Columns for HPLC - Western Analytical

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Columns for HPLCColumns for HPLCAnalytical columns packed with NUCLEOSIL ®Standard phases for RP chromatographyReversed phase silica phases are by far the most widelyused packings. Our base deactivated high performance RPmaterials are described in the preceding chapter. In thischapter you will find columns packed with standard silicaphases for RPLC. For ordering information of the correspondingbulk packings please see the chapter starting onpage 143. Octadecyl, Octyl, Butyl, Dimethyl and Phenylphases differ in retention time for a given solute. Under identicalconditions (mobile phase, flow rate, temperature etc.),e. g. the retention times on NUCLEOSIL ® C 18 are, for quite anumber of compounds, by a factor of up to 2 larger than onNUCLEOSIL ® C 8 .The mechanism of separation with all NUCLEOSIL ® andNUCLEODUR ® RP phases is the attraction between hydrophobicgroups of the test molecule and the bonded alkylgroups of the stationary phase by van der Waals forces. theless polar the sample molecules, the more they are attracted.Differences in the chemical structure of the sample moleculesresult in a stronger or weaker adsorption to the stationaryphase and hence in a separation.The more polar the sample, the shorter are the retentiontimes and the weaker are the van der Waals forces. The followingis roughly the order of increasing polarity (reverse orderof elution): aliphatic hydrocarbons, induced dipoles(CCI 4 ), permanent dipoles (CHCI 3 ), weak Lewis bases(ethers, aldehydes, ketones), strong Lewis bases (amines),weak Lewis acids (alcohols, phenols) and strong Lewis acids(carboxylic acids). Also of considerable importance for retentiontimes is the chain length of the molecules which are tobe separated.The mobile phase in RP chromatographyη[cP]at20 °C3viscosityViscosity of solvent mixturesas a function of composition3value. Higher concentrations of organic solvent will causeshorter retention times. RP separations can be optimised byutilising the specific selectivity of organic solvents.The viscosity of the eluent should not be neglected. It is oftennot realised in practice, that eluents have very different viscosities(η) and that these differences in viscosity (with thesame flow rate) cause changes in back pressure. The backpressure changes according to the following equation:ηp 11 = ----- ⋅ pη 2221watern-propanol2ethanolmethanolacetonitrile0 20 40 60 80 100% [w/w]organicThe mobile phase for reversed phase chromatography is amixture of water and an organic solvent which is misciblewith water. The time of elution of the substances to be separateddepends to a large degree on the water content of theorganic solvent and is therefore easily adjusted to a suitable1The viscosity of a solvent mixture is often higher than that ofthe single components, i.e. a higher pressure is needed toachieve the same flow rate.The graph on the left illustrates how strongly the viscosity ofthe eluent is influenced by its composition. For example theviscosity (and consequently the back pressure of the column)increases more than double when the eluent ischanged from acetonitrile – water (70:30) to methanol – water(70:30).When water is used as a solvent or as a component in gradientmixtures, it is essential that it is completely degassed.Apparent instability can be caused by air bubbles (from notproperly degassed solvents) which accumulate at the detector.The best way to remove such air is to first pump degassedsolvent through the column and then follow up withdegassed water. One can degas solvents with a vacuumpump, by passing helium through or by ultrasonic treatment.6.7.05MNwww.mn-net.com37
Columns for HPLCColumns for HPLCAnalytical columns packed with NUCLEOSIL ®Test mixture for reversed phase columnsFor reversed phase HPLC columns we supply a test mixturein acetonitrile to determine the quality of the packing. The figureon the right shows a typical test chromatogram of a columnwith the stationary phase NUCLEOSIL ® 100-5 C 18 .Ordering informationDesignation Pack of Cat. No.Test mixture for reversed phasecolumns in acetonitrile1 ml 722394Test mixture for reversed phase HPLC columns(Cat. No. 722394)Column: 250 x 4 mm NUCLEOSIL ® 100-5 C 18Sample volume: 20 µlEluent: acetonitrile – water (70:30)Flow rate: 1 ml/minPressure: 105 barTemperature: 22 °C2 3Detection: UV, 254 nm1Peaks: (concentrationin µg/ml eluent)1. Phenol (400)2. Naphthalene (180)3. Anthracene (12)4 8 min6.7.05Individual test certificate enclosed witheach NUCLEODUR ® and NUCLEOSIL ®column, respectively.38www.mn-net.comMN
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Columns for HPLC - Western Analytical

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