Kieselguhr Column Research Articles

Fractions of nucleic acids in seedlings and leaves of winter wheat in the vegetative and generative development phases

The composition of nucleic acid preparations isolated from seedlings and leaves of plants grown from vernalized and nonvernalized winter wheat grain was chromatographically investigated on a methylated albumin kieselguhr column (MAK column). The intensity of synthesis of the particular nucleic acid fractions was compared by the <sup>32</sup>P-incorporation technique and the nucleotide composition of the newly synthesized nucleic acid fractions was determined. It was observed that the rate of <sup>32</sup>P incorporation into the sRNA, rRNA+mRNA and TB-RNA fractions of the leaves of vernalized plants was about two times higher as compared with that in the nonvernalized control.

Determination of 3-chloropropane-1,2-diol in hydrolysed vegetable proteins by capillary gas chromatography with electrolytic conductivity detection

The determination of 3-chloropropane-1,2-diol in hydrolyzed vegetable protein using capillary gas chromatography is improved through the specificity of an electrolytic conductivity detector operated in the halogen mode. The hydrolysate is absorbed onto a Extrelut QE column of kieselguhr, 3-chloropropane-1,2-diol is partitioned into ethyl acetate and is quantitatively measured by gas chromatography using 1-chlorotetradecane as the internal standard. A concentration of 1 mg/kg dry substance is easily determined.

Multiple column peptide synthesis, Part 2 (1, 2).

A manually operated apparatus for parallel multiple column soli-phase peptide synthesis is described. It employs Fmoc-amino acid-O-Dhbt or -Pfp esters in the continuous flow version of the polyamide method on small packed columns of kieselguhr supported resin in a reaction block of Teflon. The solvents and deprotecting reagents are dispensed from two washers in a parallel fashion and reagent consumption is low. Activated and protected amino acids are transferred from a dispenser tray as solutions, eight at a time. The use of the method is demonstrated by the synthesis of overlapping peptides from a protein structure and of analogous protease substrates. The products have been characterized by HPLC, FAB mass spectroscopy and amino acid analysis.

Solvent and Reversed Phase Extraction Chromatographic Separation of Molybdenum and Tungsten with Quaternary Ammonium Salt Reagent, Aliquat 336

Abstract A quaternary ammonium salt reagent, Aliquat 336 has effectively been used for radioanalytical separations of molybdenum and tungsten through the techniques of solvent extraction and reversed phase extraction chromatography (RPEC). Studies on solvent extraction revealed that quantitative separations of the elements in a mixture can be achieved by extracting tungsten with ca. 10−4 mol dm−3 solution of Aliquat 336 in cyclohexane from an aqueous solution of 1 mol dm−3 H3PO4. The efficiency of separation of the elements which form varieties of metal salts of poly- and heteropolyanion species in aqueous solution is very much dependent on the types of acids as well as on the reagent concentration. In RPEC system, both the elements were found to be quantitatively extracted by an Aliquat 336 impregnated column of Kieselguhr from an alkaline solution containing a hydroxy compound like tartaric acid. The extracted species were subsequently separated by eluting molybdenum and tungsten respectively with HCl solutions of 0.05 mol dm−3 and 0.1 mol dm−3 concentrations. The purity of the separated species at various stages of their separations was verified by means of gamma-ray spectrometry.

A Method for the Determination of Tobacco-specific Nitrosamines (TSNA), Nitrate and Nitrite in Tobacco Leaves and Processed Tobacco

