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Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,*,*,Chapter 26An Introduction to Chromatographic Separations,Chromatography permit the scientist to separate closely related components of complex mixtures.In all chromatographic separations the sample is transported in a mobile phase,which may be a gas,a liquid,or a supercritical fluid.This mobile phase is then forced through an immiscible stationary phase,which is fixed in place in a column or on a solid surface.The two phases are chosen so that the components of the sample distribute themselves between the mobile and stationary phase to varying degrees.,Classification of Chromatographic Methods,Chromatographic methods can be categorized in two ways.The first classification is based upon the physical means by which the stationary and mobile phases are brought into contact.In column chromatography,the stationary phase is held in a narrow tube through which the mobile phase is forced under pressure.In planar chromatography,the stationary phase is supported on a flat plate or in the interstices of a paper;here,the mobile phase moves through the stationary phase by capillary action or under the influence of gravity.,Classification of Chromatographic Methods,A more fundamental classification of chromatographic methods is one based upon the types of mobile and stationary phases and the kinds of equilibria involved in the transfer of solutes between phases.,Three,general categories of chromatography:,(1),liquid chromatography,(2),gas chromatography,and,(3),supercritical-fluid chromatography.The mobile phases in the three techniques are liquids,gases,and supercritical fluids respectively.,Elution Chromatography on Columns,Elution involves washing a species through a column by continuous addition of fresh solvent.The sample is introduced at the head of a column,whereupon the components of the sample distribute themselves between the two phases.Introduction of additional mobile phase(the eluent)forces the solvent containing a part of the sample down the column,where further partition between the mobile phase and fresh portions of the stationary phase occurs.,Chromatograms,If a detector that responds to solute concentration is placed at the end of the column and its signal is plotted as function of time(or of volume of the added mobile phase),a series of peaks is obtained.Such a plot,called a chromatogram,is useful for both qualitative and quantitative analysis.The positions of peaks on the time axis may serve to identify the components of the sample;the areas under the peaks provide a quantitative measure of the amount of each component.,MIGRATION RATES OF SOLUTES,The effectiveness of a chromatographic column in separating two solutes depends in part upon the relative rates at which the two species are eluted.These rates are determined by the magnitude of the equilibrium constants for the reactions by which the solutes distribute themselves between the mobile and stationary phases.,Distribution Constants,The distribution equilibria involved in chromatography involve the transfer of an analyte between the mobile and stationary phases.,A,mobile,A,stationary,The equilibrium constant K for this reaction is called the distribution constant,the partition ratio,or the partition coefficient,K=c,S,/c,M,where c,s,is the molar concentration of the solute in the stationary phase and c,M,is its molar concentration in the mobile phase.K is constant over a wide range of solute concentrations.,Retention Time,The time it takes after sample injection for the analyte peak to reach the detector is called the,retention time,and is given the symbol t,R,.The time t,M,for the unretained species to reach the detector is called the dead time.The rate of migration of the unretained species is the same as the average rate of motion of the mobile phase molecules.The average linear rate of solute migration,is,=L/,t,R,where,L is the length of the column packing.The average linear rate of movement u of the molecules of the mobile phase is,u=L/t,M,Where t,M,the dead time.,The Rate of Solute Migration:The Retention Factor,The retention factor,or capacity factor,is an important parameter that is widely used to describe the migration rates of solutes on columns.For a solute A,the retention factor k,A,is defined as,k,A,=K,A,V,S,/V,M,where K,A,is the distribution constant for the species A.,k,A,=(t,R,-t,M,)/t,M,t,R,and t,M,are readily obtained from a chromatogram.When the retention factor for a solute is much less than unity,elution occurs so rapidly that accurate determination of the retention times is difficult.When the retention factor is larger than perhaps 20 to 30,elution times become inordinately long.Ideally,separations are performed under conditions in which the retention factors for the solutes in a mixture lie in the range between 2 and 10.,Relative Migration Rates:The selectivity Factor,The selectivity factor,of a column for
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