miss. rahimah binti othman (email: [email protected]) chapter 6: chromatography
TRANSCRIPT
MISS. RAHIMAH BINTI OTHMAN(Email: [email protected])
Chapter 6:Chapter 6:chromatographychromatography
COURSE OUTCOMESCOURSE OUTCOMES
CODESCRIBE and DISCUSS the basic principles and applications of chromatography process. DIFFERENTIATE the types of separation in chromatography. DEVELOP basic design of chromatography.
Basic principles and applications of
chromatography process.
Types of separation in chromatography.
Basic design of chromatography.
OUTLINESOUTLINES
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.
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.
BASIC PRINCIPLES AND APPLICATIONS OF CHROMATOGRAPHY PROCESS.
CLASSIFICATION OF CHROMATOGRAPHY
TECHNIQUES
Based on nature of mobile phase:
1. Liquid Chromatography (LC)
2. Gas Chromatography (GC)
3. Supercritical Fluid Chromatography (SFC)
Based on nature of mobile phase:
1. Liquid Chromatography (LC)
2. Gas Chromatography (GC)
3. Supercritical Fluid Chromatography (SFC)
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 crosslinked polymers;
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.
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
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
TYPES OF CHROMATOGRAPHY
1. Ion Exchange Chromatography
2. Reverse Phase Chromatography
3. Hydrophobic Interaction Chromatography
4. Affinity Chromatography
5. Size Exclusion Chromatography
1. Ion Exchange Chromatography
2. Reverse Phase Chromatography
3. Hydrophobic Interaction Chromatography
4. Affinity Chromatography
5. Size Exclusion Chromatography
ION EXCHANGE CHROMATOGRAPHY
ION EXCHANGE CHROMATOGRAPHY
resin (the stationary solid phase) is used to covalently attach anions or cations onto it.
Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.
resin (the stationary solid phase) is used to covalently attach anions or cations onto it.
Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.
TYPES OF ADSORBENTS
Silica-based resins
Polymer-based resins
Ion exchange resins
Silica-based resins
Polymer-based resins
Ion exchange resins
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
SILICA BASED RESINUncoated 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
SILICA BASED RESIN
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)
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
POLYMER BASED RESIN
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
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
ION EXCHANGE RESINCation 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
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
- 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
- typically used for protein separations- Employs derivatized polymer resins, with phenyl, butyl, or octyl ligand 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.
HYDROPHOBIC INTERACTION CHROMATOGRAPHY(HIC)
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.
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
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.
- 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
Basic principles and applications of
chromatography process.
Types of separation in chromatography.
Basic design of chromatography.
OUTLINESOUTLINES
Oven
Detector
Injection port
Nitrogen cylinder
Column
Recorder
EquipmentEquipment
Columns
• cylindrical, vertical vessels design to contain resin particles between 2 and 10 µm in diameter
Chromatographic separation involves the use of a stationary phasestationary phase and a mobile phasemobile phase.
Components of a mixture carried in the mobile phase are differentially attracted differentially attracted to the stationary phaseto the stationary phase and thus move through the move through the stationary phase at different stationary phase at different ratesrates.
Chromatographic separation involves the use of a stationary phasestationary phase and a mobile phasemobile phase.
Components of a mixture carried in the mobile phase are differentially attracted differentially attracted to the stationary phaseto the stationary phase and thus move through the move through the stationary phase at different stationary phase at different ratesrates.
COLUMNS
T=0
T=10’
T=20’
InjectorInjector DetectorDetector
MostMost Interaction with Stationary Phase Interaction with Stationary Phase LeastLeast
Flow of Mobile PhaseFlow of Mobile Phase
In gas chromatography the mobile phasemobile phase is an inert carrier gasinert carrier gas
and the stationary phasestationary phase is a solidsolid or a liquid liquid
coated on a solidcoated on a solid contained in a coiled column.
In gas chromatography the mobile phasemobile phase is an inert carrier gasinert carrier gas
and the stationary phasestationary phase is a solidsolid or a liquid liquid
coated on a solidcoated on a solid contained in a coiled column.
CHROMATOGRAPHY
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
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
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
BONDED AND CROSS-LINKED SP
Bonded and cross-linked SP provides long term stability, better reproducibility and performance.
