A simple and inexpensive high pressure liquid chromatographic system as an adjunct to gas chromatography in identification of amino acid phenylthiohydantoins.

A simple and inexpensive system has been developed in our laboratory for the identification and quantitation of amino acid phenylthiohydantoins by high pressure liquid chromatography. Isocratic methodology has been employed, so that expensive complex gradient makers can be avoided. The method is rapid, and retention times of 10 min or less are obtained in all but one case. This paper presents the rationale and details of the method, and demonstrates a comparison between gas-liquid chromatograms and high pressure liquid chromatograms of selected residues from actual automated sequence analyses of both sperm whale myoglobin and egg white lysozyme. The method is intended to complement rather than replace gas-liquid chromatography, as it cannot completely resolve all 20 phenylthiohydantoins, but rather identifies easily those residues which are difficult or impossible by gas chromatography.     

A high pressure liquid chromatographic method for the separation and quantitation of water-soluble radiolabeled benzene metabolites

The glucuronide and sulfate conjugates of benzene metabolites as well as muconic acid and pre-phenyl- and phenylmercapturic acids were separated by ion-pairing HPLC. The HPLC method developed was suitable for automated analysis of a large number of tissue or excreta samples. p-Nitrophenyl [14C]glucuronide was used as an internal standard for quantitation of these water-soluble metabolites. Quantitation was verified by spiking liver tissue with various amounts of phenylsulfate or glucuronides of phenol, catechol, or hydroquinone and analyzing by HPLC. Values determined by HPLC analysis were within 10% of the actual amount with which the liver was spiked. The amount of metabolite present in urine following exposure to [3H]benzene was determined using p-nitrophenyl [14C]glucuronide as an internal standard. Phenylsulfate was the major water-soluble metabolite in the urine of F344 rats exposed to 50 ppm [3H]benzene for 6 h. Muconic acid and an unknown metabolite which decomposed in acidic media to phenylmercapturic acid were also present. Liver, however, contained a different metabolic profile. Phenylsulfate, muconic acid, and pre-phenylmercapturic acids as well as an unknown with a HPLC retention time of 7 min were the major metabolites in the liver. This indicates that urinary metabolite profiles may not be a true reflection of what is seen in individual tissues.     

High-performance liquid chromatographic analysis of abscisic acid in plant extracts.

A HPLC assay for ABA and bound ABA is described. The method uses conventional acid-base ether extractions for partial cleanup of the plant extracts followed by Sephadex G-25 column fractionation. The ABA is separated and detected by HPLC on a 3 m × 2.1 mm i.d. column of SCX-Zipax with a uv detector. The method is sensitive to less than 10 ng of ABA. HPLC analyses of ABA have been performed on extracts of leaves, stems, and roots of soybeans, pinto beans, cotton, apple seedlings, and the albedo and flavedo sections of orange peels.     

A modified Amaranthus betacyanin bioassay for the rapid determination of cytokinins in plant extracts

Summary Pure cytokinin standards and celery seed extracts containing cytokinin activity were bioassayed using a modified Amaranthus betacyanin bioassay. The assay is very rapid and requires no special sterile precautions.     

植物挥发性物质提取的一种简易方法--液氮冷凝法

介绍了植物挥发物提取的一种简易方法——液氮冷凝法。利用液氮的制冷原理对植物挥发性物质进行提取收集,当携带植物挥发性物质的纯净空气带动供试植物组织的挥发性物质通过一置有U形玻璃管的液氮罐时,由于液氮的低温作用,挥发性物质在U形管内壁上遇冷凝结。该提取装置简单,由硅胶柱、活性碳柱、流量计、锥形瓶、U形管、液罐及各种连接管构成,容易组装。利用该装置提取的挥发性物质可以用于生测,去除水分后还可用于电生理以及挥发性物质化学组成分析等。     

A simple and inexpensive method of solid-state cultivation

Summary A simple and inexpensive procedure is described for the solid-state cultivation of fungi in plastic bags. This procedure, which provides for aeration, humidification and temperature control, may be used for extracellular enzyme production or upgrading of agricultural residues. It should be especially useful where resources are limited.     

