1. Chapter 30 Introduction to analytical Separations An important part of most analyses is dealing with foreign species that either attenuate the signal from the analyte or produce a signal that is indistinguishable from that of the analyte. A substance that effects an analytical signal is called an interference or an interferent. Several general methods are used for dealing with interferences in an analysis; i) masking, ii) chemical or electrolytic precipitation, iii) distillation, iv) solvent extraction, v) ion exchange, vi) chromatography, and vii) electrophoresis.

4. MASKING In masking, a reagent is added to the solution of the sample to immobilize, or chemically bind, the interferent as a complex that no longer contributes to or attenuates the signal from the analyte. A masking agent must not affect the behavior of the analyte significantly.

5. PRECIPITATION AND FILTRATION Precipitation, in which the analyte or an interferent is removed from a solution selectively as an insoluble species, is one of the oldest methods for dealing with interferences in an analytical procedure. SEPARATING SPECIES BY DISTILLATION Distillation is widely used to separate volatile analytes from nonvolatile interferents.

6. SEPARATING SOLUTES BY EXTRACTION The extent to which solutes, both inorganic and organic, distribute themselves between two immiscible liquids differs enormously, and these differences have been used for decades to accomplish separations of chemical species.

7. Principles The partiton of a solute between two immiscible phases in an equilibrium phenomenon that is governed by the distribution law. If the solute species A is allowed to distribute itself between water and an organic phase, the resulting equilibrium may be written as A aq A org The ratio of activities for A in the two phases will be constant and independent of the total quantity of A; that is, at any given temperature,

8. …continued… The equilibrium constant K is known as the distribution constant. The concentration of A remaining in an aqueous solution after I extractions with an organic solvent is given by the equation where, [A] i is the concentration of A remaining in the aqueous solution after extracting V aq mL of the solution having an original concentration of [A] o with I portions of the organic solvent, each with a volume of V org.

11. SEPARATING IONS BY ION EXCHANGE Ion exchange is a process by which ions held on a porous, essentially insoluble solid are exchanged for ions in a solution that is brought in contact with the solid. The ion-exchange properties of clays and zeolites have been recognized and studied since the late nineteenth century. Synthetic ion-exchange resins were first produced in 1935 and have since found widespread application in water softening, water deionization, solution purification, and ion separation.

12. Ion-Exchange Resins Synthetic ion-exchange resins are high-molecular-weight polymers that contain large numbers of an ionic functional group per molecule. Cation-exchange resins contain acidic groups, whereas anion-exchange resins have basic groups. Exchangers of the strong-acid type have sulfonic acid groups (SO 3 - H + ) attached to the polymeric matrix. Strong-base anion exchangers contain quaternary amine [N(CH 3 ) 3 + OH - ] groups. xRSO 3 - H + + M x+ (RSO 3 - ) x M x+ + xH + solid soln xRN(CH 3 ) 3 + OH - + A x- [RN(CH 3 ) 3 + ] x A x- + xOH - solid solnsolid soln

14. Applications of Ion-Exchange Methods Ion-exchange resins are used to eliminate ions that would otherwise interfere with an analysis. Another valuable application of ion-exchange resins involves concentrating ions from a very dilute solution. Thus, traces of metallic elements in large volumes of natural waters can be collected on a cation-exchange column and subsequently liberated from the resin by treatment with a small volume of an acidic solution. The total salt content of a sample can be determined by titrating the hydrogen ion released as an aliquot of the sample passes through a cation exchanger in its acidic form. Ion-exchange resins are particularly useful for the chromatographic separation of both inorganic and organic ionic species.

15. CHROMATOGRAPHIC SEPARATION Chromatography is a widely used method for the separation, identification, and determination of the chemical components in complex mixtures. No other separation method is as powerful and generally applicable as chromatography.

16. General Description of Chromatography The term “chromatography” is difficult to define rigorously because the word has been applied to several systems and techniques. Common to all these methods, however, is the use of a stationary phase and a mobile phase. Components of a mixture are carried through the stationary phase by the flow of the mobile phase, and separations are based on differences in migration rates among the mobile-phase components.

17. …continued… The stationary phase in chromatography is a phase that is fixed in place either in a column or on a planar surface. The mobile phase in chromatography is a phase that moves over or through the stationary phase, carrying with it the analyte mixture. The mobile phase may be a gas, a liquid, or a supercritical fluid.

