Tài liệu HPLC for Pharmaceutical Scientists 2007 (Part 6) pptx

A specific column can be used for separation of solutes with molecular weights that are within the molecular weight window between the exclusion and permeation limits Figure 6-2.. Hydrody

SIZE-EXCLUSION

CHROMATOGRAPHY

Yuri Kazakevich and Rosario LoBrutto

6.1 SEPARATION OF THE ANALYTE MOLECULES

BY THEIR SIZE

Size-exclusion chromatography (SEC) separates polymer molecules and bio-molecules based on differences in their molecular size The separation process

in simplified form is based on the ability of sample molecules to penetrate inside the pores of packing material and is dependent on the relative size of analyte molecules and the respective pore size of the absorbent The process also relies on the absence of any interactions with the packing material surface Two types of SEC are usually distinguished:

1 Gel permeation chromatography (GPC)—separation of synthetic (organic-soluble) polymers GPC is a powerful technique for polymer characterization using primarily organic solvents

2 Gel filtration chromatography (GFC)—separation of water-soluble biopolymers GFC uses primarily aqueous solvents (typically for aqueous soluble polymers, proteins, etc.)

Physical and chemical properties of polymers are dependent on their molec-ular weight and molecmolec-ular weight distribution.The separation principle in SEC

is based on the forced transport of the polymer molecules through the porous stationary-phase media under the conditions of suppressed interactions of the

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HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc.

polymer analyte with the surface The mobile-phase eluent is selected in such way that it interacts with the surface of packing material stronger than the polymer Under these conditions, the smaller the size of the molecule, the more

it is able to penetrate inside the pore space and the movement through the column is retarded On the other hand, the bigger the molecular size, the higher the probability the molecule will travel around the particles of the packing material and, thus, is eluted earlier The molecules are separated

in order of decreasing molecular weight, with the largest molecules eluting from the column first and smaller molecules eluting last (Figure 6-1)

Molecules larger than the pore size do not enter the pores and elute together as the first peak in the chromatogram and this is called total exclu-sion volume which defines the excluexclu-sion limit for a particular column Mole-cules that can enter the pores diffuse into the internal pore structure of the gel to an extent depending on their size and the pore size distribution of the

Figure 6-1 Illustrative description of separation in SEC.

gel The molecules will have an average residence time in the particles that depends on the molecules size and shape in the particular mobile phase.There-fore, different molecules have different total residence times in the column This portion of a chromatogram is called the selective permeation region (the effective volume in which separation can occur) Molecules that are smaller than the pore size can enter all pores, have the longest residence time on the column, and will elute all together as the last peak in the chromatogram This last peak in the chromatogram determines the total permeation limit for a par-ticular column The largest elution volume (retention volume) in any given SEC column is equal to the total mobile-phase volume in the column (known

as the void volume, V0) The exclusion range indicates the molecular weight of

solutes above which all solutes having a molecular weight greater than the exclusion limit These analytes will elute at the same retention time as a single peak A specific column can be used for separation of solutes with molecular weights that are within the molecular weight window between the exclusion and permeation limits (Figure 6-2)

Separation process in SEC is based on the actual size of the molecules, which in turn reflects the molecular weight of the polymer The resulting SEC

Figure 6-2 Elution of analytes in SEC (Reprinted with permission from reference 1.)

chromatogram reflects the size distribution of the polymer sample, and its rela-tionship with the molecular weight distribution which lays the foundation of the SEC theory

6.2 MOLECULAR SIZE AND MOLECULAR WEIGHT

A polymer molecule in solution has a certain shape that strongly depends on the type of polymer, type of solvent, temperature, and other conditions Usually a polymer forms some kind of globular species whose size is depen-dent on the degree of solvation by solvent molecules

This globe could be described by its volume (v) and hydrodynamic radius (R)

Hydrodynamic radius (radius of gyration) of the polymer in the solution could be expressed in the form

(6-1)

where [η] is intrinsic viscosity and M is molecular weight

For some polymers that are not flexible, the effective R is used to represent the radius of the sphere The parameter R is equivalent to the mechanical

behavior of the polymer in solution Viscosity is the simplest parameter of the polymer solution From the Stokes and Einstein equations, the volume of the equivalent sphere is proportional to the product of intrinsic viscosity and polymer molecular weight:

(6-2) where [η] is intrinsic viscosity, M is molecular weight, NAis Avogadro’s number,

and v is the volume of the equivalent sphere As one could see, the intrinsic

vis-cosity is an important parameter related to the molecular weight of the polymer and its molecular volume By definition, intrinsic viscosity is a limit of the ratio

of the specific viscosity of the polymer solution to its concentration at c → 0,

or it is the y-intercept of the dependence of ηspversus concentration

Polymer molecules of a different nature but with the same molecular weight usually have different hydrodynamic radii This is due to the differences in coil flexibility, intramolecular interactions, and, most importantly, the differences

in their interactions with the solvent This essentially means that if two differ-ent polymers analyzed at iddiffer-entical SEC conditions show similar peaks with

identical elution volume, it does not confirm that the molecular weights of

these polymers are identical It only indicates that at the given conditions the gyration radii of the molecules are the same, causing similar elution

