Chapter 31

1. Chapter 31 Introduction to Analytical Separations

2. A substance that affects an analytical signal or the background is called an interference or an interferent. Several methods can be used to deal with interferences in analytical procedures. Separations isolate the analyte from potentially interfering constituents. Techniques such as matrix modification, masking, dilution, and saturation are often used to offset the effects of interferents.

3. The internal standard and standard addition methods can sometimes be used to compensate for or to reduce interference effects. Separations can be complete or partial and always requires energy because the reverse process, mixing at constant volume, is spontaneous and accompanied by an increase in entropy. Separations can be preparative or analytical.

6. Separations Based on Control of Acidity • The concentration of hydrogen or hydroxide ions in a solution can be varied by • a factor of 1015 or more and can be easily controlled by the use of buffers. • Many separations based on pH control can be grouped into: • those made in relatively concentrated solutions of strong acids, • (2) those made in buffered solutions at intermediate pH values, and • (3) those made in concentrated solutions of sodium or potassium hydroxide.

8. Sulfide Separations With the exception of the alkali metals and alkaline-earth metals, most cations form sparingly soluble sulfides whose solubilities differ greatly from one another. It is relatively easy to control the sulfide ion concentration of an aqueous solution of H2S by adjustment of pH.

9. Separations by Other Inorganic Precipitants Phosphate, carbonate, and oxalate ions are often used as precipitants for cations, but they are not selective. Chloride and sulfate are useful because of their highly selective behavior. Chloride can separate silver from most other metals, and sulfate can isolate a group of metals that includes lead, barium, and strontium.

10. Separations by Organic Precipitants Organic precipitants, such as dimethylglyoxime, are useful because of their remarkable selectivity in forming precipitates with only a few ions. Other reagents, such as 8-hydroxyquinoline, yield slightly soluble compounds with many different cations.

11. Separation of Species Present in Trace Amounts by Precipitation The quantitative separation of a trace element by precipitation has some drawbacks: Supersaturation often delays formation of the precipitate. Coagulation of small amounts of a colloidally dispersed substance is often difficult. Loss of an appreciable fraction of the solid during transfer and filtration. To minimize loss, a quantity of some other ion that also forms a precipitate with the reagent (collector) is often added to the solution. The precipitate from the added ion carries the desired minor species out of solution.

12. Separation by Electrolytic Precipitation Electrolytic precipitation is a highly useful method wherein the more easily reduced species, either the wanted or the unwanted component of the sample, is isolated as a separate phase. The method is particularly effective when the potential of the working electrode is controlled at a predetermined level.

13. Salt-Induced Precipitation of Proteins A common way to separate proteins is by salting out the protein, that is, by adding a high concentration of salt. At high concentrations of salt, the repulsive effect of like charges is reduced as are the forces leading to solvation of the protein causing the protein to precipitate. At low salt concentrations, solubility is usually increased with increasing salt concentration and is called salting in effect.

14. At high concentrations, protein solubility, S is given by the following empirical equation: log S = C - K where C is a constant that is a function of pH, temperature, and the protein; K is the salting out constant that is a function of the protein and the salt used; and m is the ionic strength. Proteins are commonly least soluble at their isoelectric points. Hence, a combination of high salt concentration and pH control is used to achieve salting out. Protein mixtures can be separated by a stepwise increase in the ionic strength. Alcoholic solvents are sometimes used as they reduce the dielectric constant and reduce solubility by lowering protein-solvent interactions.

15. 31 B Separation of species by distillation Distillation is widely used to separate volatile analytes from nonvolatile interferents. It is based on differences in the boiling points of the materials in a mixture. Vacuum distillation is used for compounds that have very high boiling points. Lowering the pressure to the vapor pressure of the compound of interest causes boiling. Molecular distillation occurs at very low pressure (<0.01 torr) such that the lowest possible temperature is used with the least damage to the distillate. Pervaporation is a method for separating mixtures by partial volatilization through a nonporous membrane. Flash evaporation is a process in which a liquid is heated and then sent through a reduced pressure chamber where there is partial vaporization of the liquid.

