Lecture 8 Chromatography Mechanisms PDF

Instrumental Analysis I Lecture 8-WS24 CHROMATOGRAPHIC MECHANISMS FOR HPLC APPLICATIONS Prof. Dr. Rasha Hanafi Lecture 8_WS24 1  Competencies 2-2-2 Demonstrate understanding of mechanisms of separation. 2-2-3-2 Select resin/gel based o...

Instrumental Analysis I Lecture 8-WS24 CHROMATOGRAPHIC MECHANISMS FOR HPLC APPLICATIONS Prof. Dr. Rasha Hanafi Lecture 8_WS24 1  Competencies 2-2-2 Demonstrate understanding of mechanisms of separation. 2-2-3-2 Select resin/gel based on sample nature in ion exchange. 2-2-3-3 Compare between ion-exchange chromatography and ion-pair HPLC. 2-5-3 Correlate elution volume to size of analytes in Size Exclusion Chromatography. 2-2-4 Determine approximate mass of a compound using SEC. 2-2-3-1 Select the proper stationary phase for a given separation. Lecture 8_WS24 2  LC SEPARATIONS (Based on the type of stationary phase) Adsorption Partition Ion Exchange Ion Pair Size exclusion Lecture 8_WS24 3  Ion-Exchange Chromatography Commonly used to separate charged biological molecules such as proteins, peptides, amino acids, or What is nucleotides. exchanged? Anion for anion and cation for cation How are the ions exchanged? Ions with higher affinity displace ions of lower affinity to the Affinity stat. increases as: phase 1.Charge increases 2.Size of solvatized ions decreases Lecture 8_WS24 4 3.Polarizability increases  Stationary Phases in Ion-Exchange Chromatography "Resins" or "Gels" carry the ion-exchanger surface. Both are amorphous particles of organic material, but gels are softer. Cellulose and dextran gels: Polystyrene resins: Dextran, cross-linked to made by co-polymerization of glycerin, is called styrene and vinyl-bearing (Sephadex The TM rings of the) support are modifiedmolecules to produce cation exchange resin containing sulfonate group (SO3-) or an anion exchange resin containing ammonium Lecture 8_WS24 groups (NR +). 3 5  Stationary Phases in Ion-Exchange Chromatography Resins Gels becomes more rigid and less Cellulose and dextran porous as cross linking (polymers of glucose) possess increases. larger pore size and lower charge densities than Lightly cross linked resins : polystyrene resins. Advantage: rapid equilibration of solute  Well suited to large between the inside and molecules that may be outside of the particle. irreversibly bound to resins due to their high charge Disadvantage: they swell in (proteins). water which decreases the density of ion exchange sites and hence selectivity of the resin to different ions. Lecture 8_WS24 6  Stationary Phases in Ion-Exchange Chromatography Ion exchangers Strong Weak Looses its Remains ionized at ionization at some all pH values pH values CO2- becomes SO3- keeps its protonated at pH 4 and charge even at looses its ion exchange low pH capacity Tertiary anion exchangers Quaternary are deprotonated in ammonium keeps moderately basic its +ve charge at all solutions and hence lose pH their ability to bind anions Lecture 8_WS24 7 Examples of Ion-Exchange Resins Lecture 8_WS24 8  Gradient Elution in Ion exchange chromatography Concentration pH gradients gradient Eluents used in anion A fixed concentration of a weak exchange contain an acid (reservoir A) is mixed with anionic compound in high an increasing concentration of a concentration which strong base as NaOH (reservoir competes with the B) to produce gradient [A-] anionic analyte for sites (strong conjugate base). on the resin. HA + NaOH A- Gradient elution is accomplished by pH gradients increase the pH increasing the during an anion exchange and concentration of the hence increase the eluent anion during the concentration of the run. dissociated form of the weak acid eluent (A-) which competes with Lecture 8_WS24 9  Ion-Exchange Chromatography for Water Treatment Obtaining soft water from hard water: Water is passed through a cation-exchange resin (Na+ form). what happens to CaSO4 if present in the water? 1 Ca2+ is replaced with 2 Na+ , while SO42- is not affected.  cations of hardly soluble salts are removed (Ca2+, Mg2+). The resin can be regenerated with NaCl solution. Obtaining deionized water: Water is passed through an anion-exchange resin (OH- form) and cation-exchange resin (H+ form), what happens to Cu(NO3)2 if present in the water? 1 Cu2+ is replaced with 2 H+ and NO3- is replaced with OH-  H+ + OH- = H2O is eluted while Cu(NO3)2 is "trapped“. Separation of proteins? Lecture 8_WS24 10  Ion-Pair Chromatography Ion pair chromatography is used to separate charged analytes. It is widely used to selectively analyze acids and bases, particularly with reverse phase chromatography. Stationary phase: common RP (no ion-exchange!) Mobile phase: contains surfactant that is “loosely attached” to stationary phase and so gives ion-pair surface. Cation Also: alkyl analyt sulfonates and e perchlorates Separation: counter-ions passing the column are attracted by surfactant. Separation Example: to mechanism: partition separate a mixture + ion-pairing of cations, an anionic surfactant is added to the mobile phase. The surfactant lodges in the stationary phase and effectively transforms it into an anionic St.phase. When analyte cations pass through the column, they can associate with the stationary phase by electrostatic attraction to the surfactant anion. Lecture 8_WS24 11  Size (or Molecular) Exclusion Chromatography (SEC) Separation of mixtures according to different molecule sizes of the components. Small molecules can intrude into pores of the stationary phase easily and are retained. But less easily large molecules which are too big to enter pores and are retained least Separation of biomolecules in aqueous systems is called gel filtration chromatography (GFC). Separation of organic polymers in non-aqueous systems is called gel permeation chromatography (GPC). Lecture 8_WS24 12  Chromatographic Parameters in SEC volume of the solvent held in the pores column total volume Vt Vg  Vi  V0 volume occupied by solid free volume outside matrix particles V0 : this is the volume of the solvent needed to transport components through the whole column that are too large to enter pores. V0 + Vi : Volume of the solvent needed to transport components that are small enough to intrude into pores easilyVe is the elution volume of a substance, V V  KV e 0 i.e. the volume of mobile phase that is i required for elution of this compound. At constant flow rate, it is proportional to Lecture 8_WS24 13 retention time. Determination of Molecular Weights by SEC Calibration graph for a certain SEC column. N.B. m. wt. can only be determined approximately because of the influence of the molecular geometry on the elution volume. Lecture 8_WS24 14 Flow Chart I: Small Molecules 1. m. wt. 2. solubility Lecture 8_WS24 15 Flow Chart II: Large Molecules 1. m. wt. 2. solubility Lecture 8_WS24 16  REFERENCES 1. “Principles of instrumental analysis, 5th ed. by Skoog, Holler, Nieman” Chapter 28 and 29. 2. “Ion-Exchange Chromatography and Its Applications”, By Özlem Bahadir Acikara , DOI: 10.5772/55744 3. “Quantitative Chemical Analysis, 7th ed. By Harris” Chapter 26. 4. http://www.bio-rad.com/en-eg/application s-technologies/liquid-chromatography-pri nciples/ion-exchange-chromatography Lecture 8_WS24 17