Stepwise Extraction Research Articles

Decomposition of exponential curves in spectrometry

Two algorithms for decomposition of exponential dependences are described. One of the algorithms is an iterative generalization of the well-known method of stepwise extraction of exponential components on a semilogarithmic scale, and the other allows visualization of the set of possible solutions with interactive choice of a satisfactory approximation by user-specified criteria. The algorithms have been implemented on an IBM PC.

Comparative Measurement of Myocardial ATP and Creatine Phosphate by Two Chemical Extraction Methods and 31P-NMR Spectroscopy

Changes in ATP and creatine phosphate levels during early (up to 150 s) global ischaemia were determined in isolated rat hearts by two chemical extraction methods (a conventional direct perchloric acid extraction and a stepwise extraction using alcohol and perchloric acid solutions) and by qualitative 31P-NMR. No difference in the ATP content of normally perfused tissue was found using the two chemical extraction methods. The ATP level hardly changed up to 40 s of ischaemia when measured by the three methods, and slightly decreased at 150 s of ischaemia. In the contrast to ATP, creatine phosphate content in the normally perfused tissue was observed to be higher by the stepwise extraction (68-73 nmol/mg protein) than by direct perchloric acid extraction (55 nmol/mg protein). The creatine phosphate rapidly decreased to about 50% of normal value at 40 s of ischaemia, and the difference in the normal creatine phosphate content using the two chemical methods disappeared with the progression of ischaemia. Thus, the creatine phosphate more rapidly decreased when observed by the stepwise method than by the other two methods in this ischaemic condition. These results suggest that (1) creatine phosphate exists in an undetermined state (perhaps neither in simple soluble form nor in so-called "bound" form) in rat cardiac myocytes, and (2) the stepwise extraction method is useful to measure the content of energy metabolites and to examine the intracellular chemical state in cardiac tissues.

Improvement of juice recovery from pineapple pulp/residue using cellulases and pectinases

Abstract During stepwise extraction of pineapple juice from pineapple pulp/residue, the addition of a commercial cellulase, a pectinase or their mixture at an enzyme concentration of 0.025% at 27–30°C for 30 min, facilitated juice recovery. The percent juice recovery in some batches of pineapple after enzyme addition was in the range of 81–86% as against 72% in the untreated samples. Enzyme additions also improved the quality of juice by allowing extraction of more of the soluble solids and juice particulate. The ready-to-serve (RTS) pineapple juice obtained after enzymatic juice extraction was rated acceptable on a 5-point Hedonic scale.

Resin extraction of labile, soil organic phosphorus

SUMMARYIn order to develop a method for estimating labile, soil organic phosphorus (Po), macroporous anion‐exchange and adsorbent (XAD) resins were tested for their ability to extract Po from a permanent pasture soil. Experimental variables included: ionic form, soil‐water resin ratio, addition of cation‐exchange resin, extraction time, extraction temperature, continuous and stepwise extraction and soil pretreatment (chloroform and microwaves).The amounts of extracted Po and Pi (inorganic phosphorus) showed little variation between the anion‐exchange resins, when used in the bicarbonate form. In the chloride form, the amounts extracted were less and more variable. XAD resins extracted much less Po and very little Pi. The macroporous anion‐exchange resin, Lewatit MP500A in the bicarbonate form, was chosen for further studies. It extracted 21 mg Po kg−1 from air‐dried soil and 8 mg Po kg−1 from moist, incubated soil.The specific amounts of Po and Pi extracted were little affected by variations in the ratio between soil, water and resin, but increased with extraction time and temperature. Chloroform pretreatment of the soil mainly increased extracted Pi, whereas microwave pretreatment only increased extracted Po. The magnitude of these increases was approximately constant irrespective of extraction temperature, indicating that the increases came from killed micro organisms.

Confirmation and an unusual quality of the differentiated keratin peptide (K1) in cultured human squamous cell carcinomas

Recently K1 keratin peptide (K1, 68 kda) was found to be present in two kinds of cultured human squamous cell carcinomas (HSCs) using a low-salt aqueous solution, rather than the high-salt solution containing Triton X-100 employed by many researchers up until now. To confirm whether this phenomenon is universal in cultured HSCs we analyzed K1 peptide in four other kinds of HSCs using the same procedures. Moreover, the K1 peptide detected was a little unusual with respect to solubility versus urea concentration. Epidermal K1 peptide is usually solubilized by 6–8 M urea and reluctant; however, the K1 peptide in cultured HSCs was about 80–90% extracted by 1–2 M urea in a stepwise extraction procedure. This finding may have important implications regarding evaluation of keratin extracted from normal epidermal and cultured keratinocytes.

