PRODUCT PURIFICATION (PART I)

1. PRODUCT PURIFICATION (PART I) ERT 320 BIO-SEPARATION ENGINEERING MISS WAN KHAIRUNNISA WAN RAMLI

2. CHROMATOGRAPHY

3. INTRODUCTION CHROMATOGRAPHY • Use in separation, purification & identification of compounds before quantitative analysis is taken up. • BASIS:Selective distribution of component in a mixture between 2 immiscible phases in intimate contact with each other • 1 stationary phase & 1 mobile phase • APPLICATION: Separation of biomolecules, fine & specialty chemicals ANALITICAL TOOLS To determine chemical compositions of sample PREPARATIVE TOOLS To PURIFY & COLLECT 1/ more components of sample

4. SEPARATION TECHNIQUES

5. BASIC SEPARATION PRINCIPLES

8. SORBENT STATIONARY PHASE: LIQUID CHROMATOGRAPHY ION-EXCHANGE RESINS: Cation/ anion exchangers SILICA BASED RESINS: Uncoated/ coated silica POLYMER-BASED RESINS: Synthetic/ natural polymers

9. SILICA-BASED RESINS 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 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

10. STYRENE DIVINYLBENZENE: Very stable at pH extremes Support for ion exchange chromatography because of its stability and rigidity AGAROSE: Can be crosslinked to form a reasonably rigid bead that is capable of tolerating pressures up to 4 bar. POLYMER-BASED RESINS 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 DEXTRAN • Less rigid and used in size exclusion • Can be formed with very large pores • Capable of including antibody molecules and virus particles NATURAL POLYMERS: Used in hydrogel for a low pressure chromatography resins. Naturally hydrophillic Compatible with proteins and other biomaterials

11. Resins that have been derivatived with an ionic group • Most commonly used ionic groups: • Sulfoxyl (SO3-) - most acidic • Carboxyl (COO-) • Diethylaminoethyl (DEAE) (2C2H5N+HC2H5) • Quaternary ethylamine (QAE) (4CHN+) - most basic ION-EXCHANGE RESINS CATION EXCHANGERS: Acidic ion exchanger Carry a negative charge Attract positive counterions ANION EXCHANGERS: Basic ion exchangers Carry a positive charge Attract negative counterions

12. STATIONARY PHASE: GAS CHROMATOGRAPHY SOLID PHASE: i. Most uses for separation of low MW compounds and gases ii. Common SP: silica, alumina, molecular sieves such as zeolites, cabosieves, carbon blacks LIQUID PHASE: i. Over 300 different phases are widely available ii. Grouped liquid phases: Non-polar, polar, intermediate and special phases iii. Polymer liquid phase Non-polar phase i. Primarily separated according to their volatilities ii. Elution order varies as the boiling points of analytes iii. Common phases: dimethylpolysiloxane, dimethylphenylpolysiloxane Polar phase i. Contain polar functional groups ii. Separation based on their volatilities and polar-polar interaction iii. Common phases: polyethylene glycol Intermediate phase i. Common phase: 14% cyanopropyl phenyl polysiloxane

14. CHROMATOGRAPHY CALCULATION PARTICLE & PRESSURE DROP IN FIXED BEDS Pressure drop is given by the Darcy equation:

15. From Blake-Kozeny equation, k gives a function of resin particles size and void friction

16. Darcy equation applies for rigid particles, such as silica. • • When the stationary phase particle size is decreased, the pressure drop in the column increases as the inverse square. • • These increases requires pressure additional power in pumping, as well as more specialized requirements for the construction of the columns and its seals

17. CHROMATOGRAM DESCRIPTION

18. CHROMATOGRAM Response of a detector vs time, shown when various components come off a column RETENTION TIME, tr The time at which a component elutes from a column

19. CHROMATOGRAPHY COLUMN DYNAMICS PLATE MODELS HEIGHT OF EQUIVALENT THEORETICAL PLATE (HETP), H: Where L = Length of the column N = Number of plates

20. From Gaussian peaks: THE PLATE COUNT (N) can be expressed as the squared average retention time divided by the variance of the peak Where w = peak width at the base tr = average retention time

21. PEAK WIDTH is used in the definition of resolution, Rs  measure of the extent of separation of two peaks in chromatography Where tR1, tR2 = average retention time for separands 1 & 2 w1, w2 = peak width (time) for separands 1 & 2

22. Chromatography column mass balance with negligible dispersion • Mass balance for chromatography: • ci = concentration of solute i in the mobile phase = [C]i, • qi = concentration of solute i in the stationary phase averaged over an adsorbent particle = [CS]i, • ε = void fraction (mobile phase volume/total column volume), commonly 0.3 to 0.4 in fixed beds, • v = mobile phase superficial velocity (flow rate divided by the empty column cross-sectional area, Q/A), • Deff= effective dispersivity of the solute in the column, • t = time, • x = longitudinal distance in the column; • x = 0 at column inlet

23. Using an equilibrium isotherm relationship in the form qi =f(ci), EQ. (1) becomes: Where Where qi’(ci) is the slope of the equilibrium isotherm at concentration ci .

24. If we let: Then EQ. (2) becomes: Thus, the expression for ui given by EQ. (3) is the effective velocity of component i through the packed column.

25. SCALE-UP PRINCIPLES All process volumes are scaled-up in direct proportion to the sample volumes  Process volumes include the column bed, wash, and elution volumes. Column length is held constant  Column volume is increased by increasing column diameter or by having a number of columns operating in parallel Linear (or superficial) velocity is held constant  Because column length is held constant, volumetric flow rate increases proportionally with sample volume. Total separation time remains roughly constant in scale-up. Sample composition is held constant  Critical factors include concentration, viscosity, pH and ionic strength

26. BASIC DESIGN CALCULATIONS • Typically account for changes in bed height & diameter, linear & volumetric flow rate, and particle size. • General approach for scale up is to based on keeping the resolution, Rs constant • For linear gradient elution ion exchange & hydrophobic interaction chromatography,

27. To remove the volume term from the expression for Rs,

28. Thus, for scale-up with constant resolution from scale 1 to scale 2 for the same product and the same column void fraction, the scale-up equation is: • Thus, as the particle size increases on scale-up, the flow rate relative to the column volume must decrease and/or the gradient slope must decrease to maintain constant resolution, which seems correct intuitively.

29. Easy to develop lab scale processes that use the same resin and same gradient for the commercial process scale • In practice only the ratio between column volume and flow rate need be addressed • When the bed height can be maintained on scale-up, the mobile phase linear velocity remains the same, and the column is simply scaled by diameter

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