS resulted in 82% sequence coverage and detection of all nine phosphoserine residues. It is concluded that, being able to handle both high- and low-flow LC separations, the impulse-driven heated-droplet interface provides the flexibility to carry out MALDI analysis of peptides and proteins depending on the information sought after, analysis speed, and sample size.     

Characterization of aquatic humic substances to DBPs formation in advanced treatment processes for conventionally treated water

An advanced water treatment demonstration plant consisted of ozone/granular activated carbon processes was operated to study feasibility of the processes. Natural organic matter (NOM) from raw and process waters at the demonstration plant was isolated into humic and non-humic fractions by physicochemical fractionation method to investigate characteristics of humic fraction (i.e., humic substances, HS) as a predominant haloform reactant. Ozone did not significantly oxidize the carboxylic fraction (from 39.1 to 35.9%), while GAC removed some of the carboxylic fraction (from 35.9 to 29.1%). Formation potential of trihalomethanes (THMs) as compared to haloacetic acids formation potential (HAAFP) was highly influenced by HS. Higher yields of THMs resulted from chlorination of HS with a higher phenolic content and phenolic fraction in the HS gradually decreased from 60.5% to 15.8% through the water treatment. The structural and functional changes of HS were identified by elemental, Fourier-transform infrared (FT-IR) and proton nuclear magnetic resonance ((1)H NMR) analyses, and these results were mutually consistent. The functional distribution data obtained by using A-21 resin could be used to support the interpretation of data obtained from the spectroscopic analyses. Decreases in ratio of UV absorbance at 253 nm and 203 nm (A(253)/A(203)) and DBPFPs/DOC showed consistent trends, therefore, A(253)/A(203) ratio may be a good indicator for the disinfection by-product formation potentials (DBPFPs).     

Carbon isotope composition of dissolved humic and fulvic acids in the Tokachi River system

This study reports carbon isotopic ratios (Δ(14)C and δ(13)C) of dissolved humic and fulvic acids in the Tokachi River system, northern Japan. These acids have a refractory feature and they represent the largest fraction of dissolved organic matter in aquatic environments. The acids were isolated using the XAD extraction method from river water samples collected at three sites (on the upper and lower Tokachi River, and from one of its tributaries) in June 2004 and 2005. δ(13)C values were -27.8 to -26.9 ‰ for humic and fulvic acids. On the other hand, the Δ(14)C values ranged from -247 to +26 ‰ and the average values were -170 ± 79 ‰ for humic acid and -44 ± 73 ‰ for fulvic acid. The difference was attributed to the residence time of fulvic acid in the watershed being shorter than that of humic acid. The large variation suggested that humic substances have a different pathway in each watershed environment.     

Compositional and functional features of humic acid-like fractions from vermicomposting of sewage sludge and cow dung

The chemical changes occurring in five different substrates of sewage sludge spiked with different proportions of cow dung after vermicomposting with Eisenia foetida for 90 days were investigated. Their humic acid-like (HAL) fractions were isolated to determine the elemental and functional composition, and structural and functional characteristics using ultraviolet/visible, Fourier transform infrared (FT-IR) and fluorescence spectroscopies and scanning electron microscopy. After vermicomposting, the total organic C and C/N ratio decreased, and the total extractable C and humic acid (HA) C increased in all substrates. In the HAL fractions, the C and H contents, C/N and C/O and aliphatic structures, proteinaceous components and carbohydrates decreased, while the O and N and acidic functional group contents and C/H ratio, aromaticity and polycondensation structures increased. Further, the results suggest that the addition of cow dung to sewage sludge could improve the quality of organic matter humification of the substrates. The structures of HAL fractions in vermicomposts resembled those typical of soil HA, especially the vermicompost of cow dung alone. Scanning electron microscopy showed the microstructure of HAL fraction in final product became close-grained and lumpy. Overall results indicate that vermicomposting was an efficient technology for promoting organic matter (OM) humification in sewage sludge and cow dung alone, as well as in mixtures of both materials, improving their quality and environmental safety as a soil OM resource for utilization as soil amendments.     