16. 31 C Separation by extraction The partition of a solute between two immiscible phases is an equilibrium process 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: Aaq  Aorg The ratio of activities for A in the two phases will be constant and independent of the total quantity of A so that, at any given temperature, The equilibrium constant K is known as the distribution constant.

17. Thus, where [A]i is the concentration of A remaining in the aqueous solution after extract- ing Vaq mL of the solution with an original concentration of [A]0 with i portions of the organic solvent, each with a volume of Vorg.

20. Extracting Inorganic Species Extraction is often better than precipitation for separating inorganic species. Separating Metal Ions as Chelates Many organic chelating agents are weak acids that react with metal ions to give uncharged complexes that are highly soluble in organic solvents. Most uncharged metal chelates are nearly insoluble in water.

21. Extracting Metal Chlorides and Nitrates A number of inorganic species can be separated by extraction with suitable solvents. Example, a single ether extraction of a 6 M hydrochloric acid solution will cause better than 50% of several ions to be transferred to the organic phase, including iron(III), antimony(V), titanium(III), gold(III), molybdenum(VI), and tin(IV). Solid-Phase Extraction Liquid-liquid extractions have several limitations. With extractions from aqueous solutions, the solvents that can be used must be immisicible with water and must not form emulsions. Liquid-liquid extractions use relatively large volumes of solvent, which can cause a problem with waste disposal.

22. Solid-phase extraction, or liquid-solid extraction, can overcome several of the drawbacks of liquid-liquid extractions. Solid-phase extractions are used in determining organic constituents in drinking water by methods approved by the Environmental Protection Agency. It has the advantages of reducing extraction time and lowering solvent use. Solid-phase extraction can also be done in continuous flow systems, which can automate the preconcentra- tion process. Solid-phase microextraction helps in extracting organic analytes directly from aqueous samples or from the headspace above the samples.

23. 31 D Separating ions by ion exchange In the ion-exchange process, ions held on an ion-exchange resin are exchanged for ions in a solution brought into contact with the resin.

24. Ion-Exchange Resins Synthetic ion-exchange resins are high-molecular-mass polymers that contain large numbers of an ionic functional group per molecule. Cation-exchange resins contain acidic groups, while anion-exchange resins have basic groups. Strong-acid-type exchangers have sulfonic acid groups (--SO3-H+) attached to the polymeric matrix. Strong-base anion exchangers contain quaternary amine [--N(CH3)3+OH-] groups, while weak-base types contain secondary or tertiary amines.

25. Ion-Exchange Equilibria The law of mass action can be used to treat ion-exchange equilibria. For example, when a dilute solution containing calcium ions is passed through a column packed with a sulfonic acid resin, the following equilibrium is established: Ca+2(aq) + 2H+ (res)  Ca+2 (res) + 2H+ (aq) The equilibrium constant is give by: Ion-exchange separations are usually performed under conditions in which one ion predominates in both phases. In the removal of calcium ions from a dilute acidic solution, [Ca+2] <<< [H+]

26. The hydrogen ion concentration is essentially constant in both phases, and the equilibrium can be readjusted to: where K is a distribution constant analogous to the constant that governs an extraction equilibrium. Applications of Ion-Exchange Methods to eliminate ions that would otherwise interfere with an analysis. to concentrate ions from a dilute solution. The total salt content of a sample can be determined by titrating the hydrogen ion released as an aliquot of sample passes through a cation exchanger in the acidic form.

27. 31 E Chromatographic separations Chromatography is a technique in which the components of a mixture are separated based on differences in the rates at which they are carried through a fixed or stationary phase by a gaseous or liquid mobile phase. The stationary phase is a phase that is fixed in place either in a column or on a planar surface. The mobile phase 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. Chromatographic methods are of two basic types: Planar and column chromatography

28. 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, and the mobile phase moves through the stationary phase by capillary action or under the influence of gravity.

29. 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 termed the eluate. An eluent is a solvent used to carry the components of a mixture through a stationary phase.

30. A chromatogram is a plot of some function of solute concentration versus elution time or elution volume.

31. Improved separations can often be realized by the control of variables that either (1) increase the rate of band separation or (2) decrease the rate of band spreading.