Abstract A method for the simultaneous determination of tobacco-specific nitrosamines [TSNA], N'-nitrosonornicotine [NNN], 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone [NNK], N'-nitrosoanabasine [NAB] and N'-nitrosoanatabine [NAT], as well as nitrate and nitrite in tobacco leaves and processed tobacco was developed. The analytical procedure included aqueous extraction of tobacco leaves and processed tobacco respectively under alkaline conditions in the presence of an antibacterial agent and, for TSNA analysis, a further clean-up of the aqueous extract by liquid-liquid partition extraction (kieselguhr column extraction), removal of nicotine, and determination of the nitrosamines by gas chromatography In combination with chemiluminescence detection. It is demonstrated that N'-nitrosopentylpicolylamine [NPePicA] as well as N-nitrosodibenzylamine [NDBenzA] can be used for internal standardization. For clean-up two different extraction procedures can be utilized: alkaline extraction on kieselguhr / ascorbic acid extraction columns or acidic extraction on kieselguhr including prior destruction of nitrite with sodium azide. The recoveries of the internal standards were in a similar range (73-96 %) as those of the TSNA. The analysis of nitrite and nitrate was carried out according to standard analytical procedures using the alkaline aqueous extract. The determination of nitrate and nitrite showed high accuracy and reproducibility. It is demonstrated that, under the conditions described, artifact formation did not occur even when nitrite was present. The method described is specifically applicable for the simultaneous determination of TSNA and nitrate/nitrite in tobacco leaves.

A method for the determination of N-nitrosoalkanolamines in cosmetics.

A method für the determination of N-nitrosoalkanolamines in cosmetics and toiletries is described. The ingredients used in their manufacture N-nitroso-bis-(2-hydroxyethyl)amine (N-nitrosodiethanolamine, NDELA) and N-nitroso-bis-(2-hydroxy-propyl)amine (NBHPA) are almost exclusively the contaminants. The method has therefore been modified for their determination. N-Nitroso-(2-hydroxy-ethyl)-(2-hydroxypropyl)amine is used as the internal standard. After dilution with water, the cosmetic is adsorbed onto a kieselguhr column and extracted with n-butanol. The extract is brought to dryness, re-dissolved in chloroform/acetone (5 + 1) and transferred to a silica gel column. The column is washed and eluted with acetone. The eluate is dried and the residue is treated with N-methyl-N-trimethylsilyl-heptafluorobutyramide. Trimethylsilyl (TMS)derivatives of N-nitrosoalkanolamines are determined by gas-chromatography with a thermal energy analyzer (TEA). Recovery of the internal standard is 95% and the determination limit is 5 micrograms/kg, Repeated analyses of a foam bath, spiked with 30 micrograms/kg NDELA, gave an average content of 32 micrograms/kg NDELA (variation coefficient 8.8%; n = 10). In order to avoid artefact formation during the clean-up process, kieselguhr containing 50% sodium ascorbate has to be used when cosmetics containing free dialkanolamines are analyzed.

Rapid analysis of propylene glycol in fatty foods.

Rapid analysis of propylene glycol (PG) in fatty foods was developed by using kieselguhr column chromatography. 2-5 g of sample suspended in hot water (70-80°C) was applied to the Extrelut 20 column. After standing for 15 min, 70 ml of hexane was put on the column to remove fat. PG retained in the column was eluted with distilled water. 50 ml of aqueous eluate was collected. PG was quantitated by gas chromatography with trimetylene glycol as an intenal standard. Recoveries of PG added to margarines and mayonnaises were from 98% to 105%. Since proposed purification process is very rapid, many samples can be treated at the same time. Among commercial margarines (25 samples), shortenings (3 samples) and mayonnaises (3 samples), PG was detected in eight margarines (range 37-261 ppm).

The simultaneous determination of diacetoxyscirpenol and T-2 toxin in fungal cultures and grain samples by capillary gas chromatography.

A method for the quantitative determination of the toxic fungal products diacetoxyscirpenol and T-2 toxin is described. Initially intended for the screening of fungal cultures grown on maize, it has subsequently been used successfully for the analysis of maize and oat grain samples. The method involves extraction with aqueous methanol followed by a two-stage clean-up procedure utilizing a commercial kieselguhr column and a silica gel cartridge. The heptafluorobutyryl derivatives of the trichothecenes, including neosolaniol monoacetate used as an internal standard, are separated on a 25 m X 0.32 mm SE-30 fused silica capillary column and measured with a 63Ni electron capture detector. The quantitative detection limit was found to be of the order of 200 ng/g (ppb) with an average recovery from maize of 88% for both toxins. The method was found to be rapid and practical with the advantage that the kieselguhr packing material could be easily regenerated and re-used. The method was compared with those reported in the literature which are currently in use.

