Basic principles of HPLC

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Basic principles of HPLC ( basic-principles-hplc )

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Basic principles of HPLC Basic principles of HPLC Introduction to the theory of HPLC HPLC (High Performance Liquid Chromatography) depends on interaction of sample analytes with the stationary phase (packing) and the mobile phase to effect a separation. Fol- lowing are explanations of the separation mechanisms com- monly used in HPLC. In adsorption chromatography the stationary phase, prop- erly speaking, is the liquid-solid interface Molecules are reversibly bound to this surface by dipole-dipole interactions. Since the strength of interaction with the surface is different for different compounds, residence time at the stationary phase varies for different substances thus achieving separa- tion. Liquid-solid adsorption chromatography is most often used for polar, non-ionic organic compounds. Partition chromatography is the fundamental distribution mechanism in liquid-liquid chromatography, i. e. when both mobile phase and stationary phase are liquids. Separation by distribution is based on the relative solubility of the sam- ple in the two phases. In normal phase partition chromatog- raphy the stationary phase is more polar than the mobile phase, in reversed phase (RP) chromatography the mobile phase is more polar than the stationary phase. Stationary phases may be either coated on to a support, or they may be chemically bonded to the surface. Normal phase partition chromatography is used for very polar organic compounds, while reversed phase chromatography is commonly used for nonpolar or weakly polar substances. Ionic compounds are often better separated by ion exchange chromatography (IEC). In this case, the station- ary phase consists of acidic or basic functional groups bonded to the surface of a polymer matrix (resin or silica gel). Charged species in the mobile phase are attracted to appropriate functional groups on the ion exchanger and thereby separated. Ion pairing chromatography is an alternative to ion exchange chromatography. Mixtures of acids, bases and neutral substances are often difficult to separate by ion exchange techniques. In these cases ion pairing chromatog- raphy is applied. The stationary phases used are the same reversed phases as developed for reversed phase chroma- tography. An ionic organic compound, which forms an ion- pair with a sample component of opposite charge, is added to the mobile phase. This ion-pair is, chemically speaking, a salt which behaves chromatographically like a non-ionic organic molecule that can be separated by reversed phase chromatography. Size exclusion chromatography (SEC) or gel permeation chromatography (GPC) uses as the stationary phase a porous matrix which is permeated by mobile phase mole- cules. Sample molecules small enough to enter the pore structure are retarded, while larger molecules are excluded and therefore rapidly carried through the column. Thus size exclusion chromatography means separation of molecules by size. The chromatogram below illustrates the most important pa- rameters which characterise a separation. These parameters will be explained in the following paragraphs. w1/2 t’R1 tR2 retention time 10% of peak height AB w t0 t R1 t’R2 Explanation of the most important parameters to characterise a separation: peak widths: w1/2 = w = peak width at half height band width of the peak (intersection point of the inflectional tangents with the zero line) peak symmetry is measured at 10% or peak height symmetry parameters: dead time of a column = retention time of an unretarded substance A = B = retention times peak front at 10% of peak height to peak maximum peak maximum to peak end at 10% of peak height t0 = tR1 , tR . . = retention times of components 1, 2 . . t′R1, t′R2 . . = net retention times of components 1, 2 . . Retention: In an elution chromatographic separation sub- stances differ from each other only in their residence time in or at the stationary phase. From this the following time defini- tions arise: The total retention time ( tR1 or tR ) is the time, which is 2 needed by a sample component to migrate from column inlet (sample injection) to the column end (detector). The dead time t0 is the time required by an inert compound to migrate from column inlet to column end without any retar- dation by the stationary phase. Consequently, the dead time is identical with the residence time of the sample compound in the mobile phase. 2 174 MN Injection

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