Ultralow-volume fraction collection from NanoLC columns for mass spectrometric analysis of protein phosphorylation and glycosylation

An ultralow volume fraction collection system referred to as nano fraction analysis chip technology (nanoFACT) is reported. The system collects 25-2500-nL fractions from 75-microm nanoLC columns into pipet tips at a user-defined, timed interval, typically one fraction every 15-120 s. Following collection, the fractions in the tip dry down naturally on their own in such a way as to create a concentrated band at the very end of the interior of the pipet tip. The fractions are then reconstituted directly in the pipet tips in approximately 250 nL of solvent prior to analysis. Because the chromatography and reconstitution solvent are independent, the reconstitution solvent can be selected to maximize ionization efficiency without compromising chromatography. In the infusion analysis of the nanoLC fractions, a low-flow electrospray chip is used which consists of 400 nozzles, each with an inner diameter of 2.5 microm and yielding flow rates of approximately 20 nL/min. Therefore, when reconstituted in 250 nL, each nanoLC fraction can be analyzed for over 10 min. This increase in analysis time allows for signal averaging, resulting in higher data quality, collision energy optimization, slower scanning techniques to be used, such as neutral loss and precursor ion scanning, higher resolution scans on FTMS instruments, and improved peptide quantitation. Furthermore, the nanoLC fractions could be archived in the pipet tips for analysis at a later date. Here, the advantages of nanoFACT are shown for phosphorylation analysis using bovine fetuin and glycosylation analysis using bovine ribonuclease B (RNase B). In the phosphorylation analysis, a comparison between conventional nanoLC and a nanoFACT analysis was performed. An MS/MS spectrum of a triply phosphorylated peptide, 313-HTFSGVApSVEpSpSSGEAFHVGK-333 could only be obtained using nanoFACT, not with nanoLC. Furthermore, spectral quality for the nanoFACT analysis was significantly improved over nanoLC. This was determined by comparing the number of diagnostic ions between the nanoFACT and nanoLC spectra, and it was found that the nanoFACT spectra contained a 19% or greater number of diagnostic ions for nonphosphorylated peptides and 55% or greater for phosphorylated peptides. For the glycosylation analysis, the glycosylation site of RNase B was fully characterized using 100 fmol of tryptic digest on a three-dimensional ion trap mass spectrometer.     

Quantitation in the solid-state 13C NMR analysis of soil and organic soil fractions

13C CP-MAS and DP-MAS spin-counting experiments have been carried out on an absolute basis for a specific whole soil and its humin, humic acid, and fulvic acid fractions, as well as a sample of the soil that was treated with 2% HF(aq). The results confirm previous conclusions that a substantial fraction of the carbon content indicated by classic elemental analysis is missed in some samples, especially whole soil and humin, by both CP-MAS and DP-MAS 13C NMR methods, and that the problem is more serious for CP-MAS than for DP-MAS. This study also confirms the fact that treatment of soil organic matter with 2% HF(aq) dramatically reduces this problem but may generate some structural uncertainties associated with significant structural alterations that accompany the HF(aq) treatment, as indicated by the 13C NMR data. The relationship between the "missing carbon" problem and the concentration of paramagnetic centers, especially Fe(III) centers, is explored in substantial detail.     

Tandem-cartridge solid-phase extraction followed by GC/MS analysis for measuring partition coefficients of association of polycyclic aromatic hydrocarbons to humic acid

A tandem-cartridge solid-phase extraction system combining reversed-phase separation and dynamic ion exchange followed by GC/MS analysis was studied for measuring partition coefficients (Kdoc) of association of polycyclic aromatic hydrocarbons (PAHs) to humic acid. Time course batch experiments revealed that the association of PAHs to humic acid included a slow stage with equilibrium time of 5-7 days. The disequlibrium and retention of the PAH-humic acid associate during reversed-phase separation lead to systematic negative error for the measured partition coefficients; more accurate results were achieved with a three-cartridge tandem system in which the negative error was assessed and corrected via the PAH analytes measured on the second reversed-phase cartridge. The potential and advantages of use of C18 disk cartridges instead of conventional C18 cartridges to set up the tandem system for measuring the partition coefficients were also studied. With the tandem-cartridge system, the measured partition coefficients did not change with most experimental conditions, but a trend of decreasing partition coefficients with humic acid concentration was observed, implying possible nonlinear association of the PAHs and humic acid.     