18. Classifying Chromatographic Methods Chromatographic methods are of two basic types. In column chromatography, the stationary phase is held in a narrow tube, and the mobile phase is forced through the tube under pressure or by gravity. In planar chromatography, the stationary phase is supported on a flat plate or in the pores of a paper. Here the mobile phase moves through the stationary phase by capillary action or under the influence of gravity. Column chromatographic methods can be further subdivided according to the nature of the mobile phase, specifically liquid, gas, and supercritical fluid.

20. Elution in Column Chromatography Elution is a process in which solutes are washed through a stationary phase by the movement of a mobile phase. The mobile phase that exits the column is called the eluate. An eluent is a solvent used to carry the components of a mixture through a stationary phase.

22. Chromatograms If a detector that responds to solute concentration is placed at the end of the column during elution and its signal is plotted as a function of time (or of a volume of 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 the peaks on the time axis can be used to identify the components of the sample; the areas under the peaks provide a quantitative measure of the amount of each species.

25. Relative Migration Rates of Solutes The effectiveness of a chromatographic column in separating two solutes depends on the relative rates at which the two species are eluted. These rates are in turn determined by the rations of the solute concentrations in each of the two phases.

26. Distribution Constants All chromatographic separations are based on differences in the extent to which solutes are distributed between the mobile and the stationary phase. For the solute species A, the equilibrium involved is described by the equation A(mobile) A(stationary) The equilibrium constant K c for this reaction is called a distribution constant, which is defined as

27. Retention Times The dead time t M is the time it takes for an unretained species to pass through a column. The retention time t R is the time between injection of a sample and the appearance of a solute peak at the detector of a chromatographic column. The average linear rate of solute migration,, in centimeters per second is = L / t R where L is the length of the column packing. Similarly, the average linear velocity, u, of the molecules of the mobile phase is u = L / t M _

29. Relating Migration Rates to Distribution Constants = u x fraction of time solute spends in mobile phase _ _ _ _

30. The Retention Factor (k) The retention factor is an important experimental parameter that is widely used to compare the migration rates of solutes on columns. For solute A, the retention factor k A is defined as where K A is the distribution constant for solute A. To show how k A can be derived from a chromatogram, _

31. …continued… This equation rearranges to 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 1 and 5.

32. The Selectivity Factor The selectivity factor  of a column for the two solutes A and B is defined as  = K B / K A where K B is the distribution constant for the more strongly retained species B and K A is the constant for the less strongly held or more rapidly eluted species A. According to this definition,  is always grater than unity.  = k B / k A where k B and k A are the capacity factors

33. Quantitative Measures of Column Efficiency Two related terms are widely used as quantitative measures of chromatographic column efficiency: (1) plate height H and (2) plate count or number of theoretical plates N. The two are related by the equation N = L / H where L is the length (usually in centimeters) of the column packing. The efficiency of chromatographic columns increases as the plate count N becomes greater and as the plate height H becomes smaller.

35. Determining the Number of Plates in a Column The number of theoretical plates, N, and the plate height, H, are widely used in the literature and by instrument manufactures as measures of column performance. N can be determined from a chromatogram. The retention time of a peak t R and the width of the peak at its base W (in units of time) are measured. The number of plates can then be computed by the simple relationship. N = 16 (t R / W) 2 To obtain H, the length of the column L is measured and N = L / H Equation is applied.

37. Variables That Influence Plate Heights It has been found that plate heights can be decreased, and thus column efficiency increased by decreasing the particle size of column packings, by employing thinner layers of film (where the stationary phase is a liquid adsorbed on a solid), and by lowering the viscosity of the mobile phase. Increases in temperature also reduce band broadening in most cases.

40. Column Resolution The resolution R s of a column provides a quantitative measure of its ability to separate two analytes. The resolution is defined as A resolution of 1.5 gives an essentially complete separation of A and B, whereas a resolution of 0.75 does not. At a resolution of 1.0, zone A contains about 4% B and zone B contains about 4% A. At a resolution of 1.5, the overlap is about 0.3%. The resolution for a given stationary phase can be improved by lengthening the column and thus increasing the number of plates. An adverse consequence of the added plates, however, is an increase in the time required for separating the components.

42. Effect of Retention Factor and Selectivity Factor A useful equation is derived that relates the resolution of a column to the number of plates it contains as well as to the retention and selectivity factors of a pair of solutes on the column. The resolution is given by the equation where k B is the retention factor of the slower-moving species and  is the selectivity factor. This equation can be rearranged to give the number of plates needed to realize a given resolution:

43. Applications of Chromatography Chromatography is a powerful and versatile tool for separating closely related chemical species. In addition, it can be employed for the qualitative identification and quantitative determination of separated species.