Polymer chains
Cross-linking
Bonding
Fused silica tubing surface
COMMON STATIONARY PHASE COATING FOR CAPILLARY
COLUMNComposition Polarity Applicaitons Temp limits
100% dimethyl polysiloxane (Gum)
Nonpolar Phenols, Hydrocarbons, Amines, Sulfur compounds, Pesticides, PCBs
-60oC to 325oC
100% dimethyl polysiloxane (Fluid)
Nonpolar Amino acid derivatives, Essential oils
0oC to 280oC
5% diphenyl 95% dimethyl polysiloxane
Nonpolar Fatty acids, Methyl esters, Alkaloids, Drugs, Halogenated compounds
-60oC to 325oC
14% cyanopropyl phenyl polysiloxane
Immediate Drugs, Steroids, Pesticides -20oC to 280oC
50% phenyl, 50% methyl polysiloxane
Immediate Drugs, Steroids, Pesticides, Glycols 60oC to 240oC
50% cyanopropylmethyl, 50% phenylmethyl polysiloxane
Immediate Fatty acids, Methyl esters, Alditol acetates
60oC to 240oC
50% trifluoropropyl polysiloxane Immediate Halogenated compounds, +Aromatics
45oC to 240oC
Polyethylene glycol – TPA modified
Polar Acids, Alcohols, Aldehydes acrylates, Nitriles, Ketones
60oC to 240oC
Polyethylene glycol Polar Free acids, Alcohols, Ethers, Essential oils, Glycols, Solvents
60oC to 220oC
42
COLUMN DIMENSIONS
Column Length: 10 – 60 m
Column Internal Diameter: 0.10 – 0.53 mm
Stationary Phase Film Thickness: 0.10 –
0.25 m
Two Stage Two Stage Tank RegulatorTank Regulator
GC Flow ControllerGC Flow Controller
InjectorInjector DetectorDetector
Column in OvenColumn in Oven
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
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.
PARTICLE SIZE AND PARTICLE SIZE AND PRESSURE DROP IN FIXED PRESSURE DROP IN FIXED
BEDSBEDS 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)
From Blake-Kozeny equation, k gives a function of resin particles size and void friction
(27)
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
CHROMATOGRAPHY COLUMN DYNAMICSPlate models
Plate models seek to explain the band broadening observed in chromatography by approximating a chromatograph as a series of well-mixed tanks at equilibrium.
The terminology comes from analysis of distillation, where
plates are sometimes used to hold vapor and liquid in contact to approach equilibrium at various temperatures and compositions.
Just as distillation is often performed in a packed column but the concept of a “theoretical equilibrium plate” remains, so has it also come to symbolize resolving power of a chromatography column.
Figure 7.6 Properties of a Gaussian peak
CCmaxmax = maximum peak height; = maximum peak height;
σσ = = standard deviation; standard deviation; wwii = peak width at inflection points; = peak width at inflection points;
wwhh = peak width at half-height; = peak width at half-height;
w =w = peak width at base (base intercept); peak width at base (base intercept); ttRR = average retention time. = average retention time.
Height of the equivalent theoretical plate (HETP), H:
L = length of the column,
N = number of plates
Plate models
7.4.1
From Gaussian peaks: the plate count (N) can be expressed as the squared average retention time divided by the variance of the peak
w = peak width at the basetR = average retention time.
7.4.2
Peak width is used in the definition of resolution, Rs, which is measure of the extent of separation of two peaks in a chromatography
tR1, tR2 = average retention time for separands 1 and 2
w1,w2 = peak width (time) for separands 1 and 2
7.4.3
CHROMATOGRAPHY COLUMN MASS BALANCE WITH NEGLIGIBLE
DISPERSION Mass balance for chromatography:
7.33
ci = concentration of separand i in the mobile phase = [C]i,qi = concentration of separand 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 separand in the column, t = time, x = longitudinal distance in the column; x = 0 at column inlet
CHROMATOGRAPHY COLUMN MASS BALANCE WITH NEGLIGIBLE
DISPERSION Using an equilibrium isotherm relationship in the
form qi =f(ci)(Figure 1), Equation (7.3.3) becomes:
Where qi’(ci) is the slope of the equilibrium isotherm at concentration ci.
•If we let:
•Then Equation (7.3.6) becomes:
•Thus, the expression for ui given by Equation (7.3.7) is the effective velocity of component i through the packed column.
