MISS. RAHIMAH BINTI OTHMAN (Email: rahimah@unimap.my)

1. Chapter 6: chromatography MISS. RAHIMAH BINTI OTHMAN (Email: rahimah@unimap.edu.my)

2. COURSE OUTCOMES

3. OUTLINES • Basic principles and applications of chromatography process. • Types of separation in chromatography. • Basic design of chromatography.

4. BASIC PRINCIPLES AND APPLICATIONS OF CHROMATOGRAPHY PROCESS. Introduction • Chromatography– is a sorptive separation process useful in separation & purification. • Commonly used in separation of biologics and fine & specialty chemicals. • A specialized unit operation of adsorption-based separations. Purpose Of Chromatography • Analytical - determine chemical composition of a sample. • Preparative - purify and collect one or more components of a sample.

5. CLASSIFICATION OF CHROMATOGRAPHY TECHNIQUES • Based on nature of mobile phase: 1. Liquid Chromatography (LC) 2. Gas Chromatography (GC) 3. Supercritical Fluid Chromatography (SFC)

6. STATIONARY PHASE • The stationary phase consists of semi-permeable, porous beads with a well-defined range of pore sizes. • Solid phase is ‘stationary’ because it is packed in a fixed column. • The semi-permeable porous beads are crosslinkedpolymers; • Degree of crosslinking is controlled carefully to yield different pore sizes. • The stationary phase is said to have a fractionation range (due to the different pore sizes), meaning that molecules within that molecular weight range can be separated.

7. MOBILE PHASE • Liquid phase is often flowing past the solid phase is referred to as ‘mobile phase’. • The mobile phase contains a mixture of solutes. • Small solutes will diffuse in and out of the pores; • Their path through the column is longer • The elution time will be longer

8. TYPES OF CHROMATOGRAPHY 1. Ion Exchange Chromatography 2. Reverse Phase Chromatography 3. Hydrophobic Interaction Chromatography 4. Affinity Chromatography 5. Size Exclusion Chromatography

9. Ion Exchange Chromatography • resin (the stationary solid phase) is used to covalently attach anions or cationsonto it. • Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.

11. TYPES OF ADSORBENTS • Silica-based resins • Polymer-based resins • Ion exchange resins

12. ADSORBENT TYPE : RESIN • Two basic resin materials : polymer and silica • Silica resin – have hydrophobic coating and are used for reversed phase chromatography • Polymer resin – used in aqueous applications and are conjugated with ion exchange, hydrophobic interaction, or affinity-type ligands • Surface area is generally to 100 – 1500 m2/g

13. SILICA BASED RESIN Uncoated 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

14. SILICA BASED RESIN Coated silica • 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

15. POLYMER BASED RESIN • frequently used in industrial applications : high stability and low cost • larger (10-100 µm) than silica-based resins (1-25 µm) • less rigid • not generally suitable for high pressure applications (>4 bar)

16. POLYMER BASED RESIN Two synthetic polymer that are commonly used: styrene divinylbenzene and polyacrylamide Styrene divinylbenzene • very stable at pH extremes • support for ion exchange chromatography because of its stability and rigidity Polyacylamide • 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

17. Natural polymers • Agarose and dextran • used in hydrogel for a low pressure chromatography resins. • Naturally hydrophillic • compatible with protein 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

18. ION EXCHANGE RESINS • Resins that have been derivatized with an ionic group • most commonly used ionic groups: i) sulfoxyl (SO3-) - most acidic ii) carboxyl (COO-) iii) diethylaminoethyl (DEAE) (2C2H5N+HC2H5) iv) quaternary ethylamine (QAE) (4C2H5N+) - most basic

19. ION EXCHANGE RESIN Cation exchangers • The acidic ion exchanger • carry a negative charge • attract positive counterions Anion Exchangers • the basic ion exchangers • Carry a positive charge • Attract negative counterions

20. 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, • hydrophillic solutes bind in higher proportion in normal phase

21. Hydrophobic phases that are bonded to silica are typically actyil (C8), actyldecyl (C18), phenyl, and methyl (C1) • the 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

22. Hydrophobic Interaction Chromatography(HIC) • typically used for protein separations • Employs derivatized polymer resins, with phenyl, butyl, or octylligand groups • Protein adhere to the hydrophobic surface under high salt conditions and redissolve into the mobile phase as the salt concentration is reduced • - differs from reversed phase in that the mobile phase is kept aqueous (polar), and the salt concentration is used to effect the partitioning to the surface • - HIC is sensitive to pH, salt used, buffer type and temperature.