Supercritical fluid extraction for the analysis of liquid poly(alkylene glycol) lubricants and sorbitan ester formulations

Additives were selectively extracted from poly(alkylene glycol)(PAG) lubricant formulations using supercritical fluid extraction. The PAG matrix was adsorbed onto silica for the extractions and the extracted analytes were passed through a silica column positioned in-line in the supercritical fluid stream. The selectivity obtained by this method was compared with that obtained by the direct extraction of adsorbed and unadsorbed PAG and by the extraction of unadsorbed PAG through the in-line column. The final procedure was found to be successful in separating additives from all but the lowest molecular mass PAG oligomers. Further use of this procedure was demonstrated by a stepwise extraction of a sorbitan ester formulation. This gave a chemical-class fractionation of the product and so provided a sample preparation technique for further spectroscopic analysis.

Toxic prey discrimination in a highly specialized predator Chaetodon melannotus (Block et Schneider): visual vs. chemical cues

The specialized predator Chaetodon melannotus (Bloch et Schneider) (Vertebrata, Pisces), which feeds on highly toxic and sometimes allelopathic octocorals, initially locates its prey using visual rather than chemical cues. A novel controlled experiment was carried out to further understand the basis of food preference and selection. Organic extracts were interchanged between colonies of three species of octocoral: Alcyonium molle (Dean), Clavularia inflata (Schenck) and Sinularia flexibilis (Quoy et Gaimard0 (Coelenterata, Octocorallia). Extracts were prepared by stepwise solvent extraction of the octocoral; first with ethanol, then with mixtures containing dichloromethane in ethanol, and finally with dichloromethane. All extractable organic material was removed. Concentrated extracts (in ether) were then reintroduced into the extracted colonies in the six possible permutations. In three smorgasbord-type experiments using two octocoral species at a time, C. melannotus were offered the six types of modified colonies, including controls without any extract, yielding a balanced matrix of visual and chemical cue combinations. Responses of the fish to prey choices were nonrandom in all cases. Alcyonium colonies with original extracts reintroduced were strongly preferred over Clavularia. Clavularia colonies and any colony containing their extracts received low preference. When offered with Sinularia, Clavularia colonies devoid of organic components received high preference. Chemical cues were determined to be important in this selection process. The Sinularia colonies in this situation received moderate acceptance. Sinularia colonies with their own extract reintroduced, received high preference when offered with Alcyonium. This indicated that there might be a subtle change in appearance or texture of Alcyonium (the preferred choice in an earlier study) as a result of the extraction. Responses were significantly different from that expected if the fish were cueing solely on the basis of either visual or chemical cues. A combination of visual and chemical cueing, possibly in sequence, appears to have produced the observed results. These results suggest that C. melannotus use visual cues to locate their prey and probably further select on the basis of chemical cues. We believe that the technique used here is valuable for use in the separation of visual and chemical cues for feeding.

Drug of abuse confirmation in human urine using stepwise solid-phase extraction and micellar electrokinetic capillary chromatography.

This paper demonstrates that most common drugs of abuse can be absorbed simultaneously onto a mixed-mode bonded-phase matrix and eluted sequentially in two to three steps for subsequent analysis by micellar electrokinetic capillary chromatography (MECC). Having on-column multiwavelength UV absorption detection, this is shown to be an attractive approach for confirmation testing of barbiturates, hypnotics, amphetamines, opioids, benzodiazepines, and metabolites of cocaine in a single aliquot of human urine. For these compounds, no hydroysis of the urine specimen or sample derivatization is required. Under the examined conditions using 5 mL of urine, excellent recoveries (80-90% level) and detection limits (about 100 ng/mL) are obtained. For patient urines which tested positively for different classes of drugs using immunological screening methods, a two-step extraction scheme is shown to provide extracts suitable for rapid MECC confirmation of the drugs of abuse.

Purification and membrane topology of PSI-D and PSI-E, two subunits of the photosystem I reaction center.

