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

COURSE OUTCOMES CO DESCRIBE and DISCUSS the basic principles and applications of chromatography process. DIFFERENTIATE the types of separation in chromatography. DEVELOP basic design of chromatography.

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

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.

CLASSIFICATION OF CHROMATOGRAPHY TECHNIQUES 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

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

TYPES OF ADSORBENTS 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 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

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

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)

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

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 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

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

Hydrophobic Interaction Chromatography(HIC) 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.

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.

-used for removing small molecules from protein solution - 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

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

Equipment Injection port Oven Detector Column Nitrogen cylinder Recorder Injection port Oven Detector Column Nitrogen cylinder

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

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.

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

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

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

Stationary Phase Polymers Siloxane Arylene Polyethylene glycol

Liquid phases Non-polar phase Polar phase Intermediate 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 Polymer chains Cross-linking Bonding Fused silica tubing surface Bonded and cross-linked SP provides long term stability, better reproducibility and performance.

Common stationary phase coating for capillary column Composition Polarity Applicaitons Temp limits 100% dimethyl polysiloxane (Gum) Nonpolar Phenols, Hydrocarbons, Amines, Sulfur compounds, Pesticides, PCBs -60oC to 325oC 100% dimethyl polysiloxane (Fluid) Amino acid derivatives, Essential oils 0oC to 280oC 5% diphenyl 95% dimethyl polysiloxane Fatty acids, Methyl esters, Alkaloids, Drugs, Halogenated compounds 14% cyanopropyl phenyl polysiloxane Immediate Drugs, Steroids, Pesticides -20oC to 280oC 50% phenyl, 50% methyl polysiloxane Drugs, Steroids, Pesticides, Glycols 60oC to 240oC 50% cyanopropylmethyl, 50% phenylmethyl polysiloxane Fatty acids, Methyl esters, Alditol acetates 50% trifluoropropyl polysiloxane Halogenated compounds, +Aromatics 45oC to 240oC Polyethylene glycol – TPA modified Polar Acids, Alcohols, Aldehydes acrylates, Nitriles, Ketones Polyethylene glycol Free acids, Alcohols, Ethers, Essential oils, Glycols, Solvents 60oC to 220oC

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

Two Stage Tank Regulator GC Flow Controller

Injector Detector Column 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 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)

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 Dynamics Plate 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 Cmax = maximum peak height; σ = standard deviation; wi = peak width at inflection points; wh = peak width at half-height; w = peak width at base (base intercept); tR = average retention time. Figure 7.6 Properties of a Gaussian peak

Plate models Height of the equivalent theoretical plate (HETP), H: L = length of the column, N = number of plates 7.4.1

w = peak width at the base tR = average retention time. 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 base tR = average retention time. 7.4.2

tR1, tR2 = average retention time for separands 1 and 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

7.8.14 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 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.

Prepared by, MISS RAHIMAH OTHMAN Thank you Prepared by, MISS RAHIMAH OTHMAN