3.4 High performance liquid chromatography HPLC

Reverse phase chromatography refers to reverse low

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Unformatted text preview: to Reverse low-polarity bonded phase and more polar mobile phase. 1/11/2012 CHEM2202 WTCHAN 22 Normal phase – polar stationary phase Reverse phase – non-polar stationary phase 1/11/2012 CHEM2202 WTCHAN 23 In reverse phase chromatography, the more polar solutes in a sample have less affinity for the nonpolar stationary phase Polar components spend less time in the stationary phase and more time in the more polar mobile phase Polar components will elute first Compounds will come off the column in order of decreasing polarity 1/11/2012 CHEM2202 WTCHAN 24 The general elution problem of liquid chromatography: analytes in a sample have a wide range of k’ values for any combination of stationary and mobile phases The solution to the general elution problem is to decrease the values of k’ for the analytes while the elution is occurring. In reverse phase chromatography, the affinity of the reverse mobile phase is increased by making the mobile phase less polar. Thus, as the elution continues, an increasing amount an of a less polar solvent is mixed into the mobile phase. This is called gradient elution. gradient 1/11/2012 CHEM2202 WTCHAN 26 Common solvents for LC are water, acetonitrile, methanol, and tetrahydrofuran 1/11/2012 CHEM2202 WTCHAN 27 According to European Directive 67/548/EEC as amended. Highly flammable. Harmful by inhalation, in contact with skin and if swallowed. Irritating to eyes. 1/11/2012 CHEM2202 WTCHAN 28 Common characteristics of the solvents for LC include: low viscosity, available in high purity, UV-transparent, and miscible in each other 1/11/2012 CHEM2202 WTCHAN 29 In gradient elution, the polarity of the mobile phase is varied continuously during the elution process by combining two or more solvents in different proportion in a pre-defined program 1/11/2012 CHEM2202 WTCHAN 30 1/11/2012 CHEM2202 WTCHAN 31 Melamine 三聚氰胺 三聚 http://en.wikipedia.org/wiki/Melamine 1/11/2012 CHEM2202 WTCHAN 32 Determination of Melamine Residues in Catfish Tissue by Triple Quadrupole LC-MSMS with HILIC Chromatography – US FDA (http://www.fda.gov/Food/ScienceRes...
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