Screening for disulfide bonds in proteins by MALDI in-source decay and LIFT-TOF/TOF-MS

An automated screening method is presented that uses MALDI in-source decay (MALDI-ISD) of disulfide bonds for identification of disulfide-linked peptides in MALDI mass spectra. Peptides released by ISD of a disulfide bond can be detected at an m/z ratio that corresponds to the singly protonated peptide with a reduced cysteine residue. Therefore, screening of peak lists for signal patterns that fulfill the equation, m/z (peak A) + m/z (peak B) - m/z (H2 + H+) = m/z (peak C), facilitated identification of putative ISD fragments of disulfide-linked peptides (peaks A and B) and their precursors (peak C). Signals (peak C) from putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the existence of a disulfide bond. Using this method, we identified all 4 disulfide bonds in RNAseA and 8 two-disulfide clusters comprising 16 out of the 17 disulfide bonds in BSA. The presented screening method accelerated the identification of disulfide bonds in RNAseA and BSA, because the number of MS/MS spectra to be acquired was reduced by 1 order of magnitude. Less than 5% of the signals selected for LIFT-TOF/TOF-MS did not correspond to disulfide-linked peptides. Furthermore, the number of possible assignments for disulfide-linked peptides was reduced by 2-3 orders of magnitude, because knowledge of the mechanism of disulfide bond fragmentation by ISD permitted use of stricter rules for the interpretation of mass spectra. Therefore, interpretation of MS/ MS spectra of disulfide-linked peptides was considerably simplified in comparison to conventional approaches.     

Planar limit-assisted structural interpretation of saturates/aromatics/resins/asphaltenes fractionated crude oil compounds observed by Fourier transform ion cyclotron resonance mass spectrometry

Planar limits, defined as lines generated by connecting maximum double-bond equivalence (DBE) values at given carbon numbers, are proposed as a means of predicting and understanding the molecular structure of compounds in crude oil. The slopes and y-intercepts of the lines are determined by the DBE/carbon number ratios of functional groups defining the planar limits. For example, the planar limit generated by a serial addition of saturated cyclic rings has a slope of 0.25. The planar limit formed by the linear and nonlinear addition of benzene rings yields lines with slopes of 0.75 and 1, respectively. The y-intercepts of these lines were determined by additional functional groups added within a series of molecules. Plots of DBE versus carbon number for S(1) class compounds observed by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) showed that saturates/aromatics/resins/asphaltenes (SARA) fractions exhibited unique slopes and y-intercepts. The slope of the planar limit observed from a saturates fraction matched well with the slope of a planar limit generated by the serial addition of saturated cyclic rings. The slopes of planar limits of aromatics and resins fractions were very similar to that obtained from the linear addition of benzene rings. Finally, the slope of the asphaltenes fraction was almost identical to the slope obtained from the nonlinear addition of benzene rings. Simulated and experimental data show that SARA fractions exhibit different molecular structure characteristics. On the basis of the slope and y-intercept of the planar limit, the structures of molecules in SARA fractions were predicted and suggested. The use of planar limits for structural interpretation is not limited to crude oil compounds but can also be used to study other organic mixtures such as humic substances or metabolites.     

Association of radionuclides with colloids in soil solutions

Association of Pu and Am with variously sized particles in soil extracts is studied by ultrafiltration. In aqueous extracts, the Pu and Am concentrations depend on the soil type and aqueous solution composition. In the aqueous extracts, from 60 to 100% of the radionuclides and organic carbon are associated with fine colloidal particles (<0.05 μm). The presence of humic acid increases the recovery of radionuclides by a factor of 4–8 depending on the soil type. The Pu and Am distribution among the groups and fractions of humus acids is studied. In the group of humic acids of chernozem, from 65 to 75% of the radionuclides are associated with the fraction with MW > 100 kDa; about 25%, with the fraction with MW 50–10 kDa; and 2–4%, with the low-molecular-weight fraction with MW < 3 kDa. In the group of fulvic acids, 44–49% of the radionuclides is found in the fraction with MW > 100 kDa, and 42–51%, in the fraction with MW < 3 kDa.     

