PRODUCT PURIFICATION(PART I)

ERT 320 BIO-SEPARATION ENGINEERING

MISS WAN KHAIRUNNISA WAN RAMLI

CHROMATOGRAPHY

INTRODUCTIONCHROMATOGRAPHY

• 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 TOOLSTo determine chemical compositions of sample

PREPARATIVE TOOLSTo PURIFY & COLLECT 1/ more components of sample

SEPARATION TECHNIQUES

BASIC SEPARATION PRINCIPLES

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.

SORBENT

STATIONARY PHASE:LIQUID

CHROMATOGRAPHY

SILICA BASED RESINS:Uncoated/ coated silica

POLYMER-BASED RESINS:

Synthetic/ natural polymers

ION-EXCHANGE RESINS:

Cation/ anion exchangers

SILICA-BASED RESINS

UNCOATED SILICAi. 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 Phv. 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

COATED SILICAi. 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

POLYMER-BASED RESINS

STYRENE DIVINYLBENZENE: i. Very stable at pH extremes ii. Support for ion exchange

chromatography because of its stability and rigidity

AGAROSE: i. Can be crosslinked to form a

reasonably rigid bead that is capable of tolerating pressures up to 4 bar.

DEXTRANi. Less rigid and used in size

exclusionii. Can be formed with very large

poresiii. Capable of including antibody

molecules and virus particles

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

NATURAL POLYMERS: i. Used in hydrogel for a low

pressure chromatography resins. ii. Naturally hydrophillic iii. Compatible with proteins and

other biomaterials

ION-EXCHANGE RESINS

Resins that have been derivatived with an ionic group Most commonly used ionic groups: a. Sulfoxyl (SO3-) - most acidic b. Carboxyl (COO-) c. Diethylaminoethyl (DEAE) (2C2H5N+HC2H5) d. Quaternary ethylamine (QAE) (4CHN+) - most basic

CATION EXCHANGERS:i. Acidic ion exchanger ii. Carry a negative charge iii. Attract positive counterions

ANION EXCHANGERS:i. Basic ion exchangers ii. Carry a positive charge iii. Attract negative counterions

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

Pressure drop is given by the Darcy equation:

CHROMATOGRAPHY CALCULATION

PARTICLE & PRESSURE DROP IN FIXED BEDS

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

• 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

CHROMATOGRAM DESCRIPTION

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

CHROMATOGRAPHY COLUMN DYNAMICS

PLATE MODELS

HEIGHT OF EQUIVALENT THEORETICAL PLATE (HETP), H:

Where L = Length of the column N = Number of plates

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

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

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

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.

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.

SCALE-UP PRINCIPLES1. All process volumes are scaled-up in direct proportion to

the sample volumes Process volumes include the column bed, wash, and elution volumes.

2. Column length is held constant Column volume is increased by increasing column diameter or by having a number of columns operating in parallel

3. 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.

4. Sample composition is held constant Critical factors include concentration, viscosity, pH and ionic strength

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,

BASIC DESIGN CALCULATIONS

To remove the volume term from the expression for Rs,

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.

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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