Differential extraction of sediment phosphates with NTA solutions

An extraction technique is described for the separation of iron- and calcium-bound phosphate. The iron-bound phosphate is extracted with a 0.05 M solution of Ca-NTA under reducing conditions. Iron, also, is brought into solution, which is an advantage over the NaOH extraction. The calcium-bound phosphate is extracted with a 0.05 M solution of Na-NTA. The NTA also extracts humic compounds. Organic phosphate compounds can be measured in the NTA extracts, unlike the NaOH or H₂SO₄ extracts such as are used in the (modified) Jackson procedure.Examples of some test compound extractions and of a calcareous sediment are given.     

Available phosphorus in lake sediments in The Netherlands

The amount of phosphorus available to algae in the sediments of four lakes in the western part of the Netherlands has been assessed by means of chemical extraction and bioassay techniques. In addition to direct chemical sediment analyses, extractions were carried out with an NTA column method and a stepwise NH₄ Cl-NaOH-HCI shaking method, the latter supposedly separating the weakly bound, the Fe- and Al-bound and the Ca-bound phosphates in the sediments. Bioassays, with sediment as the sole source of P, were made withScenedesmus quadricauda in modified Skulberg's 28 medium to determine the amount of phosphates available to algae.The average total P concentration of the sediments varied from 0.8 to 3.6 mg P g^–1 dry wt and correlated well with the net external P loading of the lakes. Uptake of P by algae in the bioassays varied from 0.4 to 36% — while NTA extracted 36–69% of the total P. The ratio NH₄Cl extracted/ NaOH extracted/ HCI extracted phosphates is different from lake to lake, although in all lakes the highest extractions (27–62% of total P) are found in the NaOH fraction. However, in the peaty sediments of these lakes, the NaOH step extracted not only the Fe- and Al-bound phosphates but, also, large amounts of humus compounds. Hence, this fraction also contains non-available organic P.The results are related to soil type and chemical characteristics of the sediments, and compared with data from other authors. A positive correlation was found between phosphate available to algae and NTA- and NaOH-extractable P, but the correlation with total phosphorus was higher. Moreover, algal-extractable P proved to be positively correlated with total iron and clay content and negatively with the amount of organic matter.It is concluded that the sediments in the investigated lakes show great variability and that the chemical extraction techniques cannot replace the bioassays to assess the amount of phosphorus available to algae.     

The carbohydrate constituents of the cell wall of suspension cultures of Rosa glauca

Sequential extractions of 14-day-old Rosa glauca cell walls cultured in vitro showed that two different types of acidic polysaccharide were present. One was extracted with EDTA or ammonium oxalate solutions, and the other remained in close association with cellulose even after 4.3 N NaOH extractions or 2 N H₂SO₄ hydrolysis. The cell wall has a low content in structural protein. The behaviour of each constituent sugar was followed during the course of the various extraction steps, and a complete quantitative account of the protein, uronic acid and neutral sugar components is given at each stage.     

Development of a microporous membrane liquid–liquid extractor for organophosphate esters in human blood plasma: identification of triphenyl phosphate and octyl diphenyl phosphate in donor plasma

An extractor has been developed for microporous membrane liquid–liquid extraction (MMLLE) of lipophilic xenobiotics at trace levels in biological fluids. This new construction allows the sample phase to be stirred, while the organic phase is pumped. The extractor was evaluated using human blood plasma with added organophosphate esters. The size exclusion properties of the membrane reduced lipid co-extraction by 94% compared to ordinary liquid–liquid extraction. In combination with a solid-phase extraction (SPE) step, the method was shown to remove plasma lipids efficiently and thus allow gas chromatographic separation of the compounds. The clean-up method described, including the SPE step, showed a high level of reproducibility, and recoveries of between 72 and 83% were obtained for five of the organophosphate esters after a 200-min extraction period. Using this technique, triphenyl phosphate and an isomer of octyl diphenyl phosphate were detected in human plasma obtained from blood donors. The concentration of triphenyl phosphate ranged between 0.13 and 0.15 μg/g plasma.     

Responses of the Phytoplankton Community of a Tropical Reservoir (São Paulo,Brazil) to the Enrichment with Nitrate,Phosphate and EDTA

The effects of artificial enrichment with nitrate, phosphate and EDTA on the phytoplankton community were studied in the Lobo Reservoir (São Paulo, Brazil). After 14 days of in situ incubation, the amounts of suspended matter and chlorophyll a, the numbers of cells and the carotene/chlorophyll ratio were determined. The addition of nitrate and phosphate to water samples produced significant effects on the chlorophyll a and cell counts, while EDTA acted only on the cell production. Both nitrate and phosphate, when analysed individually, caused a decrease in the value of the carotene/chlorophyll ratio. A synergistic effect of the addition of EDTA and N on the suspended matter was observed.     