Structural studies have been conducted on polypeptides PSI-D and PSI-E, which are extrinsic but firmly bound to the photosystem I reaction center. These subunits are predicted to be involved in the correct interaction with soluble electron acceptor(s), like ferredoxin. We designed an original method to extract both polypeptides directly from thylakoid membranes and to purify them: a stepwise extraction with NaSCN followed by size fractionation and reverse-phase HPLC. Investigation of the in situ topology of PSI-D and PSI-E was undertaken using monoclonal antibody binding, controlled proteolysis, peptide sequencing and electron microscopy. The precise identification of numerous proteolytic sites indicates that the entire N-terminal regions of PSI-E (up to Glu15) and PSI-D (up to Lys15) are exposed to the medium. Partial mapping of the exposed epitopes was possible using purified fragments of each polypeptide. In the case of PSI-E, this mapping confirmed the accessibility of the N-terminal part, and suggested the need for another exposed sequence, probably located after Met39 in the second half of the protein. For PSI-D, this mapping revealed that the sequence between Met74 and Met140, including the most basic amino acid clusters, is also partly accessible. These experiments provide the first detailed informations, although still partial, on the topology of these polypeptides. They give a preliminary basis for hypotheses concerning the sites of interaction with the soluble counterparts.

Differential extractability of creatine phosphate and ATP from cardiac muscle with ethanol and perchloric acid solution

To compare the extractability of creatine phosphate with that of ATP by alcohol extraction, both compounds were extracted from normal perfused rat heart tissues by using various stepwise concentrations of ethanol and 0.4 m HClO 4. Powdered samples (6–15 mg wet wt) from the freeze-clamped tissues were homogenized in 2 ml of the ethanol solutions. After centrifugation, the supernatant was removed; each centrifuged sediment was rehomogenized with 2 ml of 0.4 m HClO 4 and centrifuged. The supernatant was neutralized with 0.4 m KHCO 3. The same powdered samples were directly homogenized with 2 ml of 0.4 m HClO 4 and treated in the same manner. Only a small amount of ATP in the tissues was extracted by an 85% or higher concentration of ethanol. Further, about 13% of the tissue ATP was not extracted by even 60% ethanol and was only slightly extractable by the subsequent perchloric acid extraction. In contrast to ATP, creatine phosphate in the tissues was partially extracted by 95% ethanol and nearly all of the tissue creatine phosphate was extracted by 70% ethanol. The total creatine phosphate obtained by 70% ethanol and by subsequent perchloric acid extraction was significantly higher than that obtained by direct perchloric acid extraction. From these results, it was concluded that the extractability of creatine phosphate in the tissue by alcohol extraction is clearly different from that of ATP. Additionally, the stepwise extraction is recommended as a useful method for the extraction of energy metabolites in perfused rat heart tissue.

Biochemical and immunochemical characterization of 125I-labeled cuticle components of Haemonchus contortus

Live Haemonchus contortus developmental stages were radioiodinated and then subjected to a stepwise extraction procedure consisting of a buffer extract (with or without detergent) to solubilize putative surface-associated antigenic macromolecules, followed by a detergent/β-mercaptoethanol (BME) extract to solubilize putative cuticle collagen proteins. A buffer-extracted iodinated 100-kDa protein was present in the free-living, infective L3(2M) stage. This labeled protein was released during in vitro exsheathment of L3(2M) and was not present in the ecdysed second molt (2M) cuticle. In addition to the 100-kDa protein, exsheathment fluid contained a 70-kDa labeled protein that was not extracted from iodinated L3(2M) with either detergent or BME. The data suggest that these proteins are components of the specialized ring portion of the 2M cuticle that is enzymatically ruptured during ecdysis. The L3(2M) and the exsheathed third-stage larvae (L3) contained 3 labeled, BME-extracted, collagenase-sensitive proteins of 108, 88 and 53 kDa. In contrast, four detergent-extracted, collagenase-insensitive, iodinated proteins (143, 81, 58 and 30 kDa) were present in adult H. contortus. The 143-kDa protein was both glycosylated and immunogenic. All 4 adult cuticle proteins were released from the cuticle surface into culture fluids. Furthermore, a cysteine protease was secreted by adults which apparently hydrolyzed the released 81-, 58- and 30-kDa surface proteins.

