1. PTT 202 ORGANIC CHEMISTRY FOR BIOTECHNOLOGY Lecture 3: Separation Methods zulkarnainidris@unimap.edu.my Zulkarnain Mohamed Idris Semester 1 2013/2014

2. Classification of Separation Methods

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

4. SEPARATION TECHNIQUES HICAC 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 Chromatography

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

6. Solutes in solution or volatiles in gas are placed in mobile phase & passed over a selected ‘adsorbent’ material (stationary phase) BASIC SEPARATION PRINCIPLES The solutes or volatiles have differential ‘affinity’ for the adsorbent material & thus separate 12 Mobile phase: Continuous flow of a carrier liquid or gas Stationary phase: A bed of solids or immobilized liquid Chromatography

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

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

9. Hydrophobic Chromatography i. Typically used for protein separations ii. Employs derivatized polymer resins, with phenyl, butyl, or octyl ligand groups iii. Protein adhere to the hydrophobic surface under high salt conditions and redissolve into the mobile phase as the salt concentration is reduced iv. 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. v. HIC is sensitive to pH, salt used, buffer type and temperature. 2 Stationary phase Ligand (e.g. phenyl group) Stationary phase SEPARATION TECHNIQUES IN CHROMATOGRAPHY LIQUID CHROMATOGRAPHY

10. Reverse Phase Chromatography i. Employs a hydrophobic phase bonded to the surface of the resin – typically silica based ii. Hydrophobic solutes bind in higher proportion in reversed phased iii. Hydrophobic phases that are bonded to silica are typically actyil (C 8 ), actyldecyl (C 18 ), phenyl, and methyl (C 1 ) iv. Different chain lengths and densities of the different bonded phases lead to more or less hydrophobicity v. Bare silica participate in separation by interacting with hydrophilic molecules, or hydrophilic domains of large molecules 3 Stationary phase Ligand (e.g. actyldecyl) Stationary phase SEPARATION TECHNIQUES IN CHROMATOGRAPHY LIQUID CHROMATOGRAPHY

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

12. Size Exclusion Chromatography i. Also referred to as gel permeation chromatography (GPC) for non-aqueous elution systems or gel filtration chromatography (GFC) for aqueous systems. ii. Separates solutes on the basis of their size iii. No binding between the solutes and the resin iv. Smaller molecules can partially or completely enter the stationary phase. v. 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 vi. Used for removing small molecules from protein solution 5 Stationary phase Pores Molecules with different sizes Smaller molecules Large molecules SEPARATION TECHNIQUES IN CHROMATOGRAPHY LIQUID CHROMATOGRAPHY

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

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

15. i. Acidic ion exchanger ii. Carry a negative charge iii. Attract positive counterions i. Basic ion exchangers ii. Carry a positive charge iii. Attract negative counterions Cation exchangers Anion exchangers ION EXCHANGE RESINS  Resins that have been derivatized with an ionic group  Most commonly used ionic groups: i.sulfoxyl (SO 3- ) - most acidic ii.carboxyl (COO-) iii.diethylaminoethyl (DEAE) (2C 2 H 5 N + HC 2 H 5 ) iv.quaternary ethylamine (QAE) (4C 2 H 5 N + ) - most basic Types of Resins (Stationary Phase)

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. 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. Selection of Mobile Phase

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

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

20. SEPARATION PROCESS IN GAS CHROMATOGRAPHY i. Separation method based on conversion of sample to vapor phase ii. The sample vapor is introduced onto a column (packed/capillary) containing stationary phase material iii. The mobile phase is typically an inert gas such helium (He), nitrogen (N 2 ) and hydrogen (H 2 ) iv. Separation occurs on the basis of interaction between the sample components and the stationary phase v. 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) Sample vapor Solvent vapor Column SEPARATION TECHNIQUES IN CHROMATOGRAPHY

21. CompositionPolarityApplicationsTemp limits 100% dimethyl polysiloxane (Gum) NonpolarPhenols, Hydrocarbons, Amines, Sulfur compounds, Pesticides, PCBs -60 o C to 325 o C 100% dimethyl polysiloxane (Fluid) NonpolarAmino acid derivatives, Essential oils0 o C to 280 o C 5% diphenyl 95% dimethyl polysiloxane NonpolarFatty acids, Methyl esters, Alkaloids, Drugs, Halogenated compounds -60 o C to 325 o C 14% cyanopropyl phenyl polysiloxane ImmediateDrugs, Steroids, Pesticides-20 o C to 280 o C 50% phenyl, 50% methyl polysiloxane ImmediateDrugs, Steroids, Pesticides, Glycols60 o C to 240 o C 50% cyanopropylmethyl, 50% phenylmethyl polysiloxane ImmediateFatty acids, Methyl esters, Alditol acetates 60 o C to 240 o C 50% trifluoropropyl polysiloxaneImmediateHalogenated compounds, +Aromatics 45 o C to 240 o C Polyethylene glycol – TPA modified PolarAcids, Alcohols, Aldehydes acrylates, Nitriles, Ketones 60 o C to 240 o C Polyethylene glycolPolarFree acids, Alcohols, Ethers, Essential oils, Glycols, Solvents 60 o C to 220 o C 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= (t R 2 -t R 1 )/0.5 (w b 1 +w b 2 ) Wb 2 Wb1Wb1

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 (t R /w b ) 2

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

25. SEPARATION TECHNIQUES IN CHROMATOGRAPHY Resolution in Chromatography (R s ) Answer: R s = (t R 2 -t R 1 )/0.5 (w b 1 +w b 2 ) = (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 10 3 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.