The nature of the solvent also has a significant effect on the polymer conformation and thus on its gyration radius and molecular volume If the

h [ ]M=2 5 N v A

R=34 M[ ]

1 3

solvent–polymer interactions are favorable or essentially prevailing over the interactions between different segments of the same polymer, then we can expect a high degree of solvation and the polymer globe will swell For instance, if polystyrene is dissolved in toluene, due to the similar nature of the solvent and polystyrene monomer, toluene will solvate polymer molecules and their gyration radius increases On the contrary, if the same polystyrene

is dissolved in tetrahydrofuran (THF), then interactions between polystyrene segments prevail over the interactions with THF As a consequence, the size

of the polymer globe in THF is relatively small, especially in comparison to that in toluene

From equation (6-2), one can conclude that intrinsic viscosity is propor-tional to the polymer molecular volume On the other hand, the effective mol-ecular volume is also the function of the molmol-ecular weight and the type of used solvent (or the nature of the solvent–polymer and polymer–polymer interac-tions) The intrinsic viscosity is an exponential function of the molecular weight with fixed coefficients for any specific polymer and solvent

(6-3)

This expression is known as the Mark–Houwink equation, and K andα are constants for any given pair of polymer and solvent These constants are tabulated and could be found for most known polymers in reference 2

6.3 SEPARATION MECHANISM

Eluent flow through the chromatographic column packed with porous packing material has a velocity distribution depending on the pathway Flow around the adsorbent particles is the fastest Flow through the pore space is much slower Since the smallest molecules can penetrate all of the pores, they can

be distributed in the whole liquid volume of the column and their average migration speed is therefore the slowest Molecules of intermediate size may penetrate into the pore space but may not come close to the pore walls, so their center of mass will be allocated closer to the center of the pores where flow velocity is higher Their average migration speed is higher The biggest molecules experience steric hindrance in permeation inside the packing pore space and move through the column primarily around the particles with the fastest possible speed As a result, the biggest molecules come out of the column first, and the smallest ones come out last

Obviously, all molecules that are not able to penetrate into the pore space, move with the same velocity Retention volume of all these molecules is the same and is called exclusion volume, also known as total exclusion The total exclusion volume is a characteristic of a particular column which determines its upper separation limit

h [ ]=K M⋅ α

6.4 CALIBRATION

SEC calibration establishes the relationship of a particular elution volume with specific molecular weight of the polymer (Figure 6-3) [3] For calibration the elution volume of the solutions of polymer standards with known narrow molecular weight distributions are measured An example of a separation is

shown in Figure 6-4 [4] In SEC, hydrodynamic volume of the polymer

mole-cules is being measured rather than the actual mass of a particular species The hydrodynamic volume is the space a particular polymer molecule occupies when it is in solution The molecular weight can be approximated from SEC data from the relationship between molecular weight and hydrodynamic volume for particular known standards However, the relationship between hydrodynamic volume and molecular weight is not the same for all polymers,

so only an approximate measurement can be obtained

A series of commercially available polystyrene standards can be used for calibration The elution volume (elution time multiplied by flow rate) corre-sponding to a particular peak in the chromatogram is related to the molecu-lar weight of a particumolecu-lar polystyrene After assignment of the molecumolecu-lar weight for each component to its elution volume, the logarithms of the mole-cular weight of the standards are plotted against their elution volumes in order

to construct a calibration curve (Figure 6-3) Each combination of column, polymer, and solvent has its own calibration curve

The same polymer molecules could have different sizes in different solvents, and two molecules of different polymers might have the same size despite their

Figure 6-3 Calibration curves for a set of AquaGel (Polymer Laboratories) columns

designed for the separation of water soluble polymers Calibration using PEO and PEG standards (Reprinted from reference 3, with permission from Polymer Laboratories Inc.)

different molecular weight So the calibration curve for the certain polymer is valid only if the standards used are of the same nature and used eluent was

of the same type

If two different polymers in the same solvent have the same intrinsic viscosity, then their molecular weights are related as

(6-4) This makes it possible to use standards of one polymer for characterization of another if the corresponding Mark–Houwink constants are known For most known polymers, Mark–Houwink constants are tabulated For example,

a polystyrene (PS)-based calibration could be used for characterization of polymethylmethacrylate (PMMA)

Retention volume in SEC is proportional to the size of the polymer molecules in solution In addition, as discussed in Section 6.2, equation (6-2), the product of the intrinsic viscosity of the polymer and its molecular weight is proportional to the hydrodynamic molecular volume These relationships allowed Benoit et al [5, 6] to introduce a universal molecular weight calibration

K1⋅M1a1=K2⋅M2a2

Figure 6-4 Example of a separation of calibration mixture of polystyrene standards.

(Reprinted from reference 4, with permission from Phenomenex.)

