Rapid Method For Separation Research Articles

A rapid method for separation of thylakoid membranes by density gradient centrifugation in percoll

Summary Thylakoid membranes were purified from isolated spinach chloroplasts and from cell-free homogenates of Chlamydomonas reinhardii in a discontinuous Percoll gradient (7.5, 15, 30%). Purity of thylakoid membrane fraction were demonstrated by LiDS-gradient PAGE and electron microscopic examination.

Rapid high-performance liquid chromatographic method for hydrocarbon class separation in environmental samples

A high-performance liquid chromatographic method for the separation of saturated and aromatic hydrocarbon mixtures, found in environmental samples, has been developed. The separation is carried out on silica. A short analysis time is achieved by using a backflush technique. Fractions collected are further analysed by high-resolution gas chromatography. The usefulness of this technique is illustrated on petroleum-polluted environmental samples.

Evaluation of the rapid micromethod for ultracentrifugal separation of labeled plasma lipoproteins.

The fractionations of plasma lipoproteins by 2 methods were compared to evaluate the rapid separation (Airfuge) method for lipoprotein distribution studies. When [125I] labeled very low density, low density, and high density lipoproteins (VLDL, LDL, HDL), were separately centrifuged in buffers at d = 1.006, 1.06 or 1.2 g/ml by the conventional ultracentrifuge and the Airfuge, separations of the fractions in the Airfuge were incomplete at both 5 C and 24 C, especially at d = 1.006. [3H] Benzo (a)pyrene, when added to plasma, associates with the plasma proteins and lipoproteins, especially LDL. Compared to the standard techniques, the Airfuge method greatly overestimated its distribution into VLDL. The distribution of [3H] vitamin D3 into the VLDL plus LDL fraction was also overestimated by the Airfuge procedure. It is concluded that caution should be observed in quantitative studies of lipoproteins in the Airfuge. A careful comparison of the distribution into or fractionation of lipoproteins by the 2 methods should always precede any quantitative determinations involving the Airfuge.

Plasma lipoprotein separation by discontinuous density gradient ultracentrifugation in hyperlipoproteinemic patients

Quantitative plasma lipoprotein separation is usually performed by sequential ultracentrifugation which because of the necessity of repeated ultracentrifugation is extremely time consuming and results in changes in lipoprotein composition. We describe a simple, reliable, and rapid method for quantitative plasma lipoprotein separation which is based on ultracentrifugation of the plasma in a discontinuous density gradient media consisting of a saline solution of different densities placed over a plasma solution of 1.250 g/ml (with potassium bromide). After 48 hr of ultracentrifugation in a swinging bucket rotor excellent lipoprotein separation was demonstrated. The lipoproteins were pure and there was significantly less loss of high density lipoprotein protein to the lipoprotein deficient plasma ( d > 1.210 g/ml) when compared to the losses incurred during the sequential flotation method. The results of separating the plasma lipoproteins from subjects with dyslipoproteinemias by this method were demonstrated. We conclude that discontinuous density gradient ultracentrifugation is the method of choice for quantitative plasma lipoprotein separation for both clinical and research use.

Quantitation of estolide triglycerides in sapium seeds by high performance liquid chromatography with infrared detection

AbstractThe kernel oil of Chinese tallow(Sapium sebiferum) seed contains tetraester triglycerides composed oftrans‐2, cis‐4‐decadienoic acid joined in an estolide linkage to 8‐hydroxy‐5, 6‐octadienoic acid. A rapid (< 10 min) method of separation and quantitation of the tri‐glyceride and estolide triglyceride fractions of the oil has been devel‐oped using high performance liquid chromatography with infrared detection.

Extraction of Ag(I), Cd(II), In(III), Sn(II), Sn(IV), Sb(III), and U(VI) from aqueous solutions by ketone solutions using single-step batch and continuous SISAK methods1,2

The extraction properties of Ag(I), Cd(II), In(III), Sn(II), Sn(IV), Sb(III), and U(VI) from aqueous KI/H2SO4 solution into a mixture of 4-methyl-2-pentanone (methyl isobutyl ketone, MIBK) and cyclohexanone (CHO) were studied. Both single-step batch and SISAK2 methods were used. The oxidation of Sn(II) to Sn(IV) by iodine and complexation of Sn(IV) by 2,3-dimercapto-propanol-1 (BAL) were also investigated. A method for rapid and continuous separation of indium from tin was developed for investigation of short-lived indium fission products.