7.3.7
7.3.8
EXAMPLE 1
Chromatographic Separation of Two Solutes
Two solutes have linear equilibrium constants of Keq,1 = 7.5 and Keq,2 = 7.8, respectively. For a flow
rate of 1.5 liter/min, in a column 63 cm in diameter, with a void fraction of 0.33, and local equilibrium, what column length is required to separate the two solutes by 5 min?
Solution
The effective velocity of solute i for negligible dispersion is given by Equation (7.3.7) as
For linear equilibrium,
The superficial velocity is
For solute 1, the effective velocity is therefore;
This same equation gives u2 = 0.08657 cm/min for solute 2.
Translating solute velocities into elution times for a constant distance traveled (L),
Solving for L gives;
** Note that four significant figures are used to calculate u1 and u2 to avoid error in calculating L.
CHROMATOGRAPHY SCALEUP
Chromatography scaleup algorithms accounts for changes in:
- bed height and diameter,
- linear and volumetric flow rate
- and particle size.
Yamamoto et al. have developed the following proportionality for resolution, Rs, of proteins in linear gradient elution ion exchange chromatography and hydrophobic interaction chromatography:
(31)
Dm = diffusion coefficient of the protein in solution;
L = column length;
g = slope of the gradient (change in concentration of gradient per volume of gradient);
V = column volume;
V0 = column void volume;
u = interstitial fluid velocity;
dp = particle diameter
The definitions can be made:
Q = inlet flow rate;
ε = column void friction;
A = column cross-sectional area
7.8.14
(31)
Thus, for scaleup with constant resolution from scale 1 to scale 2 for the same product and the same column void fraction, the scaleup equation is:
Thus, as the particle size increases on scaleup, 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.
7.8.15
easy to develop lab scale processes that use the same resin and same gradient for the commercial process scale
In practice only the ration between column volume and flow rate need be addressed
When the bed height can be maintained on scaleup, the mobile phase linear velocity remains the same, and the column is simply scaled by diameter.
7.8.16
EXAMPLE 2
Scale up of a Protein Chromatography
A column 20 cm long, with an internal diameter of 5 cm, gives sufficient purification to merit scaleup. The column produces 3.2 g of purified protein per cycle, and a cycle takes 6 h, from equilibration through regeneration. You want a throughput of
10 g/h. What are the new column’s dimensions if linear velocity is held constant?
Solution
For scale up when the linear velocity is held constant, the column diameter is increased, and the column height is maintained the same.
If the linear flow rate is held constant, then the cycle time cannot be altered.
Thus, the scaled up column must produce
6 h/cycle x 10g/h = 60g/cycle.
Since the flow rate is proportional to the throughput of protein,
From Equation (7.8.16), the scaleup relationship when the gradient and the particle size are not changed upon scaleup, and since L1= L2,
where D1 and D2 are the column diameters for columns 2 and 1, respectively.
Since D1 = 5.0 cm, we obtain
EXAMPLE 3
Scaleup of Protein Chromatography Using Standard Column Sizes
Consider the case given in Example 2. Available standard column diameters are 20 and 25 cm. What flow rates and bed depths would apply to each of these columns?
Solution
The column volumes for both columns are still 18.75 times that used in the laboratory
column. Thus,
For a column 20 cm in diameter,
and for column the 25 cm in diameter,
Note that:
the gradient is also expressed in column volumes.
The total gradient volume, that is, the total volume of eluent used to go from the leanest mobile phase condition to the richest, is expressed in terms of column volumes, and this is held constant on scaleup.
EXAMPLE 4
Consideration of Pressure Drop in Column Scaling
Determine the minimum diameter possible for the columns analyzed in Example 3.The flow rate is 40 ml/min. The column pressure should not exceed 300 kPa (28.8 psig), the maximum solution viscosity is 1.1 cp, and the void fraction in the column is 0.35. The resin particle size is 100 µm.
Solution
Pressure drop can be calculated using Darcy’s law [Equation 26] by knowing µ, v, k and L. We can calculate k from the Blake—Kozeny equation (7.6.2):
The column volume must be 7359 ml (from Example 3), and the flow rate is 40 ml/min times the scaleup factor of 18.75 (= 750 ml/min).
We calculate the pressure drop for the column 20 cm in diameter and 23.4 cm in length, which would give the higher pressure drop of the two standard column sizes (20 and 25 cm):
From Equation (26) the pressure drop is
Thus, the standard 20 cm diameter column would operate at well below the maximum allowable pressure.
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