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
Solutes in solution/ volatiles in gas are placed in MOBILE PHASE & passed over a selected adsorbent material [stationary phase]
MOBILE PHASE:
Continuous flow of a carrier liquid/ gas
STATIONARY PHASE:
A bed of solids/ immobilized liquid
The solutes/ volatiles have differential AFFINITY for the adsorbent material & thus, separation occurs.

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. POLYMER-BASED RESINS 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 LIQUID PHASE: 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
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

30. THANK YOU