23. Affinity Chromatography • This is the most selective type of chromatography employed. • It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized 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 extracted by changing the ionic strength or pH.

27. Immobilized Metal Affinity Chromatography (IMAC) • Some proteins have high affinities for specific metals such as nickel and copper. • The affinity may either be structural (metalloproteins) – require metal centers for their biological activities • or based on the content of specific amino residues such as histidine and cysteine • - immobilize metal ions onto polymer resins (IMAC resins) • - Used to purify proteins that have one of two characteristics mentioned above

28. Size Exclusion Chromatography (SEC) • also called gel permeation or gel filtration • chromatography • separates solutes on the basis of their size • no binding between the solutes and the resin • -The pores are normally small and exclude the larger • solute molecules, but allows smaller molecules to • enter the gel, causing them to flow through a larger • volume.

29. - resins are hydrophilic polymer gels with a broad distribution of pore sizes • molecules larger than the largest pores in the gel cannot enter the gel and are eluted first, • - smaller molecules enter the gel to varying extents, depending on their size and shape, and retarded on their passage through the bed • -used for removing small molecules from protein solution

31. OUTLINES • Basic principles and applications of chromatography process. • Types of separation in chromatography. • Basic design of chromatography.

32. Equipment Recorder Injection port Oven Detector Column Nitrogen cylinder

33. Columns • cylindrical, vertical vessels design to contain resin particles between 2 and 10 µm in diameter

34. COLUMNS • Chromatographic separation involves the use of a stationary phase and a mobile phase. • Components of a mixture carried in the mobile phase are differentially attracted to the stationary phase and thus move through the stationary phase at different rates.

35. Flow of Mobile Phase Injector Detector T=0 T=10’ T=20’ Most Interaction with Stationary Phase Least

36. CHROMATOGRAPHY In gas chromatography • the mobile phase is an inert carrier gas and • the stationary phaseis a solid or a liquid coated on a solid contained in a coiled column.

37. Stationary Phases • Solid phase • Most uses for separation of low MW compounds and gases • Common SP: silica, alumina, molecular sieves such as zeolites, cabosieves, carbon blacks • Liquid phase • Over 300 different phases are widely available • grouped liquid phases • Non-polar, polar, intermediate and special phases • Polymer liquid phase

38. Stationary Phase Polymers • Siloxane • Arylene • Polyethylene glycol

39. Liquid phases • Non-polar phase • Primarily separated according to their volatilities • Elution order varies as the boiling points of analytes • Common phases: dimethylpolysiloxane, dimethylphenylpolysiloxane • Polar phase • Contain polar functional groups • Separation based on their volatilities and polar-polar interaction • Common phases: polyethyleneglycol • Intermediate phase

40. Bonded and Cross-linked SP Polymer chains • Bonded and cross-linked SP provides long term stability, better reproducibility and performance. Cross-linking Bonding Fused silica tubing surface

41. Common stationary phase coating for capillary column

43. Column Dimensions • Column Length: 10 – 60 m • Column Internal Diameter: 0.10 – 0.53 mm • Stationary Phase Film Thickness: 0.10 – 0.25 mm

46. Two Stage Tank Regulator GC Flow Controller

47. Injector Detector Column in Oven

48. Detector • Most common detection techniques: pH, conductivity and light absorbance • Conductivity and pH : to check the performance of the gradient, the loading of the column and the regeneration • Light absorbance (280, 254, 229, 214 nm, depending on the application) : used to monitor the effluent for evidence of the target molecules • Other common detection methods in use in large-scale chromatography: refractive index, electrochemical detection & light scattering

49. Chromatography system fluidics • Pumps and tubing are the most important. • Pumps: typically positive displacement pumps – have a low shear, so do not pose a problem for sensitive biomolecules. • comes in two varieties: peristaltic and rotary lobe • fluid is pumped downflow through chromatography columns.

50. Particle Size and Pressure Drop in Fixed Beds • Pressure drop is given by the Darcy equation: Δp = pressure drop over column length L; µ = viscosity of the mobile phase; v = superficial velocity; k = constant (26)