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Food Analysis Lecture 20 (4/8/2005)

Food Analysis Lecture 20 (4/8/2005). Basic Principles of Chromatography (3). Qingrong Huang Department of Food Science Read Material: Chapter 27, page 437 Final Exam: April 29. Chromatography. Physicochemical Principles of Separation.

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Food Analysis Lecture 20 (4/8/2005)

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  1. Food Analysis Lecture 20 (4/8/2005) Basic Principles of Chromatography (3) Qingrong Huang Department of Food Science Read Material: Chapter 27, page 437 Final Exam: April 29

  2. Chromatography

  3. Physicochemical Principles of Separation • Adsorption (solid-liquid) chromatography: oldest, Tsvet in 1903 • The stationary phase is a finely divided solid (to maximize the • surface area), • The mobile phase can be either gas or liquid • The stationary phase (adsorbent) is chosen to permit differential • Interaction with the components of the sample to be resolved. • The interaction forces include: • - Van der Waals forces • - Electrostatic forces • - Hydrogen bonds • - Hydrophobic interactions • Typical stationary phases: silica (slightly acidic), alumina (slightly • Basic), charcoal (nonpolar).

  4. Mechanism of AC • The solute and solvent molecules are competing for active sites • on the stationary phase. • As the relative adsorption of the mobile phase increases, adsorption • of the solute must decrease. • A solvent strength (or polarity) scale is called an eluotropic series.

  5. Ion-Exchange Chromatography • Ion-exchange Chromatography: a separation/purification process • occurring naturally, e.g. in soils, and is utilized in water softeners and • deionization. Three types of separation: • Ionic from nonionic; • Cationic from anionic; • Mixtures of similarly charged species. • Similar to adsorption chromatography • nature of interactions - electrostatic • Cationic exchangers: contain covalently bound negatively charged • functional groups. • Anionic exchangers: contain bound positively charged groups.

  6. Ion-Exchange Chromatography (2)

  7. Ion-Exchange Chromatography (3) • The strongly acidic sulfonic acid moieties (RSO3-) of “strong” cation • exchangers are completely ionized at pH>2; • Strongly basic quarternary amine group (RNR’3+) on “strong” anion • exchangers are ionized at pH<10; • Since maximum negative or positive charge is maintained over a broad • pH range, the exchange or binding capacity of these stationary phases is • essentially constant; • “Weak” cation exchangers contain RCOO-, their exchange capacity • varies considerably between 4-10; • “Weak” anion exchangers contain RNR’2+, which are deprotonated in • moderately basic solution, thereby losing their positive charge and the • ability to bind anions; • One way to elute solutes is to change the mobile phase pH; a second way • is to increase the ionic strength (e.g. use of NaCl) of the mobile phase, to • Weaken the electrostatic interactions.

  8. Ion-Exchange Chromatography (4) • Factors that govern selectivity: ionic valence, radius, concentration, • the nature of the exchanger, the composition and pH of the mobile • Phase; • Materials used must be ionic and highly permeable: • - Crosslinked Polyelectrolytes like polystyrene crosslinked with divinyl • benzene; • Polysaccharide-based, like cellulose, dextran, or agarose for separation • and purification of large molecules, such as proteins and nuclei acids, with • the advantage of being able to derivatized with strong or with weakly acidic • or basic groups via OH moieties on the polysaccharide backbone.

  9. Size-Exclusion Chromatography (SEC) • SEC, also known as Gel Permeation Chromatography (GPC), can • be used for the resolution of macromolecules, such as proteins and • carbohydrates, as well as for the fractionation and characterization • of synthetic polymers; • Ideal SEC, separated based on size, no interaction occur between • Solutes and the stationary phase; • Column void volume (V0) – the volume of the mobile phase in the • Column, measured by running a very large molecules, e.g MW=2x106; • Total permeation volume (Vt)-column void volume V0 + the volume • of liquid inside the sorbent pores Vi; measured by running a low MW • (e.g.glycyltyrosine);

  10. Size-Exclusion Chromatography (SEC) • The available partition coefficient Kav: • Ve: elution volume of solute; • SEC packing materials: hydrophobic media like crosslinked PS and • Hydrophilic gels like polysaccharide-based packings (Fig. 27-5a).

  11. Chromatography

  12. Affinity Chromatography • Affinity Chromatography: separation is based on the specific, reversible • interaction between a solute molecule and a ligand immobilized on the • chromatographic stationary phase. • The ultimate extension of adsorption chromatography • Involved biological materials as the stationary phase, including antibodies, • enzyme inhibitors,

  13. A: The support presents the immobilized ligand to the analyte To be isolated; B: The analyte makes contact with the ligand and attaches itself; C: The analyte is recovered by the introduction of an eluent, which dissociates the complex Holding the analyte to the ligand; D: The support is regenerated, ready for the next isolation.

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