Harris: Quantitative Chemical Analysis, Eight Edition CHAPTER 25: CHROMATOGRAPHIC METHODS AND CAPILLARY ELECTROPHORESIS

CHAPTER 25: Opener Aa

CHAPTER 25: Opener Ab

CHAPTER 25: Opener B

25-1 Ion-Exchange Chromatography

25-1 Ion Exchangers Three classes of ion exchangers : Resins : amorphous (noncrystalline) particles of organic material : for the separation of small molecules (MW < 500) Gels : cellulose and dextran ion exchangers : much softer, larger pore size and lower charge density than resin : for the separation of large molecules (proteins and nucleic acids) Inorganic Exchangers : hydrous oxide of Zr, Ti, Sn, W

25-1. Ion Exchangers Resins : amorphous (noncrystalline) particles of organic material : polystyrene crosslinked by divinylbezene (1~16%) :modification of benzene ring, high charge density :pore size shrinks as crosslinking increases. increased selectivity but slower equilibration : charge density is so great that highly charged macromolecules (proteins) may be irreversibly bound (disadvantage) R-SO - 3 ; strongly acidic cation exchanger R-CO - 2 ; weakly acidic cation exchanger R-N + R 3 ; strongly basic anion exchanger R-N + R 2 H ; weakly basic anion exchanger

Fig. 25-1 Structures of styrene-divinylbenzene cross-linked ion exchange resins.

25-1 Ion Exchangers Resins : modification of benzene ring, high charge density 1) R-SO - 3 ; strongly acidic cation exchanger 2) R-CO - 2 ; weakly acidic cation exchanger 3) R-N + R 3 ; strongly basic anion exchanger 4) R-N + R 2 H ; weakly basic anion exchanger

Fig. 25-1 Structures of styrene-divinylbenzene cross-linked ion exchange resins.

Gels : cellulose and dextran ion exchangers ( polymers of glucose) : because softer than resins, larger pore sizes and lower charge density good for protein separation :

25-1 Ion-Exchange Selectivity R - Na + + Li + R - Li + + Na + Selectivity coefficient: K = [R [R Li + Na ][Na + ][Li + + ] ] (25-1) - Larger K for i) higher charge, ii) smaller hydrated radius, iii) larger polarizability. - Selectivities tend to increase with the extent of cross-linking, because the pore sizes of resin shrinks. - Large hydrated ion such as Li +, do not have as much access to the resin as smaller hydrated ions such as Cs+. - Ions can be removed by using excess amount of weakly binding ions.

Selectivities tend to increase with the extent of cross-linking, because the pore sizes of resin shrinks.

25-1 Donnan Equilibrium Definition: When an ion exchanger is placed in an electrolyte solution, the concentration of electrolyte is higher outside the resin than inside it. The equilibrium between ions in solution and ions inside the resin is called Donnan Equilibrium R - Na + Cl - H 2 O Resin phase Na + Cl - H 2 O Solution At equilibrium, the chemical potentials are equal in both phases, µ o NaCl + RT ln{a Na+ a Cl- } R = µ o NaCl + RT ln{a Na+ a Cl- } S continued

25-1. Donnan Equilibrium Under ideal conditions (activity coefficients 1) [C Na+ ] R [C Cl- ] R = [C Na+ ] S [C Cl- ] S (R : Resin, S : Solution) Because of electrical neutrality [C R- ] R + [C Cl- ] R = [C Na+ ] R [C Na+ ] S = [C Cl- ] S Combination of eq. & [C Cl- ] R ([C Cl- ] R + [C R- ] R ) = [C Na+ ] S [C Cl- ] S Substitution of eq. into gives S R [ C ] [ C ] [C Cl- ] R [C R- ] R + ([C Cl- ] R ) 2 = ([C Cl- ] S ) 2 Cl R or = + 1 R R [ C ] [ C ] - From eq., [C Cl -] S must be greater than [C Cl -] R Cl Cl

25-1 Conducting Ion-Exchange Chromatography Gradient Elution : analogous to a solvent or temperature gradient ( increasing ionic strength or changing ph) - Let s consider an anion exchange resin where the order of binding strength: A - > B - - As the concentration of C - (A- > B- > C - ) in the eluent is increased, B - is eventually displaced and moves down the column. - At a still higher concentration of C -, the anion A - is also eluted.