Phosphate adsorption on bottom sediments of the Rio de la Plata

A study of the phosphate adsorption onto the bottom sediments of the Rio de la Plata has been made with the aim to understand the dynamics of this compound in the fluvio-marine system. Previous to the chemical analysis of the phosphate content, an extraction of the different adsorbed phosphate fractions have been made. In addition to phosphate, calcium, iron and aluminium in the sediment samples were determined. The phosphate is associated to the fine fractions of the sediments and good correlations with Al and Fe content in the bottom sediments were found. There is a relative decrease of the adsorbed phosphate on the bottom sediment in the areas where the influence of the marine water is more conspicuous; it is explained by the pH increase of the estuary waters due to the mixture with the marine waters. A hypothesis about the role of the ionic strength and the pH on the phosphate adsorption process is suggested.     

An improved phycobilin extraction method

One unique feature in cyanoprokaryotes, rhodophytes, and cryptophytes is the presence of phycobilin pigments-these water soluble pigments can absorb red, orange, yellow, and green light enhancing the spectral range available for cellular conversion to chemical energy. The presence of phycobilin pigment complexes can be detected using fluorescence, or absorbance measures. Efficient detection of these compounds is essential for use in calibrating absorbance in remote sensing or in physiological studies. The standard procedure for phycobilin analysis involves sonication, extraction in buffer potentially coupled with additional digestion steps using enzymes, repeated freeze and thawing cycles, followed by filtration, and spectrophotometric analyses. An alternative method, using asolectin-CHAPS ((3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid – AC)) solution for extraction, and three cycles of freeze/thawing/sonication, was compared to the phosphate buffer (PB) standard procedure. Cultures of both coccoid and filamentous cyanoprokaryota had improved extraction efficiency (38–80%) using AC. After two complete extractions, no pigment was detectable in AC and near baseline fluorescence was observed in the cell pellet, whereas the PB extraction method removed <90% of the phycobilins after two extractions. Phycocyanin concentration measured by AC extraction was better correlated to lipophilic pigment concentration than using phosphate buffer extraction. AC buffered to pH 6.7 was more effective than AC 3.75. One potential source of experimental error was determined to be the use of a baseline correction for the extraction buffer, not the sample.     

The distribution of phosphate in sediments and its relation with eutrophication of a Mediterranean coastal lagoon

A major problem of the Mediterranean coastal lagoons is an excessive input of nutrients (i.e. N and P), causing eutrophic conditions in summer. The sediments of these lagoons can serve as a reservoir by fixing phosphate, or as a source when this phosphate is released under certain conditions. Knowledge of nutrient sources and fluxes is needed if coastal lagoons are to be protected against eutrophication. Therefore, we have evaluated the total pool of phosphate in the lagoon sediments, and the quantity of phosphate which may be released.Sediment profiles have been analysed at two stations of the Lagune de Thau both in and outside the oyster-bank zone. A sequential fractionation, using chelating agents was performed to extract the inorganic (iron and calcium bound phosphate) and the organic phosphate fractions. A statistical analysis of the data set has revealed several significant factors which explain the fluctuations of the concentrations of each phosphate fraction. These factors are: the time of year (seasons), the depth (5 cm layers of sediment), and the site (station).A spatial and a temporal variation of the concentration of Tot-P was found. The largest variation between the two different zones appeared only in the first five cm of sediment. There is only a slight seasonal variation in the amount of phosphate at other depths at the two different zones. Season and station are the factors which control the variations in distribution of phosphate fractions. The spatial and temporal variations of the iron and calcium bound phosphate are explained by the redox potential and pH in the top layer of the sediment.Abbreviations In principle the general abbreviations are used (see page vi). Futhermore Fe(OOH)-P= Ferric hydroxide bound phosphate - CaCO₃-P= Calcium carbonate bound phosphate - ASOP= Acid soluble organic phosphate - ROP= Residual organic phosphate     

The evaluation of inorganic phosphate species in salt water lake sediments

Abstract

Surface sediments drawn from 10 shallow bays have been subjected to selective extraction in order to sub-divide the total P content into sub-categories such as water soluble P, Ca-P, Al-P and Fe-P. The reagents selected were similar to systems used in soil analysis, but evaluation of the procedures showed that the species values varied with time of extraction, weight of sediment taken, volume of extradant and chemical nature of the sediment. In water extractions, the P levels appeared to be determined by saturation with a sparingly soluble salt, while in acidic media P extract levels peaked (using different experimental conditions) due to loss of extracted P as a new phase (e.g. CaHPO₄) or through re-adsorption on other components.