Separation of Prostanoids by One-Dimensional Thin-Layer Chromatography

Abstract We have developed a simple and rapid method for separation of 7 prostanoids and arachidonic acid by one-dimensional thin-layer chromatography. For this separation we employ commercially prepared thin-layer plates that have a preadsorbant (celite) area to which samples are applied. Due to the inert characteristics of the celite no separation occurs until the sample reaches the preadsorbant-silica gel junction. Since all the material moves with the solvent front as a sharp, narrow band to the preadsorbant (celite)-silica gel boundary, a high resolution of the separated compounds is achieved. The time required to apply one sample to the preadsorbant (celite) area is considerably less (2 min) than that required for application of a sample to silica gel (10 min). This method is suitable for the separation of major prostaglandins (PGE2, PGF2α), the major enzymatically formed metabolites of these prostaglandins, and the stable, nonenzymatically formed product of thromboxane A2 (TXB2).

A rapid rosette technique for quantitation and separation of mononuclear cell subsets using monoclonal antibodies

We present a rapid and simple method for simultaneous quantitation and separation of mononuclear cell (MNC) subsets. When lymphoid cells are sensitized with monoclonal antibodies of the OK and Leu series, they rapidly form rosettes with ox erythrocytes (ORBC) coated with affinity-purified rabbit IgG against mouse IgG. Rosette-forming cells (RFC) may then be counted and separated from non-rosetting MNC by Isopaque-Ficoll gradient centrifugation. The yield and viability are close to 100% after ORBC lysis. Adherent cells do not interfere. Isolated T8 + and Leu3a + cells were further tested: the purity was 97–99%, and the cells were functionally intact with respect to their modulating activity on the generation of immunoglobulin-secreting cells by MNC after stimulation with pokeweed mitogen.

Isolation and Identification of Human Fetal Adrenal Medullary Cellsin Vitro*

A rapid method for the partial separation of human fetal adrenal medullary cells is described. Enzymatically dissociated human fetal adrenal cells were centrifuged on a preformed gradient of colloidal Silica particles (Percoll) in isotonic Earle's balanced salt buffer. This procedure leads to the formation of five fractions containing nonviable cells and debris, predominantly fetal zone cells, predominantly definitive zone cells and clumps of medullary cells, isolated medullary cells, and red blood cells. The medullary cells were then pooled and plated on plastic culture dishes coated with an extracellular matrix produced by cultured bovine corneal endothelial cells. Medullary cells plated on extracellular matrix demonstrated extensive neurite formation, facilitating their identification. Further identification of the cells as medullary in origin was confirmed using a specific catecholamine fluorescence technique.

Phospholipid and neutral lipid separation by one-dimensional thin-layer chromatography

A simple and rapid method for separation of six major phospholipids and four major neutral lipids from cell extracts by one-dimensional preadsorbant thin-layer chromatography was developed. Due to the inert characteristics of the preadsorbent layer (Celite) no separation occurs until the sample reaches the preadsorbent (Celite)—silica gel junction. The compounds were applied to the preadsorbent (Celite) area (625 mm 2) in 10-μl aliquots (total volume of 0.15 ml). By this method, samples can be applied rapidly in large volumes without respotting any area of the preadsorbent layer. The time required to apply one sample was reduced considerably (2 min) compared to conventional methods (10 min). Since all the compounds move with the solvent front as a sharp, narrow band to the preadsorbent (Celite)—silica gel boundary, excellent separation was achieved when up to 650 μg of lipid material was applied on each lane (25 mm wide). Thus, this method is suitable for the separation of relatively large amounts of radiolabeled and non-radiolabeled lipids and free fatty acids from extracts of biological fluids, tissues, or cells maintained in monolayer culture.

Analysis of a Mixture of Polychlorinated Biphenyls, DDT and its Analogues by High-Performance Liquid Chromatography

Abstract A simple and rapid method for separation and quantitative analysis of polychlorinated biphenyis (PCBs) and chlorinated pesticides (DDT and its analogues DDE and DDD in their o, o'-and p, p'-isomers) is described. The procedure consists of two steps: a) transformation of DDT and its analogues in o, p'-and p, p'-dichlorobenzophenone (DCBP); b)determination of the amount of PCBs and ∑DDT as DCBP by HPLC. Results obtained confirm that HPLC can be considered as an alternative or a supplementary methodology to conventional methods such as gas chromatography. The method is applied to marine organisms.

