PTT 202 Organic Chemistry for Biotechnology

1. Lecture 3: Separation Methods Zulkarnain Mohamed Idris PTT 202 Organic Chemistry for Biotechnology zulkarnainidris@unimap.edu.my Semester 1 2013/2014

2. Classification of Separation Methods

3. 1. Chromatography is adsorption based separation process Used to separate biomolecules, fine & specialty chemicals Analytical tools: To determine chemical compositions of sample Preparative tools: To purify & collect one/more components of a sample

4. Chromatography SEPARATION TECHNIQUES AC HIC SF: Super Critical; GS: Gas-Solid; GL: Gas-Liquid ; A: Affinity ; HI: Hydrophobic/Hydrophilic; NP: Normal-Phase; RP: Reverse-Phase; IE: Ion Exchange; SE: Size Exclusion; GP: Gel Permeation; GF: Gel Filtration; TL: Thin-Layer. C: Chromatography

5. SEPARATION TECHNIQUES: Choice of MethodsMolecular Size /Polarity

6. Chromatography BASIC SEPARATION PRINCIPLES Mobile phase: Continuous flow of a carrier liquid or gas 1 2 Solutes in solution or volatiles in gas are placed in mobile phase & passed over a selected ‘adsorbent’ material (stationary phase) The solutes or volatiles have differential ‘affinity’ for the adsorbent material & thus separate Stationary phase: A bed of solids or immobilized liquid

7. Chromatography BASIC SEPARATION PRINCIPLES Example 1: Hydrophobic interaction Example 2: Ionic interaction

8. SEPARATION TECHNIQUES IN CHROMATOGRAPHY LIQUID CHROMATOGRAPHY • The most selective type of chromatography employed. • Utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized (ligand) on a stationary phase. • For example, the immobilized molecule may be an antibody to some specific protein. • When solute containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. • This protein is later eluted by changing the ionic strength or pH. 1 • Affinity Chromatography • “Lock-and-key concept” Ligand (e.g. antibody) Specific protein binds to ligand Stationary phase Non-specific proteins

9. SEPARATION TECHNIQUES IN CHROMATOGRAPHY LIQUID CHROMATOGRAPHY • Typically used for protein separations • Employs derivatized polymer resins, with phenyl, butyl, or octyl ligand groups • Protein adhere to the hydrophobic surface under high salt conditions and redissolve into the mobile phase as the salt concentration is reduced • By increasing the salt concentration of the solvent, these hydrophobic patches of the protein become more exposed and can interact with hydrophobic ligands on the HIC packing. • HIC is sensitive to pH, salt used, buffer type and temperature. 2 • Hydrophobic Chromatography Stationary phase Ligand (e.g. phenyl group) Stationary phase

10. SEPARATION TECHNIQUES IN CHROMATOGRAPHY LIQUID CHROMATOGRAPHY • Employs a hydrophobic phase bonded to the surface of the resin – typically silica based • Hydrophobic solutes bind in higher proportion in reversed phased • Hydrophobic phases that are bonded to silica are typically actyil (C8), actyldecyl (C18), phenyl, and methyl (C1) • Different chain lengths and densities of the different bonded phases lead to more or less hydrophobicity • Bare silica participate in separation by interacting with hydrophilic molecules, or hydrophilic domains of large molecules 3 • Reverse Phase Chromatography Stationary phase Ligand (e.g. actyldecyl) Stationary phase

11. SEPARATION TECHNIQUES IN CHROMATOGRAPHY LIQUID CHROMATOGRAPHY • Biomolecules generally have charged groups on their surfaces, which change with the pH of the solution • Molecule reversibly binds to an oppositely charged group of the packing material • Molecules with a higher charge density bind more strongly to the packing • The bound sample may be selectively removed from the stationary phase by changing the pH or salt concentration of the mobile phase • It is particularly effective for proteins because they are amphoteric >Charge density 4 • Ion Exchange Chromatography Stationary phase Stationary phase Ligand (e.g. actyldecyl) <Charge density Stationary phase Stationary phase Competing ions

12. SEPARATION TECHNIQUES IN CHROMATOGRAPHY LIQUID CHROMATOGRAPHY • Also referred to as gel permeation chromatography (GPC) for non-aqueous elution systems or gel filtration chromatography (GFC) for aqueous systems. • Separates solutes on the basis of their size • No binding between the solutes and the resin • Smaller molecules can partially or completely enter the stationary phase. • Because these smaller molecules have to flow through both, the interparticle space, as well as through the pore volume, they will elute from the column after the excluded sample components • Used for removing small molecules from protein solution 5 • Size Exclusion Chromatography Molecules with different sizes Stationary phase Pores Smaller molecules Large molecules

13. Types of Resins (Stationary Phase) SILICA-BASED RESINS Uncoated silica Coated silica • Compatible with water or organic solvent • Serves as a good reversible adsorbent for hydrophilic compounds • Organic solvent used as mobile phase, and water is added as the chromatography progresses • Not typically stable at extremes of pH • Available with high surface area and small particle size; being very rigid; does not collapse under high pressures • Denature some proteins and irreversibly bind others • Used for purification of many commercial biotechnology products • Particles coated with long-chain alkanes • Has a high affinity for hydrophobic molecules, which increases as the chain length of the bonded alkane increases. • Many varieties of the same chain length phase – polymerized, simple monolayer and end-capped

