3.4 High performance liquid chromatography HPLC

1112012 chem2202 wtchan 11 the point of sample

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Unformatted text preview: M2202 WTCHAN 11 The point of sample injection is at high pressure. Syringe injection of sample is not practical. Instead, a sample loop is generally used. http://www.ecomsro.com/en/s32‐products/c24‐sampling Interchangeable loops of sample size 5 – 500 µL are available 1/11/2012 CHEM2202 WTCHAN 12 Sample injection in HPLC 1. 2. 1/11/2012 Sample loading into the sample loop – sample volume determined by volume of the loop Rotating the injector by 60° to connect the sample loop to the flow of mobile phase CHEM2202 WTCHAN 13 Detection methods include light absorbance, fluorescence, refractive index, coulometry and other electrochemical techniques, and mass spectrometry Requirements of the detector are : 1. Detector volume smaller than the mobile phase volume in a single theoretical plate (to avoid loss of resolution) 2. Sufficient sensitivity to respond to the very small amounts sensitivity to of analyte eluting from the column H= 1/11/2012 σ t2 L 2 tR wt2 L ( fwhm) t2 L = = 2 2 16t R 5.546t R CHEM2202 WTCHAN 14 UV-visible detector 1/11/2012 CHEM2202 WTCHAN 15 Refractive index detector Optical beam position sensing using 4‐quadrant photodiode arrays 1/11/2012 CHEM2202 WTCHAN 16 HPLC types 1/11/2012 CHEM2202 WTCHAN 18 In partition chromatography, analytes partition between two liquid phases To minimize kinetic transfer broadening minimize (the Cu term), the columns use: Cu 1. Thin liquid coating on the stationary phase beads 2. Small beads of similar size (larger surface area) 1/11/2012 CHEM2202 WTCHAN 19 The stationary phase is chemically bonded to the silica surface to produce stable and uniform coating (Bonded phase) Adsorption of the immobile liquid phase on the silica bead surface to form the stationary phase is not satisfactory because not of non-uniform coating thickness and poor adhesion of the non coating to the beads. The column performance deteriorates with time. 1/11/2012 CHEM2202 WTCHAN 20 The solubilizing nature of the bonded phases can be adjusted using different functional groups in the silylating agent Nonpolar (saturated or aromatic hydrocarbons) and polar functional polar groups (amino, cyano, or alcohol termini) are available Nonpolar stationary phases are used with more polar mobile phases and vice versa vice 1/11/2012 CHEM2202 WTCHAN 21 Normal phase & Reverse Phase Chromatography Normal phase chromatography refers to polar stationary phase and nonpolar solvent. Reverse phase chromatography refers...
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This note was uploaded on 08/17/2013 for the course CHEM 2202 taught by Professor Wt during the Fall '11 term at HKU.

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