HPLC determination of N-nitrosomethylurea in biological fluids by reaction with 4-nitrothiophenol

A simple and sensitive method for the determination of N-nitrosomethylurea (NMU) in biological fluids was developed. The principle is based on the formation of 4-nitrothiophenol methyl thioether (NTP-Me) from 4-nitrothiophenol (NTP) and diazomethane formed from MNU by adding alkali. After sodium hydroxide solution was added to the mixture of NMU in aqueous solution and diethyl ether, the aqueous layer was removed and NTP was added to the ether layer in which diazomethane had been trapped. NTP-Me thus formed was determined by HPLC. This method was successfully applied to the analysis of NMU in biological samples with some clean-up. Stomach and intestine contents and blood samples from guinea-pigs were cleaned up by use of a kieselguhr column followed by heptane-water partition. The recoveries of NMU from the stomach and intestine contents were about 95% and that of NMU from blood samples was about 80%. The detection limit of NMU was 0.5 nmol/g for the stomach contents and 1 nmol/g for the intestine contents and blood samples.

Removal of sodium-24 by chromatographic extraction with a Kieselguhr column and a crown ether solution

Sodium-24 was readily removed from aqueous solutions by a chromatographic process based on an isotopic exchange reaction. The reaction was carried out with a granular Kieselguhr column and a chloroform solution of crown ether-sodium picrate. The process took about 1 h and can be utilized in neutron activation analysis of many elements, Cl, Br, Sc, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Ru, In, Sn, Ce and W. It cannot be used, however, for alkali and alkaline earth elements. The amount of /sup 24/Na in a treated sample was 10/sup -2/ of the original amount. The Compton background was reduced in the range of ..gamma..-ray energy after the removal of sodium-24. 3 figures, 3 tables.

A rapid isotope dilution procedure for estimating the relative proportion of mitochondrial DNA in yeast

A method is described for estimating rapidly the relative proportion of total DNA that is of mitochondrial origin in small quantities of the yeast, Saccharomyces cerevisiae. This procedure involves the mechanical disruption of cells followed by the addition of small amounts of radioactively labeled yeast nuclear and mitochondrial DNA to the lysate. Both labeled and unlabeled DNAs are then co-extracted from the mixture and separated into nuclear and mitochondrial DNA components by poly( l-lysine) Kieselguhr column chromatography. The resulting specific radioactivities of each species of DNA, when compared to the amount of labeled DNA initially added, are related to the relative proportion of unlabeled nuclear and mitochondrial DNA in the original cell sample. The isotope dilution procedure reported here is shown to be both reproducible and to reflect the true relative concentrations of each species of DNA in this yeast.

Alterations in molecular size of messenger RNA from ultraviolet-irradiated T-4-phage.

T4-phages were irradiated by UV-light and phage-specific mRNAs were extracted from Escherichia coli BB-1 cells at various periods of infection. The RNAs were fractionated by methylated albumin-coated kieselguhr (MAK) column chromatography into characteristic sizeclasses. With UV-irradiated phages the last-eluted mRNA component of high molecular weight disappeared preferentially after the middle stage of infection but not at the early stage. However, quite the contrasted changes in the last-eluted, phage-specific component in chromatographic profiles were observed in E. coli cells irradiated by UV-light before infection of normal phages. Some unstable proteins might be responsible for the UV-induced, selective inhibition of high molecular weight mRNA synthesis in bacterial cells.

Separation of140Ba−140La by reversed phase chromatography using high molecular weight amine

A method has been described for the isolation of radiochemically pure140La from140Ba. The tracer140Ba−140La is mixed with 6M LiNO3 solution to make an anionic complex. The solution is then fed into a column (1 cm×0.4 cm) of Kieselguhr impregnated with Aliquat-336.140La is adsorbed in the column while140Ba is eluted with 6M LiNO3. After complete removal of140Ba,140La is eluted with 0.002M HNO3 solution. The purity of140La is established by both its half-life and γ-spectrum.