顺酐改性乙丙三元胶／尼龙的共混研究

对极性差别较大的乙丙三元橡胶和尼龙1010体系,首次通过官能化反应及动态全硫化共混方法制得了一种耐温性较高的热塑性弹性体,并对相容性的表征及分散相粒径问题进行了研究。实验表明,上述共混体系仍是非均相的不相容体系,尼龙相的玻璃化转变温度向低温移动的程度可以作为相容程度的判断。此时,在两相界面间是橡胶向尼龙基质的渗透,渗透方向应该也对改性作用有其特有的贡献。同时分散相的最大填充体积φ_(max)可以用来表征相容性的优劣。     

Exploitation of endogenous protease activity in raw mastitic milk by MALDI-TOF/TOF

The proteic profiling of bovine milk produced by cows with subclinical mastitis was obtained by MALDI mass spectrometry. A simple procedure of chemical fractionation of raw milk was developed, whereby less complex fractions of proteins were obtained prior to mass spectrometric and SDS-PAGE analysis. The profiles of milk proteins thus obtained could allow the identification of either early markers of the acute phase of mastitis or endogenous peptide of innate immune response. The activity of the endogenous proteases in raw milk produced from each quarter of healthy and mastic cows was therefore assayed over 24-, 48-, 96-, and 216-h incubation at 37 degrees C at both physiological and acid pH. Sequence-specific peptides were identified for each fraction by MS/MS experiments, and all tandem mass spectra were evaluated using MASCOT database searching. The results show a specific proteolytic activity of endogenous enzyme toward beta-casein precursor (P02666), alpha-S2-casein (P02663), alpha-S1-casein (P02662), and kappa-casein (P02668).     

Practical implementation of 2D HPLC scheme with accurate peptide retention prediction in both dimensions for high-throughput bottom-up proteomics

We describe the practical implementation of a new RP (pH 10 - pH 2) 2D HPLC-ESI/MS scheme for large-scale bottom-up analysis in proteomics. When compared to the common SCX-RP approach, it provides a higher separation efficiency in the first dimension and increases the number of identified peptides/proteins. We also employed the methodology of our sequence-specific retention calculator (SSRCalc) and developed peptide retention prediction algorithms for both LC dimensions. A diverse set of approximately 10,000 tryptic peptides from the soluble protein fraction of whole NK-type cells gave retention time versus hydrophobicity correlations, with R (2) values of 0.95 for pH 10 and 0.945 for pH 2 (formic acid) separation modes. The superior separation efficiency and the ability to use retention prediction to filter out false-positive MS/MS identifications gives promise that this approach will be a method of choice for large-scale proteomics analyses in the future. Finally, the "semi-orthogonal" separation selectivity permits the concatenation of fractions in the first dimension of separation before the final LC-ESI MS step, effectively cutting the analysis time in half, while resulting in a minimal reduction in protein identification.     

Selective gas-phase cleavage at the peptide bond C-terminal to aspartic acid in fixed-charge derivatives of Asp-containing peptides

