Nitrogen, detection purification

Relative purity measurement and the relative purity-based reaction optimization have long been used in combinatorial synthesis. In order to make high-through-put purification a success, the yield-based optimization is essential. Chemiluminescent nitrogen detection (CLND) [4] with HPLC determines the quantitative yield after each reaction step during the library feasibility and rehearsal stages. The yield of each synthetic step provides guidance for the final library synthesis. 

Searle, P Hochlowski, J. High Throughput Purification of Parallel Synthesis Samples An Integrated Preparative-LC/MS System with Quantitation by Chemiluminescent Nitrogen Detection, presented at The HPLC 2002, Montreal, Canada, June, 2002. 

Purification of the conjugates may be done by reverse phase HPLC separation. Dry the reaction solution under a nitrogen stream and reconstitute in a minimum volume of acetonitrile/water (1 1, v/v). Apply the sample to a 5 pm Cig-silica HPLC column (250 X 4.6 mm, Nucleosil). Elute with a gradient of water to acetonitrile at a flow rate of 1 ml/minute over a time course of 30 minutes. Free BNAH and BNAH-glycan derivatives can be monitored by absorbance at 275 nm. The conjugate peak also will be positive for carbohydrate by reaction with orcinol, which can be detected by spray after spotting a small eluted sample on a TLC plate. 

Another difficulty in the gas chromatographic separation of amino acids is the choice of detector and it may be necessary to split the gas stream and use two different detectors. The flame ionization detector, which is commonly used, is non-specific and will detect any non-amino acid components of the sample unless purification has been performed prior to derivatization. In addition the relative molar response of the flame ionization detector varies for each amino acid, necessitating the production of separate standard curves. As a consequence, although gas chromatography offers theoretical advantages, its practical application is mainly reserved for special circumstances when a nitrogen detector may be useful to increase the specificity. 

Kirkbride (1987) described the estimation of diazinon in human omental tissue (fatty tissue) after a fatal poisoning. In this method, the tissue was pulverized and extracted with acetone. After extract concentration and purification by sweep co-distillation and Florisil fractionation, diazinon was measured by gas chromatography (GC) with nitrogen-phosphorus detection (NPD). After another fatal diazinon poisoning, diazinon was quantified by GC/electron capture detection (ECD) and GC/flame ionization detection (FID) by Poklis et al. (1980). The diazinon in human adipose, bile, blood, brain, stomach contents, kidney, and liver was recovered by macerating the sample with acetonitrile followed by the addition of aqueous sodium sulfate and extraction into hexane. Following an adsorption chromatography clean-up, the sample was analyzed. 

Low-temperature adsorption systems continue to find an increasing number of applications. For example, systems are used to remove the last traces of carbon dioxide and hydrocarbons in many air-separation plants. Adsorbents are also used in hydrogen liquefaction to remove oxygen, nitrogen, methane, and other trace impurities. They are also used in the purification of helium suitable for liquefaction (grade A) and for ultrapure helium (grade AAA, 99.999% purity). Adsorption at 35 K will, in fact, yield a helium with less than 2 ppb of neon, which is the only detectible impurity in helium after this treatment. 

The purification procedure using this immunoaffinity column has been optimized as follows After extraction with a Sep-Pak PS2 cartridge, the extract is dissolved in 25 mM Tris-HCl buffer (pH 7.2) containing 1 mM EDTA, 0.15 M sodium chloride, and 0.1% sodium azide (Tris-HCl buffer A) with 0.1 % BSA (Tris-HCl buffer B) (5 mL), and the solution is loaded onto an immunoaffinity column, which is preconditioned with Tris-HCl buffer B (10 mL). After washing with Tris-HCl buffer A (10 mL) and distilled water (10 mL), the microcystin fraction is eluted with 100% dimethylformamide (DMF) (2.5 mL). The eluate is then dried on a hot block (60 °C) under a constant stream of nitrogen. The residue is dissolved in 30% methanol-water (0.5 mL) and then subjected to HPLC-photodiode array detection (HPLC-PDA) and LC/ MS analysis. The immunoaffinity column is regenerated by washing with Tris-HCl buffer B (10 mL) before each reuse. 

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