In conventional molecular weight calibration, the dependence of the reten-tion volumes of a series of narrow molecular weight distribureten-tion standards with known average masses is plotted against the logarithms of their molecu-lar weights In universal calibration, the logarithm of the product of the intrin-sic viscosity [η] and molecular weight M (essentially hydrodynamic volume)

is plotted against retention volume Hydrodynamic molecular volume is directly related to the retention volume if only the steric separation mecha-nism is involved Benoit et al [5, 6] found that plots of the logarithm of hydro-dynamic volumes versus corresponding retention volumes for a series of narrow standards of different polymers in different solvents resulted in a single calibration curve, as shown in Figure 6-5

The combination of the differential refractive index (RI) detector and on-line viscometer allows the direct use of the universal calibration and thus true molecular weight determination The RI detector is concentration-sensitive, and the viscometer records specific viscosity The ratio of the specific viscosity to the concentration is equivalent to intrinsic viscosity (as discussed

in Section 6.1), and the continuous dependence of this ratio versus the reten-tion volume could be related to the universal calibrareten-tion curve, thus allowing the correlation of each point on the chromatogram with the true molecular weight

More detailed information on this type of GPC analysis can be found in

the book by Yau et al., Modern Size-Exclusion Liquid Chromatography [7].

Figure 6-5 Universal Benoit calibration (Reprinted from reference 5, with

permission.)

6.5 COLUMNS

Polymer-based packing materials are the main type of adsorbent used in size-exclusion HPLC Most SEC analyses of synthetic organic polymers are performed on rigid or semirigid crosslinked polystyrene gel materials (styrene–divinylbenzene copolymers with different degree of crosslinkage) These materials require careful selection of separation conditions and solvent, since they can shrink and swell in different solvents and temperatures, thus changing their separation power A major requirement of size-exclusion separation is the complete absence of any interactions between analyzed com-ponents and the surface of packing material in the column Polymeric pack-ings do not have any active surface groups and could be synthesized with controlled porosity For the separation of biopolymers in aqueous media, gel-filtration chromatography is used with stationary phases that have a mildly hydrophilic surface, which is usually required to avoid noticeable hydropho-bic interactions with the surface These types of stationary phases for GFC include (a) dextrans, agaroses, polyacrylamide, or mixtures of these compo-nents which are suitable for low- or medium-pressure chromatography and (b) porous silica-based media which are more suitable for higher-pressure applications

The separation range in size-exclusion chromatography for a particular column is relatively narrow, and it lies between the total volume of the liquid phase in the column (void volume) and the exclusion volume, Ve The differ-ence between these two volumes is the total pore volume of the packing mate-rial in the column Indeed, if some molecules of studied polymers are small enough to penetrate inside all pores of the packing material, they will elute with the column void volume On the other hand, polymers with significant molecular size that cannot penetrate inside the particles will all travel together around the particles and elute early with exclusion volume

To obtain wider separation range, longer and wider column dimensions are used The greater the amount of packing material in the column, the higher the total pore volume and the higher the difference between void and exclu-sion volumes

Another important parameter is the ability of the column to discriminate different ranges of molecular masses This range is dependent on the size of the pores of packing material and pore size distribution Column manufac-turers usually provide the standard calibration curves of polystyrene standards

in THF for each column, as shown in Figure 6-6 [8]

One of the most important requirements for the GPC column is the absence

of the specific interactions with the studied polymer The more inert the surface of the packing material, the better Early applications of GPC separa-tion were sometimes performed on porous glass particles with controlled porosity [9] The ease of the manufacturing of the controlled porosity par-ticles had determined this choice, but it was not always possible to find an

eluent that would suppress interactions of the polymer with the glass surface Modern GPC columns are primarily made of styrene–divinylbenzene copolymer Increased technological advances allow preparation of the rigid porous particles with relatively narrow pore size distribution from this copolymer

The presence of the aromatic rings in the body of the packing material is prone to π–π-type interactions, which could be a problem for the separation

of polymers with significant aromaticity, like polyimids

Traditional reversed-phase columns appear to offer the most inert surface since the alkyl-type bonded ligands at high bonding density can only partici-pate in weak dispersive interactions, which could be suppressed by practically any organic solvent An example of the calibration curve made on a commer-cial reversed-phase column is shown in Figure 6-7

For the analysis of water-soluble polymers (such as surfactants, oligosac-charides, PEGs, lignosulfonates, polyacrylates, polysacoligosac-charides, PVA, cellulose derivatives, PEO, polyacrylic acids, polyacrylamides, hyaluronic acids, CMC, starches, gums) and for separations of oligomers and small molecules, columns that are comprised of macroporous material with hydrophilic functionalities may be used The requirement for these columns in SEC mode is to eliminate

or minimize ionic and hydrophobic effects that make aqueous SEC (otherwise known as GFC) very demanding The interaction of analytes with neutral, ionic, and hydrophobic moieties must be suppressed It is often necessary to modify the eluent (addition of salt) in order to avoid sample-to-sample and sample-to-column interactions that can result in poor aqueous SEC separa-tions and low recoveries

Figure 6-6 Typical calibration curves for a set of SEC columns (Reprinted from

reference 8, with permission.)