This study focuses on the molecular level interpretation of the selective gas-phase cleavage at aspartic acid residues (Asp) in protonated peptides. A phi3P+CH2C(=O)group (phi = 2,4,6-trimethoxyphenyl) is attached to the N-terminal nitrogen of the selected peptides LDIFSDF and LDIFSDFR, via solid-phase synthesis, to "mimic" the tightly held charge of a protonated arginine (Arg) residue. Collision-induced dissociation in a quadrupole ion trap instrument and surface-induced dissociation in a dual quadrupole instrument were performed for electrospray-generated ions of the fixed-charge peptide derivatives. Selective cleavages at Asp-Xxx are observed for those ions with charge provided only by the fixed charge or for those with a fixed charge and one Arg plus one added proton. This supports a previously proposed mechanism which suggests that the cleavages at Asp-Xxx, initiated by the acidic hydrogen of the Asp residue, become significant when ionizing protons are strongly bound by Arg in the protonated peptides. It is clear that the fixed charge is indeed serving as a "mimic" of protonated Arg and that a protonated Arg side chain is not required to interact with the Asp to induce cleavage at Asp-Xxx. When the number of protons exceeds the number of Arg in a peptide containing Arg and Asp, nonselective cleavages occur. The fragmentation efficiency of the peptides is consistent with the idea that these nonselective cleavages are promoted by a mobile proton. The peptide with a fixed charge and one added proton, [phi3P+CH2C(=O)-LDIFSDF + H]2+, fragments much more efficiently than the corresponding peptide with a fixed charge, an Arg and one added proton, [phi3P+CH2C(=O)-LDIFSDFR + H]2+; both of these fragment more efficiently than the peptide with a fixed charge and no added proton, phi3P+CH2C(=O)-LDIFSDF. MS/MS/MS (i.e., MS3) experimental results for bn ions formed at Asp-Xxx from phi3P+CH2C(=O)-LDIFSDF and its H/D exchange derivative, phi3P+CH2C(=O)-LDIFSDF-d11, are consistent with the bn ions formed at Asp-Xxx having a succinic anhydride cyclic structure. MS/MS experiments were also carried out for phi3P+CH2C(=O)-AAAA, a peptide derivative containing active hydrogens only at amide nitrogens plus the C-terminus, and its active H/D exchange product, phi3P+CH2C(=O)-AAAA-d5. The results show that a hydrogen originally located at an amide nitrogen is transferred away in the formation of a cyclic charge remote b ion.     

Specular reflection and diffuse reflectance spectroscopy of soils

Studies on the occurrence and effects of specular reflection in midinfrared spectra of soils have shown that distortions due to specular reflection occur for both organic (humic acid) and non-organic fractions (carbonates, silica, ashed fraction of soil). The results explain why the spectra of CaCO(3) in limed soils do not match published spectra and offer an explanation as to why the presence of inorganic C interferes with the development of calibrations for organic C. These results may also have implications for the use of mid-infrared spectra for quantitative and qualitative analysis of soils. For example, libraries of spectra collected by means other than diffuse reflectance would be largely useless for comparing mineral spectra to soil spectra. To obtain the best results with forages and grains, it is necessary to develop separate calibrations for different products, but this has not seemed to be a problem for diverse sets of soil samples with C contents of 0 to 5%. Mid-infrared calibrations have also appeared to be more robust than the corresponding near-infrared calibrations in that fewer outliers are found. However, the results discussed here indicate that at least for some soil types (e. g., large differences in mineralogy or C contents), separate calibrations may be necessary.     

Chromium fractionation in semi-arid soils amended with chromium and tannery sludge

We studied Cr fractionation in three semi-arid soils (cultivated, under-the-canopy, and outside-the-canopy soils). The soils were amended with: Cr3+, Cr6+, tannery sludge, Cr3++tannery sludge, and Cr6++tannery sludge and all soils were incubated for 30 and 120 days at 25 degrees C. The Cr in three semi-arid soils was fractionated using sequential extraction (Tessier scheme). Data of Cr fractionation were used to find the correlations with microbial activities determined in previous work. The microbial activities: CO2-C evolved, dehydrogenase activity and nitrification were determined in the same soils amended with the same treatments. Tannery sludge was added at 0.0125 g g(-1) soil and Cr3+ or Cr6+ at 250 microg g(-1) soils. After 120 days of incubation, higher values of concentration of Cr were found in the residual fraction in the three soils amended with all the treatments, except cultivated and outside-the-canopy soils amended with Cr6++tannery. The non-residual fraction tended to decrease with time except in cultivated and outside-the-canopy soils amended with Cr6++tannery sludge. CO2-C evolved was significantly correlated (p<0.05 and p<0.001) with fractions bound to: Mn oxides, Fe oxides organic matter at 30 and 120 days of incubation (from r=0.827 to 0.979). Dehydrogenase activity was correlated with fractions bound to Fe oxides and bound to organic matter, and nitrification with fraction bound to organic matter at 30 days of incubation (r=0.874, 0.959, and 0.803, respectively). These results suggest that even in a sparingly available Cr fraction in semi-arid soils has effect on microbial activities.     