Analysis of the Primary Structure of the Strongly Hydrophobic Brain Myelin Proteolipid Apoprotein (Lipophilin). Isolation and Amino Acid Sequence Determination of Proteolytic Fragments

Proteolipid aproprotein (lipophilin) and DM-20 protein from bovine brain white matter proved to be identical in polyacrylamide gel electrophoresis and automated Edman degradation of the N-terminal end over 20 cycles. Lipophilin can be hydrolysed by trypsin, thermolysis, chymotrypsin and subtilisin. We describe here a new, effective and rapid high-performance liquid chromatographic separation method for hydrophilic polypeptides according to molecular mass on an analytical and preparative scale. Three large and several small peptides have been isolated from the tryptic and thermolysinolytic hydrolysate and purified by combined molecular sieve and high-performance chromatographic separation and purification for automated Edman degradation. 40 amino acid residues of the large tryptic fragment and sequences of 43 and 22 amino acids of two thermolysinolytic fragments have been determined. These three polypeptides are partial structures of the l4 kDa large tryptophan fragment 1 or the cyanogen bromide fragment I (18-19 kDa). Thermolysin also releases a polypeptide from incompletely reductively carboxymethylated lipophilin which is cleaved into the large thermolysin fragment mentioned, 22 residues of which were analysed, and a 14 amino acids long sequence of tryptophan fragment IV, described in the previous paper. Reductively carboxymethylated liprophilin, the lysine side chains of which were blocked with maleic anhydride, can be cleaved at arginine specific sites. Bio-Gel P-150 and high-performance chromatographic purification yielded a polypeptide, which upon performic acid oxidation was split into a 15 kDa and a 7.8 kDa polypeptide. The 15 kDa polypeptide resembles the N-terminal end as proven by 31 cycles in Edman degradation. The 7.8 kDa polypeptide corresponds to the 72 amino acid C-terminal sequence, which equals cyanogen bromide fragments II, III and IV and embraces tryptophan fragment IV.

Vitamin B12 binders (transcobalamins) in serum.

The measurement of plasma or serum vitamin B12 (B12) binders is important in the diagnosis of congenital megaloblastic anemias, the myeloproliferative disorders and perhaps in other malignancies. Two classes of binders circulate in plasma, Transcobalamin II (TC II) and R-binders. The latter have been divided by some authors into transcobalamin I (TC I) and transcobalamin III (TC III), and the validity of this division is discussed. R-binders and TC II differ in their apparent molecular weight on gel filtration chromatography, by which method they can be separated reliably. However, the technique is time-consuming and cumbersome and a variety of rapid separation methods have been described in the literature. These are discussed and compared to the standard gel filtration separation method. TC I and III are separable on the basis of their differing charges, and the methods which have been described for accomplishing this are compared and critically reviewed. There has been some controversy in the literature as to whether plasma or serum should be used to measure circulating B12 binders. Leukocytes, which contain R-binders, release these in vitro and this release is greatest when blood is allowed to clot and the serum separated. Consequently, the use of plasma, sometimes with agents added to prevent leukocytic release of binder, has been advocated. Yet, none of the agents used eliminates artifact totally, and this aspect, too, is reviewed. Lastly, several techniques for the purification of B12 binders have been described. Some techniques result in pure binder preparations, while others result in preparations which are free of other binders but contaminated with non-B12 binding proteins. Both approaches have advantages and disadvantages, and these are discussed.

Gas-liquid chromatographic separation and determination of the components of maltitol syrups

Relative retention times and detector responses of trimethylsilyl and trifluoroacetyl derivatives of the components of maltitol (maltotriitol) syrups on Dexsil GC 300 liquid phase are reported. Glucose, sorbitol, maltose, maltitol, maltotriose and maltotriitol gave a single main peak, whereas as trifluoroacetylated derivatives two or three separated peaks could be observed, which probably showed products of incomplete trifluoroacetylation. A method for rapid separation and quantitative estimations obtained by gas-liquid chromatography for the constituents of maltitol (maltotriitol) syrups are given.