Thermodynamics of solvent extraction of metal picrates with crown ethers: enthalpy–entropy compensation. Part 2. Sandwiching 1 : 2 complexation

Thermodynamics of sandwich complexation in solvent extractions were investigated for the first time. Quantitative solvent extractions over a wide range of ligand concentration in the water-dichloromethane system at 10–25 °C gave the overall and stepwise extraction equilibrium constants and the thermodynamic quantities for both 1 : 1 and 1 : 2 complexations of some alkali, alkaline earth, and/or heavy metal picrates with 15-crown-5 (1), benzo-15-crown-5 (2), and cis-cyclohexano-15-crown-5 (3). The overall sandwich complexation process was deemed as being composed of the first stoicheiometric and the subsequent sandwiching complexation equilibrium. The feasibility of the second, sandwiching complexation in the solvent extraction and in the homogeneous solutions is discussed. Global treatments of the thermodynamic parameters obtained in the solvent extraction and of those reported in the homogeneous phase led to a comparable linear relationship between the enthalpy and entropy changes of complexation. The large slope (α) of the ΔH°vs. TΔS° plot indicates the substantial conformational change of not only the second but also of the originally ligating crown ether molecule upon the second, sandwiching complexation.

Extractability of glutenins with water/alcohol mixtures under reducing conditions

After the extraction of albumins, globulins and gliadins from defatted wheat flour, the glutenin-containing residue was further subjected to extraction under reducing conditions with water/alcohol mixtures. The protein pattern of the extracts and of the residues was characterized by reversed-phase high-performance liquid chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis. A rather high amount of middle-molecular-weight glutenins and of low-molecular-weight glutenins was extracted at 4°C with 30%–70% and 50%–70% aqueous ethanol, respectively. The high-molecular-weight glutenins were subdivided into two groups. The larger subunits (band nos. 1–5) were extracted with 40%–60% aqueous ethanol at 4°C, whereas the smaller subunits (bands nos. 8–11 according to Krause et al.) were not extracted. The same results were obtained with (n-10)% aqueous 2-propanol. By stepwise extraction with 35%, 70% and 50% aqueous ethanol at 4°C, a rather high yield in the glutenins was obtained only with the first solvent. The reduced glutenins appeared to be almost completely soluble in 70% ethanol at 60°C and at pH 3.5.

Identification of Hemoglobin and Two Serine Proteases in Acid Extracts of Calcium Containing Kidney Stones

By stepwise extraction of kidney stones, first with 10% acetic acid then 0.1 M EDTA, three pools of protein material have been obtained. The soluble material from the acid extract showed well defined bands when analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis, and amino acid sequence analysis of peptides identified hemoglobin as a common constituent. From one stone neutrophil elastase has been identified along with another serine protease of unknown origin. The second pool of material which was soluble in EDTA showed a smear on SDS gel electrophoresis while the third pool consisted of the insoluble matrix. The finding of proteolytic enzymes opens up the possibility that limited proteolysis might be involved in the stone forming process.

Nuclear matrix bound terminal deoxynucleotidyl transferase in rat thymus nuclei. II. Effect of ATP on free and matrix bound TdT.

Endogenous nuclease digestion of thymus nuclei from 3-4 week old rats followed by a step wise extraction with low salt, 0.5 M salt and 1 M salt removed approximately 70-85% of total nuclear terminal deoxynucleotidyl transferase (TdT) whereas approximately 15-30% of the enzyme remained tightly bound to the residual nuclear matrix. The cytoplasmic TdT as well as the bulk of nuclear TdT extracted in low salt and 0.5 M salt was found to be strongly inhibited at low concentration of ATP whereas matrix bound TdT and a significant portion of the enzyme in 1 M salt extract was completely insensitive to this nucleotide. The ATP resistant enzyme in the 1 M salt extract was unstable and slowly converted to ATP sensitive form upon prolonged preincubation on ice whereas under similar conditions it remained unaffected in the matrix bound form. These observations lead us to suggest that ATP resistant matrix bound TdT being capable of discriminating unnatural rNTPs against the natural dNTP substrates, may be the functionally organized form of the enzyme and that free TdT having lost the capability to distinguish between dNTP and rNTP may be the nonfunctional form of the enzyme in the thymus gland.

Solvent extraction separation of cerium(III) from transition elements with 15-crown-5 with picrate as the counter ion

Cerium(III) was quantitatively extracted between pH 6.0 and 8.0 with 0.05 M 15-crown-5 in dichloromethane from 0.01 M picric acid with picrate as the counter ion. Cerium(III) was stripped with 1 M perchloric acid and determined spectrophotometrically at 655 nm as its complex with Arsenazo III. Cerium was separated from a large number of elements. Stepwise extraction of cerium permits its separation from thorium, uranium, scandium, yttrium and strontium. The method was applied to the determination of cerium in monazite sand.