雷电流组合波形下碳纤维/环氧树脂基复合材料层压板雷击损伤试验

为了评估碳纤维/环氧树脂基复合材料位于不同飞机雷击分区下的雷击损伤程度及损伤特征,采用A+B、D+B+C和A+B+C+D 3种不同的雷电流波形组合,对两类不同尺寸复合材料层压板试验件（Type1和Type2）进行了模拟雷电流冲击试验,通过目视损伤观察和超声损伤扫描,分析评估了复合材料位于飞机1A区、2A区及1B区时的雷击损伤程度及特征,同时还对含铜网防护碳纤维/环氧树脂基复合材料层压板在不同雷击分区下的雷击防护效果进行了评估。结果表明：不同雷击分区下,碳纤维/环氧树脂基复合材料在雷电流作用下的损伤模式基本一致,包括纤维断裂、基体烧蚀及分层损伤;对于相同类型试验件,1B区对应雷击损伤程度最严重,其次为2A区,1A区损伤程度最小;在相同雷击分区下,长宽比较小的Type1型试验件雷击损伤程度大于长宽比较大的Type2型试验件;0.25 mm厚铜网能够有效对碳纤维/环氧树脂基复合材料进行雷击防护,位于1A区、2A区及1B区的含防护与无防护Type2型试件,前者雷击损伤程度较后者分别下降88.9%、53.9%和68.7%。     

A Study of the Fractionation Dose Effect on the Radiation Response of Windose B3 Dosimeter

Windose B3 dosimeters have been investigated using UV–vis spectrophotometry to determine the impact of the frequent interruptions during gamma irradiation on the absorbed dose. To achieve this purpose, a set of experiments were conducted by examining the behavior of these dosimeters as result of these accidents. Experiments were conducted based on varying four influencing factors: the storage temperature, the number of fractions, the total delivered dose and the storage time. The chosen parameters (dose and fraction time) are selected from pre-studies and in accordance with conditions of industrial irradiation. The obtained results for one fraction or five fractions had bigger specific absorbance than the dosimeters receiving its dose without fractionation and that the effect of five fractions on the response of dosimeters is more significant than the effect of one fraction. The most important over response (15%) is obtained for 10 kGy as absorbed dose with five fractions, 40 °C as a storage temperature, and 24 h as storage time. The influence quantities have an effective effect on the Windose B3 dosimeter response and suitable corrections are indispensable.     

Thermoluminescence response of calcic bentonite subjected to conditions of high nuclear waste underground storage

Bentonite is regarded as a backfilling material for underground storage facilities of highly radioactive nuclear waste built on granite formations. In these facilities, bentonite will be subjected to a gradient of temperature and dose rate, achieving a very high integrated dose and, therefore, changes in its structure and physical properties may take place. Two experiments to discriminate between the thermal and the irradiation effect were performed. In the first (named BIC 2A), samples were subjected to temperature while in the second (named BIC-2B) the combined effect of temperature and irradiation was studied. The experimental conditions were: a thermal gradient between 130 degrees C and 90 degrees C, a maximum dose rate of 3.5 kGy.h(-1) and a gradient of the integrated dose between 1.75 MGy and 10 MGy. Both experiments lasted a total of 124 days. An irradiation source of 60Co with an activity close to 300,000 Ci, and bentonite samples of 200 mm in length and 50 mm in diameter were used. After the experiment, the samples were ground and two fractions were obtained: a fine fraction (<2 microm) enriched in montmorillonite clay mineral and a coarse fraction (>80 microm). The results are described of thermoluminescence analyses on the two fractions obtained which showed that the coarse fraction can be 100 times more sensitive to radiation than the fine fraction. On the other hand, the heated and irradiated samples showed a thermoluminescence response around 50 times greater than the samples that were only heated. In addition to this, the temperature and dose rate conditions are relevant parameters in the generation and stabilisation of radiation induced defects. Finally, the response of samples heated and irradiated for two months was quite similar to that obtained on samples heated and irradiated for four months, indicating a saturation phenomenon.