Isocratic chromatography method development

Cholesterol is one of the main components of cell membranes and has several functions in the body, including the synthesis of certain hormones such as vitamin D and bile acid. Over the years, gas chromatography has been used to characterize cholesterol and its derivatives. Thiam et al. [123] developed an isocratic CEC method that allows baseline separation of a complex mixture of cholesterol and 12 ester derivatives in less than 40 min. The use of a polymeric surfactant, poly(sodium N-undecanoyl-L-glycinate), in the CEC buffer reduced migration time and improved resolution of the analytes. 

Therefore, if a fast, automated, routine method for the measurement of multi-species/elements is the desired analytical goal, it is often best to attempt an isocratic separation method first, because of the complexity of method development and the low sample throughput of gradient elution methods. In fact, a simultaneous method for the separation of As, Cr, and Se species in drinking water samples was demonstrated by Neubauer and coworkers they developed a method to determine inorganic forms of arsenic (As, As+ ), chromium (Cr , Cr ), and selenium (Se, Se and SeCN ) by reverse-phase ion-pairing chromatography with isocratic elution. Details of the HPLC separation parameters/conditions they used are shown in Table 18.2. 

A liquid chromatography-mass spectrometry (LC-MS) method that can quantitatively analyze urinar y normal and modified nucleosides in less than 30 min with a good resolution and sufficient sensitivity has been developed. Nineteen kinds of normal and modified nucleosides were determined in urine samples from 10 healthy persons and 18 breast cancer patients. Compounds were separ ated on a reverse phase Kromasil C18 column (2.1 mm I.D.) by isocratic elution mode using 20 mg/1 ammonium acetate - acetonitrile (97 3 % v/v) at 200 p.l/min. A higher sensitivity was obtained in positive atmospheric pressure chemical ionization mode APCI(-i-). 

Reverse phase liquid chromatography has typically been used for the separation of PFCs, employing either Cg or Cig columns [96], although the use of perfluoroalkyl columns has also been reported [115]. Mobile phases are typically mixtures of methanol-water or acetonitrile-water and are often modified with ammonium acetate to improve chromatographic separation and MS sensitivity. Both isocratic and gradient elution methodologies have been employed [96]. LC-MS/MS methods [116, 117] have also been developed for the separation of PFSA and PFCA isomers and generally employ linear perfluorooctyl stationary phases and acidified mobile phases. 

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