High-performance liquid quantitative purity

High performance liquid chromatography is used to determine the purity of calcitriol, and to separate it from related compounds. Using a 10 micron silica column of 25 cm length, and a mobile phase of spectroquality heptane ethyl acetate. methanol (50 50 1) at a flow rate of 1.7 ml/ minute, separation and quantitation are achieved. p-Dimethyl-aminobenzaldehyde may be used as an internal standard to compensate for variations in injection technique and instrumental conditions. With a 254 nm ultraviolet absorbance detector, 0.01 ug of calcitriol may be detected (3). 

Identification. Chromatographic methods (interpretation of fluorescing spots in thin layer chromatography or of UV absorption peaks and fluorescence light emission peaks in high performance liquid chromatography) allow fairly rapid and certain identification of FWAs [158-164], The same methods can be used for qualitative and quantitative characterization of the FWA purity in terms of fluorescent or nonfluorescent byproducts. 

Thin-layer chromatography (TLC) is mainly applied in micropreparative taxoids separation [2-4]. Silica gel 6OF254 preparative plates are usually applied for this purpose. The problem of taxoids separation involves not only their similar chemical structure (e.g., paclitaxel versus cephalomannine) but also, due to different coextracted compounds usually encountered in crude yew extracts (polar compounds such as phenolics and nonpolar ones such as chlorophylls and biflavones), the separation is very difficult. The common band of paclitaxel and cephalomannine was satisfactorily resolved from an extraneous fraction in isocratic elution with ethyl acetate as a polar modifier [4] and n-heptane-dichloromethane as the solvent mixture and it was of suitable purity for high-performance liquid chromatography (HPLC) quantitative determination. 

The purity control and the structure verification of compound collections from automated synthesis and combinatorial chemistry play an essential role in the success of medicinal chemistry programs. High performance liquid chromatography (HPLC), mass spectrometry (MS), and liquid chromatography-mass spectrometry (LC-MS) techniques are generally accepted as the most appropriate means of characterization (1,2). While these analytical methods are fast and easy to automate, they do not provide sufficient structural and quantitative data about the desired products. 

Instmmental methods of analysis provide information about the specific composition and purity of the amines. Qualitative information about the identity of the product (functional groups present) and quantitative analysis (amount of various components such as nitrile, amide, acid, and determination of unsaturation) can be obtained by infrared analysis. Gas chromatography (gc), with a liquid phase of either Apiezon grease or Carbowax, and high performance Hquid chromatography (hplc), using silica columns and solvent systems such as isooctane, methyl tert-huty ether, tetrahydrofiiran, and methanol, are used for quantitative analysis of fatty amine mixtures. Nuclear magnetic resonance spectroscopy (nmr), both proton ( H) and carbon-13 ( C), which can be used for qualitative and quantitative analysis, is an important method used to analyze fatty amines (8,81). 

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