The optimum conditions for P speciation in sediments must be determined from a series of preliminary studies because each of the five sediments studied in detail displayed individual characteristic behaviour.     

Kinetic Extractions of Nickel and Lead from Some Contaminated Calcareous Soils

The mobility of heavy metals in contaminated soils is dependent on the kinetics release from soils. Metal extraction over time is commonly used to distinguish two or more fractions of metal based on differences of extraction or release rates. Kinetic studies using 0.01 M CaCl₂, 0.01 M malic acid, and 0.01 M EDTA extractions were performed to characterize nickel (Ni) and lead (Pb) kinetic release in 10 contaminated calcareous soils. Proportions of Ni and Pb extracted with EDTA were higher than when using malic acid and CaCl₂, respectively. The release of Ni and Pb was characterized by an initial fast rate followed by a slower rate and could best be described by a two first-order reactions model with rate constants k₁ and k₂ and two metal pools: readily labile (Q ₁) and less labile (Q ₂). In an EDTA extractant, different Q₁ /Q₂ ratios for Ni and Pb were observed, indicating binding energies to soil constituents is not comparable. The k₁ of the model for Ni (average of 10 soils: 0.2204 h^?1 and 0.2359 h^?1 for 0.01 M CaCl₂ and 0.01 EDTA, respectively) was higher than Pb (0.1044 h^?1 and 0.1631 h^?1 for 0.01 M CaCl₂ and 0.01 M EDTA, respectively), indicating a higher potential of Ni for leaching and groundwater contamination in contaminated calcareous soils. Relationships between the fraction of Ni and Pb determined with the two first-order reactions model and the soil composition and Pb fractions were established. The results indicated that the efficiency of the extractions Ni and Pb from soils depends both on the Ni and Pb content and soil composition. Overall, the results indicated that the use of a 0.01 M malic acid washing solution would be preferred in the field condition.     

Experimental Assessment of Using Soluble Humic Substances for Remediation of Heavy Metal Polluted Soils

Since heavy metals are nondegradable and strongly bonded in soils, remediation of heavy metal polluted soils by extraction is difficult and current extraction techniques require harsh chemicals such as ethylenediaminetetraacetic acid (EDTA). However, use of EDTA is environmentally problematic because of costs, persistence, toxicity and deterioration of soil structure. Therefore, the potential of soluble natural humic substances (HS) to extract heavy metals from contaminated soils is tested as an environmentally friendly substitute for EDTA. A strongly polluted, calcareous urban soil (CRC soil) and a moderately polluted agricultural soil (CUP soil) were extracted at neutral pH in batch mode by three HS solutions from beech and Norway spruce litter (Beech-HS and Spruce-HS) and processed cow slurry (Bio-HS), all containing 25 mM dissolved organic carbon (DOC). After 10 extractions with a solution to soil ratio of 5:1 (L/kg), 8% to 39% of the total Cd, Cu, Ni and Pb soil contents, lowest for Ni and highest for Cu/Pb, were extracted. Natural and processed HS samples had comparable capacities to extract the heavy metals. A comparison of 100 mM DOC of Bio-HS and EDTA as extractants for Cu from the CRC soil showed extraction of 67% by EDTA and 41% by Bio-HS, indicating somewhat higher efficiency of EDTA than of HS. Sequential extraction of the CRC soil after Bio-HS and EDTA extraction showed removal of exchangeable, carbonate- and metal oxide-bound Cu but also of some residual Cu. It is therefore concluded that HS appears to be an attractive and promising alternative to EDTA as remediation agent for heavy metal polluted soils provided cheap HS of good quality is easily available.     

Glucose dehydrogenase of a rhizobacterial strain of <Emphasis Type="Italic">Enterobacter asburiae</Emphasis> involved in mineral phosphate solubilization shares properties and sequence homology with other members of enterobacteriaceae

Glucose dehydrogenase (GDH) of Gram-negative bacteria is a membrane bound enzyme catalyzing the oxidation of glucose to gluconic acid and is involved in the solubilization of insoluble mineral phosphate complexes. A 2.4 kb glucose dehydrogenase gene (gcd) of Enterobacter asburiae sharing extensive homology to the gcd of other enterobacteriaceae members was cloned in a PCR-based directional genome walking approach and the expression confirmed in Escherichia coli YU423 on both MacConkey glucose agar and hydroxyapatite (HAP) containing media. Mineral phosphate solubilization by the cloned E. asburiae gcd was confirmed by the release of significant amount of phosphate in HAP containing liquid medium. gcd was over expressed in E. coli AT15 (gcd::cm) and the purified recombinant protein had a high affinity to glucose, and oxidized galactose and maltose with lower affinities. The enzyme was highly sensitive to heat and EDTA, and belonged to Type I, similar to GDH of E. coli.     