32. Migration Rates of Solutes The effectiveness of a chromatographic column in separating two solutes depends in part on the relative rates at which the two species are eluted. These rates in turn are determined by the ratios of the solute concentrations in each of the two phases. All chromatographic separations are based on differences in the extent to which solutes are distributed between the mobile and the stationary phase. A (mobile)  A (stationary) The equilibrium constant Kc for this reaction is called a distribution constant, Kc = (aA)s/(aA)M where (aA)S is the activity of solute A in the stationary phase and (aA)M is the activity in the mobile phase.

33. Substituting cS, the molar analytical concentrations of the solute in the stationary phase, for (aA)S and cM, the molar analytical concentration in the mobile phase, for (aA)M Kc = cS/cM Retention times The small peak on the left is for a species that is not retained by the stationary phase. The time tM after sample injection for this peak to appear is called the dead or void time.

34. The time required for this zone to reach the detector after sample injection is called the retention time and is given the symbol tR. The retention time is tR = tS + tM The average linear rate of solute migration, v (usually cm/s), is v = L/tR Similarly, the average linear velocity, u, of the mobile phase molecules is u = L/tM

35. The mobile phase flow is usually characterized by the volumetric flow rate, F (cm3/min), at the column outlet. For an open tubular column, F is related to the linear velocity at the column outlet uo by F = u0A = u0 r2 where A is the cross-sectional area of the tube (r2). For a packed column, the entire column volume is not available to the liquid, thus F = r2u0

36. The rate of migration of a solute can be related to its distribution constant as v = u  fraction of time solute spends in mobile phase v = u  no. of moles of solute in mobile phase/ total no. of moles of solute Similarly, the number of moles of solute in the stationary phase is given by the product of cS, the concentration of the solute in the stationary phase, and its volume, VS. v = u  cMVM/cMVM + cSVS Substitution gives, v = u  1/1 + KcVS/VM

37. For solute A, the retention factor kA kA = KAVS/VM where KA is the distribution constant for solute A. Substitution gives, v = u 1/1 + kA kA can be calculated from a chromatogram,

38. The selectivity factor, a, for solutes A and B is defined as the ratio of the distribution constant of the more strongly retained solute (B) to the distribution constant for the less strongly held solute (A).  = KB/KA where KB is the distribution constant for the more strongly retained species B and KA is the constant for the less strongly held or more rapidly eluted species A. The relationship between the selectivity factor for two solutes and their retention factors:  = kB/kA  = (tR)B – tM (tR)A - tM

39. Band Broadening and Column Efficiency The amount of band broadening that occurs as a solute passes through a chromatographic column strongly affects the column efficiency. The rate theory of chromatography describes the shapes and breadths of elution bands in quantitative terms based on a random-walk mechanism for the migration of molecules through a column.

40. Two related terms are widely used as quantitative measures of chromatographic • column efficiency: • plate height, H, and (2) plate count or number of theoretical plates, N. • N = L/H • where L is the length (usually in centimeters) of the column packing. • Chromatographic bands are often Gaussian and because the efficiency of a column is reflected in the breadth of chromatographic peaks, the variance per unit length of column is used by chromatographers as a measure of column efficiency. • H = 2/L

42. The number of theoretical plates, N, and the plate height, H can be determined from the chromatogram. The number of plates can be computed by the relationship: N = 16(tR/W)2

45. Theory of Band Broadening The efficiency of capillary chromatographic columns and packed chromatographic columns at low flow velocities can be approximated by the expression where H is the plate height in centimeters and u is the linear velocity of the mobile phase in centimeters per second; B is the longitudinal diffusion coefficient, while CS and CM are mass-transfer coefficients for the stationary and mobile phases, respectively. At high flow velocities in packed columns where flow effects dominate diffusion, the efficiency can be approximated by

46. The longitudinal Diffusion term, b/u.  Diffusion is a process in which species migrate from a more concentrated part of a medium to a more dilute region. The rate of migration is proportional to the concentration difference between the regions and to the diffusion coefficient DM of the species.

47. Zone broadening in the mobile phase is due in part to the multitude of pathways by which a molecule (or ion) makes its way through a packed column.

50. Column Resolution The resolution, Rs, of a column tells us how far apart two bands are relative to their widths. The resolution provides a quantitative measure of the ability of the column to separate two analytes. The resolution of each column is defined as