A simple and sensitive DNA assay for plant extracts

A sensitive fluorimetric method was developed for the quantitative determination of DNA in plant (Zea mays L. and Medicago sativa L.) extracts. This method takes advantage of the specific increase in fluorescence intensity of the complex of DNA and the dye 4′,6′-diamidino-2-phenylindole (DAPI). Recovery of DNA and dissociation of histones from DNA were maximized by the addition of 2.0 molar NaCl to the homogenates. Treatment of the homogenate with chloroform to remove pigments and proteins decreased the quenching of fluorescence of the DAPI-DNA complex. The fluorescence intensity of RNA with DAPI was less than 2% of that produced by an equivalent weight of DNA. Comparisons were made between this fluorimetric DNA method and the commonly used diphenylamine assay for DNA. The diphenylamine DNA assay was more timeconsuming, less sensitive, and consistently resulted in lower estimates of DNA concentrations than did the fluorimetric DNA assay.     

A simple method for separating cells of Microcystis aeruginosa for counting

Heat was used to break up the mucilage-bound colonies of the blue-green alga, Microcystis aeruginosa Kütz. emend. Elenkin, for cell counting. Samples were heated in a water bath at 80°C for 5–10 min, then agitated in a Vortex mixer for 30–60 s. The treatment separated cells completely and reaggregation of cells did not occur. There was no evidence that heating distintegrated the cells.     

A sensitive high performance liquid chromatographic method for determining small amounts of glycosylproteins

We developed a simple isocratic high performance liquid chromatography (HPLC) method for the quantitative determination of 5-hydroxymethyl-2-furfuraldehyde (5-HMF) liberated by mild hydrolysis of small amounts of glycosyl proteins. The absorbance of hydrolysate components after HPLC separation was recorded at 280 nm. To detect substances possibly interfering with the 5-HMF peak we always recorded the ratio of the peak heights A280 nm/A254 nm which was a constant value of 4.4. For each sample the blank was obtained by reduction with NaBH4 before hydrolysis with oxalic acid 1 mol/l. The best NaBH4/protein ratio was found to be 4 mg/mg. With this method we measured the nonenzymatic glycosylation (glycation) as 5-HMF in samples with a protein concentration as low as 0.8 mg/ml. 5-HMF produced per milligram of protein was independent from protein concentration for a wide range (0.8-10 mg/ml). The mean coefficient of variation for within assay and between precision was 6.8 and 11.6%, respectively. The 5-HMF measured on plasma proteins from normal subjects (n = 7) was 0.16 +/- 0.04 nmol/mg. Protein from insulin-dependent diabetic patients was 0.31 +/- 0.07 nmol/mg. With this method we succeeded in detecting an excessive glycation of platelet membrane proteins in 13 type-I diabetic patients.     

A simple and inexpensive method for producing fluorescently labelled size standard

Current methods of automated genotyping offer many advantages over traditional gel‐based approaches, including reduced handling and processing times, and increased accuracy and consistency. Unfortunately, these advances have come at a substantial cost; at present, roughly one‐half of the cost of automated genotyping is due to fluorescently labelled internal size standard. Here we describe detailed methodologies for generating a highly consistent, fluorescently labelled, internal size standard using polymerase chain reaction (PCR). The methods are simple and the required reagents are inexpensive, making the in‐house production of fluorescently labelled size standards a more widely accessible alternative to commercially available size standards.     

A simple, reliable and inexpensive method for the collection of rat urine.

Aluminum foil attached to the bottom of hanging-type rat cages approximately 10 cm from the front of the cage was used for collecting rat urine. Most urine samples were obtained within 1 hour of placing the rat in the cage.