Three-step preparation and purification of phosphorus-33-labeled creatine phosphate of high specific activity

Rabbit heart mitochondria were used as a source of enzymes for the synthesis of phosphoruslabeled creatine phosphate. This method is based on the coupled reaction between mitochondrial oxidative phosphorylation and mitochondrial-bound creatine kinase. It is possible to convert more than 90% of the inorganic phosphate (P_i) to creatine phosphate. The method used only small amounts of adenine nucleotides which led to a product with only slight nucleotide contamination. This could be removed by activated charcoal extraction. For further purification, a method for the removal of residual P_i is described.     

Ammonium chloride: a novel effective and inexpensive salt solution for phycocyanin extraction from <Emphasis Type="BoldItalic">Arthrospira</Emphasis> (<Emphasis Type="BoldItalic">Spirulina</Emphasis>) <Emphasis Type="BoldItalic">platensis</Emphasis>

Phycocyanin, a blue pigment, is a type of phycobiliproteins. Because of its various potential properties, phycocyanin is applied to various fields, such as nutraceutical, pharmaceutical, medicine, cosmetics, and biotechnological research. The cost and application of phycocyanin are highly dependent on its purity index. In this study, ammonium chloride is presented as a novel, effective, and inexpensive salt for phycocyanin extraction. Compared with sodium phosphate, which is commonly used during phycocyanin extraction process, ammonium chloride solution efficiently extracted phycocyanin with high purity from Arthrospira platensis FACHB-314. In addition, ammonium phosphate solution is also presented as an alternative precipitation agent in phycocyanin purification that may replace the widely used ammonium sulfate. Statistical analysis shows that there is no significant difference in phycocyanin concentration between crude extracts (overall mean of 0.208 and 0.215 for extraction using sodium phosphate and ammonium chloride, respectively). However, the difference in phycocyanin purity ratio (A₆₂₀/A₂₈₀) between these two extractions is significant (overall mean of 0.742 and 1.428 for extraction using sodium phosphate and ammonium chloride, respectively). With ammonium chloride, the purity indexes of phycocyanin are 1.5 and 2.81 after the optimum extraction step, and precipitation used as the primary purification step, respectively. The present study describes a novel purification method to achieve phycocyanin with analytical grade without multiple purification steps.     

Bone protein extraction without demineralization using principles from hydroxyapatite chromatography

Historically, extraction of bone proteins has relied on the use of demineralization to better retrieve proteins from the extracellular matrix; however, demineralization can be a slow process that restricts subsequent analysis of the samples. Here, we developed a novel protein extraction method that does not use demineralization but instead uses a methodology from hydroxyapatite chromatography where high concentrations of ammonium phosphate and ammonium bicarbonate are used to extract bone proteins. We report that this method has a higher yield than those with previously published small-scale extant bone extractions, with and without demineralization. Furthermore, after digestion with trypsin and subsequent high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) analysis, we were able to detect several extracellular matrix and vascular proteins in addition to collagen I and osteocalcin. Our new method has the potential to isolate proteins within a short period (4 h) and provide information about bone proteins that may be lost during demineralization or with the use of denaturing agents.     

Sequential extractions of inorganic and org-Phosphate from fish pond sediments (Deroua station,Beni Mellal,Morocco) by different fractionation methods

The carp ponds of Deroua station (Beni Mellal, Centre of Morocco) receive a large quantity of fertilizers, particularly urea and triple super phosphate during the entire period of culture. Only a small part of that is necessary to promote an adequate development of phytoplankton, the base of the food chain. A large part of the phosphate added to the ponds is stocked in pond soils and can be released under specific conditions. Although some phosphate is necessary for the development of aquatic populations, too large of an input causes problems. Therefore, knowledge of the external load and the mobility of the internal pool is essential for the purpose of management against eutrophication. In order to assess the inorganic and organic fractions of phosphate present in sediments, different methods of P fractionation were compared. The methods do not give the same results. In the Deroua sediments, calcium-bound phosphate represents the major part of total phosphate between 86% and 91% according to the different methods. The org-Phosphate represents a small part and varies considerably depending on the different methods between 0.87 and 5%. EDTA extractions (Golterman, 1996) have an advantage over other methods such as the Sedex, the Hieltjes &; Lijklema and the Bonzongo procedures.     