Differences in the binding of methylated albumin to non-replicating, replicating and denatured DNA from Ehrlich ascites cells

The binding of methylated albumin to DNA, the basis of the chromatography on columns of kieselguhr coated with methylated albumin (MAK chromatography), was investigated. Scatchard plots revealed only one mode of interaction with fully double-stranded DNA. The complexes should be completely dissociated by raising the NaCl concentration of the solution to 0.8 M, indicating a binding by electrostatic attraction between the oppositely charged protein and DNA molecules. In complexes with denatured and partially single-stranded replicating DNA an additional kind of binding was found which made these complexes more stable against salt dissociation. These secondary interactions were stronger at 23°C than at 0°C and could be weakened by the addition of 6 M urea. It was therefore concluded that apolar forces were involved in these interactions.

Mammalian tRNA sulfurtransferase: properties of the enzyme in rat liver.

Transfer RNA sulfurtransferase activity was detected in 105,000 x g supernatant preparations from rat liver and several other rat tissues. Sulfur is transferred from [35S] cysteine to tRNA in a reaction which also requires ATP, Mg2+, and supernatant protein. While [35S] beta-mercaptopyruvate appeared to be a substrate for this enzyme, the reaction product was sensitive to deacylation and the reaction was inhibited by [32S] cysteine. Of the various nucleic acids tested, only tRNAs were effective sulfur acceptors, with rat liver tRNA being the poorest substrate. The [35S] reaction product was sensitive to ribonuclease, cochromatographed with tRNA on methylated-albumin kieselguhr columns, and was converted to nucleotide material after alkaline hydrolysis. DEAE-cellulose chromatography of the neutralized [35S] nucleotide digest revealed a single thionucleotide peak. These studies demonstrate that tRNA sulfurtransferase is present in various rat tissues, and that the requirements of the liver enzyme are similar to those of bacterial enzymes.

Incorporation of label from radioactive uridine into the stylar nucleic acids of Lilium longiflorum thunb. as affected by heat, 6-methylpurine and actinomycin D

5-(3)H-uridine injected into the stylar canal of detached lily stigma-styles was taken up initially into the rapidly-labeled-RNA of the nucleic acid profile of a methylated albumin kieselguhr (MAK) column but with increasing time was found in all portions of the RNA profile, but not in the DNA. Heat treatment of the style before injection of 5-(3)H-uridine greatly reduced the rate of incorporation of label into and the ultimate amount of label found in the RNA species of the lily style. Translocation of 5-(3)H-uridine through the ovary into heattreated pistils and the injection of 5-(3)H-uridine into styles which had been incubated for 1 or 2 days after heat treatment resulted in stylar nucleic acids more highly labeled than nucleic acids in control styles, with an incorporation pattern different than control styles. Heat treatment of lily pistils resulted in detectable changes in the proportion of stylar RNA species as separated on MAK columns and measured as absorbance units. Actinomycin D and 6-methylpurine treated styles incorporated label from a stylar injection of radioactive uridine in patterns different than each other, different than heat-treated styles and different than non-treated styles. 6-methylpurine and heat treatment of styles only slightly reduced the rate at which 5-(3)H-uridine was removed from the stylar canal into the stylar tissue.

Study of messenger-like RNA extracted from HeLa cells polysomes. II. Identification of the types of RNA eluted from methylated albumin kieselguhr columns.

Together with the elution pattern of pure messenger RNA molecules of various origin, the labelling kinetics of rapidly labelled heterogeneously sedimenting RNA (HSRNA) extracted from polysomes of HeLa cells have been studied by chromatogrphy on columns made of methylated bovine serum albumin adsorbed on kieselguhr. HSRNA is eluted within three peaks-IP, Q2P and TDP-following in that order the increase of NaC1 concentration in the eluting buffer. Besides peak TDP which results from an experimental artefact, our data suggest that the appearance of peaks IP and Q2P reflects the absence and presence respectively of polyadenylic acid stretches in these molecules. Within peak Q2P, the critical factor affecting the order of elution is the size of the polyadenylic acid stretch.

Deoxyribonucleic acid strandedness. Partial characterization of the antigenic regions binding antibodies in lupus erythematosus serum.