25-1 Conducting Ion-Exchange Chromatography Gradient Elution : increasing ionic strength or ph

25-1 Applications of Ion Exchange Water Softeners : remove Mg 2+ or Ca 2+ Deionized Water Example: elimination of copper nitrate Cu 2 H + ion exchanger 2H + 2NO - 3 OH - ion exchanger 2OH - pure H 2 O Salt Conversion R + X - anion exchanger R + OH - Pre-concentration : OH - example 1) Chelex 100 : collects trace transition metal then the metals are eluted with 2M HNO 3

Pre-concentration example 2)

25-1 Simultaneous Separation of Anions and Cations on one column Using zwitterionic bonded stationary phase and a mixed organic-aqueous mobile phase (next slide)

Hydrophilic Interaction Chromatography (HILIC) * Stationary phases for HILIC are strongly polar

Hydrophilic Interaction Chromatography (HILIC) - HILIC is most useful for small molecules that are too polar to be retained by reversed-phase columns. - The mobile phase typically contains CH 3 CN (25-97 vol %) or other organic solvent mixed with aqueous buffer. - HILIC is useful for separating peptides and saccharides (sugars) which are soluble in water.

25-2 Ion Chromatography (IC) - IC: a high-performance version of ion-exchange chromatography - Example) Used in the semi-conductor industry to monitor anions and cations at 0.1 ppb levels in deionized water. Suppressed-Ion Anion Chromatography - A mixture of anions (NO - 3 + SO 2-4 ) is separated by ion exchange and detected by electrical conductivity. - Unwanted electrolyte is suppressed (or removed) prior to conductivity measurement. - Separator column; separating the analytes Suppressor column; replacing the ionic eluent with a nonionic species.

Figure 25-6 Suppressed-Ion Anion Chromatography. -From the separator column, KNO 3 and K 2 SO 4 are eluted. - They can not be easily detected because a high concentration (high conductivity) of KOH obscures that of the analytes. -Through a cation exchange membrane in a suppressor, cations (K + ) are replaced by H + resulting in H 2 O which has low conductivity.

Figure 25-7 IC of Pond water. Eluent : NaHCO 3 /Na 2 CO 3 buffer Upper; standards, Lower; pond water (ug/ml)

25-3 Molecular Exclusion Chromatography (= Size exclusion chromatography) - Molecules are separated according to their size -Small molecules penetrates the small pores, but large molecules do not. - Large molecules are eluted first - Gel Filtration Chromatography (GFC) (mobile phase : water, hydrophilic) - Gel Permeation Chromatography (GPC) (mobile phase : organic solvent, hydrophobic) a) Large molecule, b) small molecule

25-3 Molecular Exclusion Chromatography But in molecular exclusion chromatography, V t = V o + V i + V g V t : total volume of column V o : free volume outside the gel particles (void volume) V i : the volume of solvent inside the gel particles V g : volume occupied by the solid matrix of the gel * V o, V i, V g are of the same order of magnitude

25-3 The Elution Equation V r = V o + K V i, K = C C i) For molecules too large to enter the gel pores, C s = 0, K = 0, V r = V o s m K; Partition coefficient (no adsorption between solute and gel surface is assumed!!) ii) iii) For molecules that can enter the pores unhindered, C s = C m, K = 1, V r = V o + V i K > 1, if adsorption occurs For molecules of intermediate size, they are able to enter into some fraction of the solvent held in the pores 0 < K < 1, V r = V o + K V i K av V = r V V i where V i = the solvent volume of o inside the gel

25-3 The Elution Equation Exclusion Limit : M.W. of species beyond which no retention occurs. All species of M.W. > Exclusion limit are not retained and elute together to give peak, A. Permeation Limit : M.W. below which the solute molecules can permeate into pores completely. All species of M.W. < Permeation limit are so small that they elute as the single ban, D.

25-3 Stationary Phase Gels for open column : 1) Sephadex 2) Sephacryl 3) Sepharose 4) Bio- Gel

25-3 Stationary Phase Sephadex G-10 : The smallest pore sizes in highly cross linked gels : Exclude molecules with a molecular mass > 700

25-3 Molecular Mass Determination - For each stationary phase, there is a range over which there is a logarithmic relation between molecular mass and elution volume. - We can estimate the molecular mass of an unknown by comparing its elution volume with those of standards. - For proteins, it is important to use an ionic strength high enough (> 0.05M) to eliminate electrostatic adsorption of solute by occasional charged sites on the gel.

25-3 Molecular Mass Determination Res Pore size of stationary phase = 5 nm ~ 100 um

25-3 Separation of Nanoparticles by molecular exclusion chromatography Quantum Dots : the wavelength of their visible emision depends on their size Fig. 25-18 CdSe quantum dots

Fig. 25-18 Triangles; CdSe, Squares; polystyrene calibration standards.