PTT 202 ORGANIC CHEMISTRY FOR BIOTECHNOLOGY Lecture 3
PTT 202 ORGANIC CHEMISTRY FOR BIOTECHNOLOGY Lecture 3: Separation Methods Sharifah Zati Hanani sharifahzati@unimap. edu. my Semester 1 2014/2015
Classification of Separation Methods
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
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-
SEPARATION TECHNIQUES: Choice of Methods Molecular Size /Polarity
Chromatography BASIC SEPARATION PRINCIPLES 1 Solutes in solution or volatiles in gas are placed in mobile phase & passed over a selected ‘adsorbent’ material (stationary phase) Mobile phase: Continuous flow of a carrier liquid or gas Stationary phase: A bed of solids or immobilized liquid 2 The solutes or volatiles have differential ‘affinity’ for the adsorbent material & thus separate
Chromatography BASIC SEPARATION PRINCIPLES Example 1: Hydrophobic interaction Example 2: Ionic interaction
SEPARATION TECHNIQUES IN CHROMATOGRAPHY LIQUID CHROMATOGRAPHY 1 i. iii. iv. Affinity Chromatography The most selective chromatography employed. type of 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. “Lock-and-key concept” Ligand Specific protein (e. g. antibody) binds to ligand Stationary phase Non-specific proteins
SEPARATION TECHNIQUES IN CHROMATOGRAPHY LIQUID CHROMATOGRAPHY 2 i. ii. iv. Hydrophobic Chromatography Typically used for protein separations Employs derivatized polymer resins, with phenyl, butyl, or octyl ligand groups Stationary phase Protein adhere to the hydrophobic Ligand surface under high salt conditions and (e. g. phenyl group) 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 p. H, salt used, buffer Stationary phase
SEPARATION TECHNIQUES IN CHROMATOGRAPHY LIQUID CHROMATOGRAPHY 3 i. iii. iv. Reverse Phase 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 (C 8), actyldecyl (C 18), phenyl, and methyl (C 1) 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 Stationary phase Ligand (e. g. actyldecyl) Stationary phase
SEPARATION TECHNIQUES IN CHROMATOGRAPHY LIQUID CHROMATOGRAPHY 4 i. Ion Exchange Chromatography Biomolecules generally have charged groups on their surfaces, which change with the p. H of the solution ii. iv. 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 p. H or salt concentration of the mobile phase It is particularly effective for proteins because they are amphoteric >Charge density Stationary phase Ligand <Charge (e. g. actyldecyl) density Stationary phase Competing ions
SEPARATION TECHNIQUES IN CHROMATOGRAPHY LIQUID CHROMATOGRAPHY 5 i. ii. iii. iv. Size Exclusion referred to as gel permeation Chromatography Also chromatography (GPC) for non-aqueous elution systems or gel filtration chromatography (GFC) for aqueous systems. Molecules with different sizes Stationary phase Separates solutes on the basis of their size No binding between the solutes and the resin Smaller molecules can partially completely enter the stationary phase. Smaller molecules or 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 Large molecules Pores
Types of Resins (Stationary Phase) SILICA-BASED RESINS Uncoated silica i. iii. iv. vi. Coated silica Compatible with water or organic solvent Serves as adsorbent compounds a good reversible for hydrophilic Organic solvent used as mobile phase, and water is added as the chromatography progresses Not typically stable at extremes of p. H Available with high surface area and small particle size; being very rigid; does not collapse under high pressures Denature some proteins and i. iii. 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 endcapped
Types of Resins (Stationary Phase) POLYMER-BASED RESINS Synthetic polymers Styrene divinylbenzene : i. ii. Very stable at p. H extremes Support for ion exchange chromatography because of its stability and rigidity Polyacrylamide: i. ii. 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 Natural Polymers o o o Used in hydrogel for a low pressure chromatography resins. Naturally hydrophillic 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
Types of Resins (Stationary Phase) 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) (2 C 2 H 5 N+HC 2 H 5) iv. quaternary ethylamine (QAE) (4 C 2 H 5 N+) - most basic i. Cation exchangers Acidic ion exchanger i. Anion exchangers Basic ion exchangers ii. Carry a negative charge ii. Carry a positive charge iii. Attract positive counterions iii. Attract negative counterions
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 p. H 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.
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
SEPARATION TECHNIQUES IN CHROMATOGRAPHY GAS CHROMATOGRAPHY (GC) Gas chromatographic equipment Chromatographic separation system Packed column Capillary column
SEPARATION TECHNIQUES IN CHROMATOGRAPHY CROSS-SECTIONAL VIEWS OF PACKED & CAPILLARY COLUMNS
SEPARATION TECHNIQUES IN CHROMATOGRAPHY i. SEPARATION PROCESS IN GAS CHROMATOGRAPHY Separation method based on Liquid film of conversion of sample to vapor phase ii. iv. solvent + samples The sample vapor is introduced onto a column (packed/capillary) containing stationary phase material Carrier gas (He) Column The mobile phase is typically an inert gas such helium (He), nitrogen (N 2) and hydrogen (H 2) Separation occurs on the basis of interaction between the sample components and the stationary phase Sample vapor Solvent vapor
TYPES OF ADSORBENTS (EXAMPLES: STATIONARY PHASE FOR GAS CHROMATOGRAPHY) Composition Polarity Applications Temp limits 100% dimethyl polysiloxane (Gum) Nonpolar Phenols, Hydrocarbons, Amines, -60 o. C to Sulfur compounds, Pesticides, 325 o. C PCBs 100% dimethyl polysiloxane (Fluid) Nonpolar Amino acid derivatives, Essential oils 0 o. C to 280 o. C 5% diphenyl 95% dimethyl polysiloxane Nonpolar Fatty acids, Methyl esters, Alkaloids, Drugs, Halogenated compounds -60 o. C to 325 o. C 14% cyanopropyl phenyl polysiloxane Immediate Drugs, Steroids, Pesticides -20 o. C to 280 o. C 50% phenyl, 50% methyl polysiloxane Immediate Drugs, Steroids, Pesticides, Glycols 60 o. C to 240 o. C 50% cyanopropylmethyl, 50% phenylmethyl polysiloxane Immediate Fatty acids, Methyl esters, Alditol acetates 60 o. C to 240 o. C 50% trifluoropropyl polysiloxane Immediate Halogenated compounds, +Aromatics 45 o. C to 240 o. C Polyethylene glycol – TPA modified Polar Acids, Alcohols, Aldehydes acrylates, Nitriles, Ketones 60 o. C to 240 o. C Polyethylene glycol Polar Free acids, Alcohols, Ethers, Essential oils, Glycols, Solvents 60 o. C to 220 o. C
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 (wb 1+wb 2) W b 1 Wb 2
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/wb)2
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).
SEPARATION TECHNIQUES IN CHROMATOGRAPHY Resolution in Chromatography (Rs) Answer: Rs= (t. R 2 -t. R 1)/0. 5 (wb 1+wb 2) = (385 -370)/0. 5(17. 0+16. 0) = 0. 91
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
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.
Dialysis (Low concentrated)
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
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