This study shows that tritiated thymidine labeled DNA prepared from mammalian cells by the Marmur technique is a pure preparation of nucleic acid that is composed essentially of two populations of molecules. One molecular population consists of primarily double-standed nucleic acid, while the other population is of double-stranded nucleic acid with significant single-stranded regions. The double-stranded DNA with single-stranded regions can, depending upon the length of the single strand, behave as "native" DNA or "denatured" DNA on methylated albumin kieselguhr (MAK) column chromatography, Using MAK chromatography we have separated the DNA into a saltelutable fraction composed of primarily double-stranded molecules and an alkaline-elutable fraction containing double-stranded nucleic acid with variable length, single-stranded regions. Endonuclease enzyme removal of the single-stranded regions from the alkaline fraction DNA yield nucleic acid that behaves identically to the salt elutable DNA. Exonuclease removal of the single-stranded regions suggests they are located primarily at the ends of the molecules. Our data show that the alkaline-elutable DNA differs from salt-elutable DNA only in that the former has significant single-stranded regions. Sera of patients with systemic lupus erythematosus (SLE) selected for anti-DNA by hemagglutination bind significantly less to the alkaline fraction DNA than the sale fraction DNA. This difference in binding clearly does not represent simply an affinity for double-stranded vs. single-stranded nucleic acid since the alkaline fraction DNA contains predominately double-stranded nucleic acid. A model for antibody-DNA binding is suggested from the present data and information contained in the literature.

CHANGES IN LABELLING OF RIBONUCLEIC ACID SPECIES FRACTIONATED FROM THE RAT VENTRAL PROSTATE IN RESPONSE TO TESTOSTERONE

ABSTRACT The total RNA isolated at various times up to 24 h after testosterone administration from the ventral prostate of castrated rats, was labelled either by injecting 3H-orotic acid directly into the ventral prostate 6 h before the animals were killed, or by incubating prostatic tissue in vitro with 3H-uridine for 20 to 60 min. The isolated RNA was separated into tRNA, ribosomal RNA (Q1 RNA) and two DNA-like RNA fractions (Q2) and TD RNA) by chromatography on methylated albumin kieselguhr (MAK) columns, and the fractions were further analysed by sucrose gradient centrifugation. Testosterone given into castrated animals for 12 h, stimulated the labelling of all main fractions. The radioactivity of TD RNA after a 60-min incubation period in vitro with 3H-uridine was approximately twice that seen in the castrated rat, while there was a 3.1- and 2.2-fold increase in the radioactivity of the Q1 and Q2 RNA fractions respectively. Kinetics of incorporation of 3H-uridine into different RNA fractions revealed that the hormone facilitated the labelling of the TD RNA fraction relatively more than that of the Q2 fraction. The injection of 3H-orotic acid into the ventral prostate labelled the Q1 RNA preferentially. More than 60 % of the recovered radioactivity was found in Q1 RNA (as 18 and 28 S). Testosterone increased markedly (9.4-fold) the labelling of this fraction. It was concluded that testosterone has an activatory effect on the production of ribosomal RNA, and the bulk of the testosterone effect on the total RNA labelling is to be found in this fraction. Furthermore, it seems likely that testosterone also stimulates both the synthesis and processing of DNA-like RNA. When actinomycin D was given 2 h before the hormone administration in a dose of 25 μg per 100 g of body weight, there was no noticeable increase in the labelling of any fraction above the level seen in the untreated castrated rat. There is evidence that testosterone exerts some effects on the labelling of proteins with radioactive amino acids and 14C-glucose metabolism in the absence of that fraction of the total RNA synthesis which is sensitive to a low dose (25 μg per 100 g of body weight) of actinomycin D (Isotalo & Santti 1972). In this way it may be concluded that the major changes of the RNA synthesis after testosterone administration are likely to be secondary to the protein synthesis and glucose metabolism, or the hormone exerts its anabolic effect on prostatic cells at different sites and by different modes of action, each of which can be operated independently.

Heterogeneity of the DNA preparation from light-anaerobically grown Rhodopseudomonas spheroides cells as revealed by methylated albumin kieselguhr column chromatography

Heterogeneity of the DNA preparation from light-anaerobically grown Rhodopseudomonas spheroides cells as revealed by methylated albumin kieselguhr column chromatography