Metabolite profiling of Calvin cycle intermediates by HPLC-MS using mixed-mode stationary phases

SUMMARY: A sensitive and robust mixed-mode high performance liquid chromatography-tandem mass spectrometry method was developed for the qualitative and quantitative determination of sugar phosphates, which are notoriously difficult to separate using reversed-phase materials. Sugar phosphates were separated on a Primesep SB column by gradient elution using aqueous ammonium formate and acetonitrile as mobile phases. Target analytes were identified by their precursor/product ions and retention times. Quantitative analysis was performed in negative ionization/multiple reaction monitoring mode with five different time segments. The method was validated by spiking authentic sugar phosphate standards into complex plant tissue extracts. Standard curves of neat authentic standards and spiked extracts were generated for concentrations in the low picomole to nanomole range, with correlation coefficients of R(2) > 0.991, and the degree of ion suppression in the presence of a plant matrix was calculated for each analyte. Analyte recoveries, which were determined by including known quantities of authentic standards in the sugar phosphate extraction protocol, ranged from 40.0% to 57.4%. The analytical reproducibility was assessed by determining the coefficient of variance based on repeated extractions/measurements (<20%). The utility of our method is demonstrated with two types of applications: profiling of Calvin cycle intermediates in (i) dark-adapted and light-treated tobacco leaves, and in (ii) antisense plants expressing reduced levels of the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase or ribulose-1,5-bisphosphate carboxylase/oxygenase (comparison with wild-type controls). The broader applicability of our method is illustrated by profiling sugar phosphates extracted from the leaves of five taxonomically diverse plants.     

Determination of estramustine phosphate and its metabolites estromustine, estramustine, estrone and estradiol in human plasma by liquid chromatography with fluorescence detection and gas chromatography with nitrogen-phosphorus and mass spectrometric detection

Bioanalytical methods for the determination of estramustine phosphate by liquid chromatography and its four main metabolites estromustine, estramustine, estrone and estradiol by gas chromatography are described. For the estramustine phosphate assay the plasma was purified by protein precipitation followed by a C₁₈ solid-phase extraction. For the metabolite assay the plasma samples were purified by a C₁₈ solid-phase and liquid–liquid extraction procedure and derivatised by silanization. Thereafter, estramustine and estromustine were quantified by gas chromatography with nitrogen-phosphorus detection and estradiol and estrone were quantified by gas chromatography with selected ion monitoring. The methods were validated with respect to linearity, selectivity, precision, accuracy, limit of quantitation, limit of detection, recovery and stability. The limit of quantitation was 2.3 μmol/l for estramustine phosphate, 30 nmol/l for estromustine and estramustine, 12 nmol/l for estrone and 8 nmol/l for estradiol. The results showed good precision and accuracy for estramustine phosphate and the four metabolites. The intermediate precision was 6.2–13.5% (C.V.) and the accuracy was 91.8–103.9%.     

Sequential fractionation of sediment phosphate

By means of sequential extractions with Ca-NTA and EDTA, a separation was performed between Fe(OOH) P and CaC0₃P in a few sediments; the remaining fraction, considered to be organic phosphate, was quantified as well. We found that with the commonly used method of extraction with NaOH and H₂S0₄, less Fe(OOH) P and much more CaC0₃ P was found than with the chelating extractants. The organic phosphate pool in live and dead algal material and in some mud samples was partly hydrolysed and therefore recovered as inorganic phosphates with classical extractions. The difference between chelating extractants and the classical ones is discussed.Abbreviations o-P: ortho phosphate (or its concentration) - org-P: organic phosphate - extr-P: extractable sediment bound phosphate - extr-Fe: extractable sediment bound iron - Fe(OOH) P: iron bound, sediment phosphate - CaCO₃ P: calcium bound, sediment phosphate - org-C: organic sediment bound carbon     

KINETICS OF PHOSPHATE TRANSPORT BY SYNECHOCOCCUS LEOPOLIENSIS (CYANOPHYTA): EVIDENCE FOR DIFFUSION LIMITATION OF PHOSPHATE UPTAKE1

The kinetics of phosphate transport by Synechococcus leopoliensis (Racih.) Komarek was investigated. Deviations from Michaelis-Menten kinetics were observed at law concentrations of phosphate and the deviations were consistent with diffusion limitation of transport. Activation energy analysis of the transport process at two concentrations, one at carrier saturation and the other at zero added phosphate yielded activation energies of 11.9 and 5.6 kcal/mole respectively at 25° C. The first is consistent with an enzyme limited process and the second with diffusion limitation through the unstirred layer.