1. Food Analysis Lecture 18 (03/27/2012) Basic Principles of Chromatography (3) Qingrong Huang Department of Food Science Read Material: Chapter 27, page 473-489

2. Characteristics of Different Chromatographic Techniques

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. Applications of Adsorption Chromatography Separate aromatic or aliphatic nonpolar compounds, such as lipids, primarily according to the type and number of functional groups present. Carotenoid pigments from plants; Analysis of fat – soluble vitamins, etc…

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: (1)Ionic from nonionic; (2)Cationic from anionic; (3)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 (RSO 3 - ) 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.

11. 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 (V 0 ) – the volume of the mobile phase in the Column, measured by running a very large molecules, e.g MW=2x10 6 ; Total permeation volume (V t )-column void volume V 0 + the volume of liquid inside the sorbent pores V i ; measured by running a low MW (e.g.glycyltyrosine);

12. Size-Exclusion Chromatography (SEC) The available partition coefficient K av : V e : elution volume of solute; SEC packing materials: hydrophobic media like crosslinked PS and Hydrophilic gels like polysaccharide-based packings (Fig. 27-5a).

13. 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,

14. 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.