Separation of plant polyphenolics by chromatography on a boronate resin

m-Aminophenylboronic acid attached to polyacrylamide beads by a short aliphatic chain strongly and selectively binds vicinal dihydroxyphenyl substituted compounds at pH 7–8. At lower pH these substances are released quantitatively thus affording a simple and rapid method for separation and purification of catecholic materials.

Production of Carrier-free 38K by means of photonuclear reactions

The simultaneous 40 Ca(γ, pn) 38 K and the 40 Ca(γ, 2n) 38 Ca ⇀ 38 K reactions have proved to be useful for the production of carrier-free 38K. The production rates of 38K and contaminant were determined as a function of the maximum bremsstrahlung energies between 30 and 60 MeV. As a result, the production rate of 38K at an electron energy of 60 MeV was found to be 840 μCi μAh −1 g −1 of Ca. In addition, the rapid chemical separation method of carrier-free 38K was studied in detail by using 43K and 47Ca tracers. Carrier-free 38K for medical uses was separated with a 70% yield within 8 min of the end of irradiation.

Selective Separation of Molybdenum as Volatile Halides from Deep-Sea Ferromanganese Nodules

Abstract A simple and rapid method for a selective separation of Mo from deep-sea ferromanganese nodules is described. Sulfation of the ground nodule material with a gas mixture of SO2 and O2 at elevated temperatures results in the sublimation of the Mo therein. The sublimates are mainly composed of pure MoCl4 and MoO2Cl2 which are readily soluble in water. Addition of alkali metal halides to the nodules prior to sulfation increases the Mo recovery to over 95% in 30-min sulfation at 400 to 500 [ddot]C. The sulfation process, developed primarily for the selective separation of Mn, Cu, Ni, and Co, can be effectively used for Mo separation without complication.

Isocratic high-performance liquid chromatography separation of phosphoglycerides and lysophosphoglycerides

An isocratic, rapid method for separation of phospho- and lysophospholipids by high-performance liquid chromatography employing acetonitrile, methanol and water as the mobile phase is described. Separation is achieved on a silica based cation-exchange column with individual components monitored directly by UV absorbance at 203 nm. In solutions of phospho- and lysophospholipid standards and in extracts of rabbit myocardium, recovery of individual components exceeds 95%. With the exception of phosphatidyl serine and phosphatidyl ethanolamine that could not be resolved other major phospho- and lysophospholipid constituents found in extracts of cell membranes are separated completely within 40 min. Retention time for selected moieties can be shortened even further with flow programming.

Revised Method for Aflatoxins in Cottonseed Products, and Comparison of Thin Layer and High Performance Liquid Chromatography Determinative Steps: Collaborative Study

Abstract Modifications to save analyst time and reagents have been made in the rapid method for separation and quantitation of aflatoxins in cottonseed products. A collaborative study of the revised method, including optional high performance liquid chromatography (HPLC) in addition to thin layer chromatography for the determinative step, showed no change in the method precision or accuracy due to the modifications and no significant difference in the values obtained by either determinative procedure. The HPLC technique provided no significant difference in analysis repeatability, but did reduce the betweenlaboratory error component. The method has been adopted as official first action.

Distribution of Metabolites between Chloroplast and Cytoplasm during the Induction Phase of Photosynthesis in Leaf Protoplasts

A method for rapid separation of the chloroplast and cytoplasmic fractions from isolated leaf protoplasts of wheat and spinach has been used to determine the distribution of (14)C-labeled products during photosynthesis. In the dark, CO(2) fixation was only 1 to 2% of that in the light and the products were mainly in the cytoplasmic fraction suggesting fixation by phosphoenolpyruvate carboxylase. Label appeared rapidly in the chloroplast fraction following illumination but the amount leveled off after 4 to 5 minutes reflecting the buildup of intermediates to steady state levels. There was only a slight lag before label appeared in the cytoplasmic fraction and it continued to increase at a constant rate reflecting synthesis of neutral products. In the light, the percentage of label in the chloroplast fraction decreased rapidly in the first minute of illumination and was only 10 to 20% in the steady-state. It is suggested that the chloroplast phosphate transporter promotes a rapid transfer of sugar phosphates from the chloroplast to the cytoplasm, even during the induction phase of photosynthesis.