Cell-wall polysaccharides of kiwifruit ( Actinidia deliciosa): Chemical features in different tissue zones of the fruit at harvest

Cell-wall material (CWM) was isolated from cryo-milled (− 196°) powders prepared from 4 different tissue zones of kiwifruit ( Actinidia deliciosa). Polysaccharides were solubilised by stepwise extraction with cyclohexane- trans-1,2-diaminetetra-acetate (CDTA), 0.05 m Na 2CO 3, 6 m guanidinium thiocyanate (GTC), and 4 m KOH. A heterogeneous mixture of pectic galactans accounted for 40–50% of the CWMs, while hemicelluloses, the bulk of which were xyloglucans, accounted for 15–25%. Each tissue zone contained similar types of polysaccharide. Variability in their amount and sugar composition are thought to reflect different stages in the physiological development of the fruit at harvest, in the 4 zones. Polymers from the outer pericarp tissue were fractionated by anion-exchange chromatography and subjected to methylation analysis. The CDTA- and Na 2CO 3-soluble polymers were rhamnogalacturonans substituted to varying degrees with galactan and arabinogalactan side-chains containing 4-, 2,4-, 3,4- and 4,6-linked galactose and 5- and 3,5-linked arabinose. Side chains were terminated by galactose and arabinose and lesser amounts of rhamnose, fucose, xylose, and galacturonic acid. The pectic polysaccharides of the GTC- and KOH-soluble fractions had more highly branched rhamnogalacturonan backbones than the CDTA- and Na 2CO 3-soluble polymers and contained hemicellulosic elements. The major hemicellulose was a xyloglucan, but lesser amounts of a 4- O-methylglucuronoxylan and a branched mannan were partially characterised. Several polymers were associated with proteins low in hydroxyproline. Evidence is presented that a polysaccharide of the rhamnogalacturonan II type is associated with the pectic polymers of kiwifruit.

Extraction of coal through dilute alkaline hydrolytic treatment at low temperature and atmospheric pressure

Assam coal was extracted with ethylenediamine (EDA) using batch as well as Soxhlet techniques. It was found to be further extractable in EDA after a first extraction step. Stepwise extraction of coal in EDA resulted in 44% extraction. Alkali treatment enhanced the extractability of coal in EDA. Two step (1.5%) aqueous alkali treatment followed at each step by EDA extraction, resulted in 50% extraction of coal. Various solvents were tried in the aqueous alkali treatment of coal. EDA, tetralin, phenol, quinoline, ethanol and butanol were found to give good results. The use of Na 2CO 3 and Ca(OH) 2 along with NaOH in the alkali treatment did not improve the extractability of the coal. Nonaqueous alkali treatment of coal in different solvents also resulted in an enhanced extraction. Phenol, tetralin and pyridine were found to be good reaction solvents for the treatment of coal using solid NaOH in these solvents.

Evaluation of component composition of complex structural units of petroleum disperse systems

A variant of a fractionation procedure for high-molecular-weight petroleum stocks, based on stepwise extraction with a set of solvents, was developed for the isolation of core and solvate shell components for complex structural unit determinations of petroleum disperse systems. A vacuum resid from West Siberian crude, asphalts, and a product obtained by coking the stock were investigated. Samples were pretreated with hot isooctane on a paper filter and placed in an extractor, then treated with isopropanol-benzene mixtures of different ratios as well as with straight benzene. It was possible to recover concentrates of the solvate shells and cores from resids, asphalts, coker residues, and pitches by the method.

Analysenschema zur Untersuchung der Bleiverteilung in und auf kontaminierten Pflanzen

The solubility of lead upon contaminated common grass is determined by stepwise extraction with n-butanol, dest. water and 0.1 or 1 mol/l HCl. Quantitative analyses are carried out by means of flameless AAS. Compartimentations of lead in leaves can be obtained by filtration and centrifugation in cellular walls, vacuole liquid (and soluble proteins) and cellular organelles. Cellular walls and organelles are also analysed by stepwise extraction. This scheme for analysis provides the following distribution of lead (the average content of total lead is 14 ppm in 10 samples): 64% upon the surface of leaves (thereof 66% soluble in water), 28% within the cellular walls (thereof 80% soluble), 5% within the vacuole liquid and 3% as mainly phytotoxically active part within the cellular organelles.