14. Types of Resins (Stationary Phase) POLYMER-BASED RESINS Synthetic polymers Natural Polymers Styrene divinylbenzene : • Very stable at pH extremes • Support for ion exchange chromatography because of its stability and rigidity Polyacrylamide: • used less often, not used as a polymer solid but as hydrogel and used as a size exclusion gel • The crosslinking in polyacrylamide can be controlled by the amount of bisacrylamide added in suspension mixture • Used in hydrogel for a low pressure chromatography resins. • Naturally hydrophillic • Compatible with proteins and other biomaterials Agarose : • can be crosslinked to form a reasonably rigid bead that is capable of tolerating pressures up to 4 bar. Dextran: • Less rigid and used in size exclusion • Can be formed with very large pores • Capable of including antibody molecules and virus particles

15. Types of Resins (Stationary Phase) ION EXCHANGE RESINS Cation exchangers Anion exchangers • Resins that have been derivatized with an ionic group • Most commonly used ionic groups: • sulfoxyl (SO3-) - most acidic • carboxyl (COO-) • diethylaminoethyl (DEAE) (2C2H5N+HC2H5) • quaternary ethylamine (QAE) (4C2H5N+) - most basic • Acidic ion exchanger • Carry a negative charge • Attract positive counterions • Basic ion exchangers • Carry a positive charge • Attract negative counterions

16. Selection of Mobile Phase • 1. Compatibility with stationary phase: • -must no react chemically with the stationary phase or break the bond linking it to the supporting materials. • -Extreme pH or strong oxidizing agents should normally be avoided. • 2. Compatibility with detection system: • -the mobile phase must not interfere with the detection system. • -the solvent used must not absorb significantly at the wavelength used. • -e.g: absorption at 280 nm is frequently used to detect protein but some solvents such as acetone absorb at this wavelength, so this solvent must be avoided as a mobile phase.

17. Selection of Mobile Phase • 3. Polarity: • -the major factor in selecting a mobile phase is the polarity of the solute or analyte (or molecules that are going to be separated). • -the polarity of the mobile phase should be such that there is an effective partition of the solute or analyte between the two phases (stationary and the mobile phases). • -gradient elution is required in which the solvent strength (or the ability the solvent to break adsorptive bonds and elute the solute from the adsorbent) of the mobile phase is gradually changed during the separation process by altering the solvents in the mixture. • 4. Pressure consideration: • -solvents chosen should achieved the desired separation without requiring pressures too high for the system.

18. SEPARATION TECHNIQUES IN CHROMATOGRAPHY GAS CHROMATOGRAPHY (GC) Gas chromatographic equipment Chromatographic separation system Packed column Capillary column

19. SEPARATION TECHNIQUES IN CHROMATOGRAPHY CROSS-SECTIONAL VIEWS OF PACKED & CAPILLARY COLUMNS

20. SEPARATION TECHNIQUES IN CHROMATOGRAPHY SEPARATION PROCESS IN GAS CHROMATOGRAPHY • Separation method based on conversion of sample to vapor phase • The sample vapor is introduced onto a column (packed/capillary) containing stationary phase material • The mobile phase is typically an inert gas such helium (He), nitrogen (N2) and hydrogen (H2) • Separation occurs on the basis of interaction between the sample components and the stationary phase • Because the sample must be maintained in the vapor phase, the column is contained within an oven. Liquid film of solvent + samples Carrier gas (He) Column Sample vapor Solvent vapor

21. TYPES OF ADSORBENTS (EXAMPLES: STATIONARY PHASE FOR GAS CHROMATOGRAPHY)

22. SEPARATION TECHNIQUES IN CHROMATOGRAPHY Resolution in Chromatography: To measure the ability of column to separate two peaks Rs= (tR2-tR1)/0.5 (wb1+wb2) Wb1 Wb2

23. SEPARATION TECHNIQUES IN CHROMATOGRAPHY The separation efficiency of a column can be expressed in term of the number of theoretical plates in the column, (N): N= 16 (tR/wb)2

24. SEPARATION TECHNIQUES IN CHROMATOGRAPHY Resolution in Chromatography (Rs) Example: Ethanol and methanol are separated in a capillary GC column with retention times of 370 and 385 s, respectively, and base width (wb) of 16.0 and 17.0 s. Calculate the resolution (Rs) and the number of plates (N).

25. SEPARATION TECHNIQUES IN CHROMATOGRAPHY Resolution in Chromatography (Rs) Answer: Rs= (tR2-tR1)/0.5 (wb1+wb2) = (385-370)/0.5(17.0+16.0) = 0.91

26. SEPARATION TECHNIQUES IN CHROMATOGRAPHY The number of plates (N) Answer: Use the longest eluting peak to calculate N: N= 16 (385/17.0)2 = 8.21 x 103 plates

27. 2. Dialysis • A technique for separating macromolecules from small solute molecules. • Refers to diffusion of the solute molecules through a membrane which restricts the movement of large molecule (depends on the pore size). • The passage of the small molecules is due to a concentration gradient across the membrane. • Cellophane is frequently used for dialysis and it has a pore size of approximately 4-8 µm, makes it impermeable to molecules with relative molecular mass in excess about 10 000 Da.

28. Dialysis (Low concentrated)

29. 3. Ultrafiltration • The solvent and solute are forced through the membrane under pressure and the movement of large molecules is restricted by the pore size. • Various cellulose and polycarbonate membranes are available with pore size down to 5 nm which are capable of excluding molecules with a relative molecular mass of 50 Da.