Chromatography, size-exclusion

Pore Size (A) Globular Molecules (Molecular Weight) Linear Molecules (Molecular Weight) 

The matrices used in SEC are either polymeric or silica-based particles with a hydrophilic coating. The disadvantage of silica particles is that they tend to retain solutes by adsorption and may catalyze the degradation of solute molecules. To reduce adsorption, the surfaces of these particles are often modified by reaction with organic substituents. 

Size exclusion chromatography (SEC), also called gel permeation chromatography (GPC) or gel filtration chromatography (GFC), is a technique for separating dissolved species on the basis of their size. 

At Los Alamos National Laboratory in New Mexico the Analytical Chemistry Group (C-AAC) supports the Pu-238 Heat Source Project that fabricates heat sources for use in the space industry. These heat sources have been used on NASA s deep-space probes and on instruments exploring the surface of Mars. The chemical and isotopic purity of the heat sources are critically controlled to ensure dependable service. The Radiochemistry Task Area performs analyses of the heat source material for four radioisotopes americium-241, plutonium-238, neptunium-237, and uranium-235. 

All of the work is performed in a negative-pressure glove box or open-front hood in a radiological control area. Proper personal protective equipment (PPE) is used, including a lab coat, booties, safety glasses, disposable sleeves, and gloves. 

Lisa Townsend, a technician in the Radiochemistry section of the Actinide Analytical Chemistry Group, analyzes bulk components and impurities in plutonium-238 materials used to fabricate heat sources used in space exploration. She utilizes a combination of ion exchange and solvent extraction techniques and determines component concentrations using alpha and gamma radio-counting instrumentation. 

FIGUREll.il An illustration of size exclusion chro matography. 

Size-exclusion chromatography (SEC) separates polymer molecules and biomolecules 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  

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

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 molecular weight and molecular 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 

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

Size-exclusion chromatography is basically different from all other chromatographic methods in that a simple molecule size classification process rather than any interaction phenomena forms the basis of separation. 

A porous material is used as the column packing. The only space available to sample molecules that are too large to diffuse into the pores is that between the individual stationary phase particles hence they become excluded. The column then appears to be filled with massive impenetrable beads and, as no attractive forces are apparent, i.e. there is no interaction between beads and sample molecules, the latter are transported by the mobile phase through the column within the shortest possible time. 

However, if molecules that are small enough to penetrate all the pores are present, then the whole of the mobile phase volume becomes available to them. As the mobile phase stagnates in the pores, diffusion is the only way in which molecules can escape and as a result they move more slowly than the excluded molecules. They are the last to appear in the detector, namely at the breakthrough time known from all the other column chromatographic methods. The solvent itself does not interact with the stationary phase but passes through all the pores, and solvent molecules are the first to appear in the detector in all other modes of 

Practical High-Performance Liquid Chromatography, Fifth edition Veronika R. Meyer 

No pore volume accessible Available pore volume is a 

Size-exclusion chromatography (SEC) differs from the other methods in that the separation is based on physical sieving processes and not on chemical phenomena. The stationary phase is chemically inert and there is selective diffusion of solute molecules into and out of the mobile phase-filled pores in a three-dimensional network which may be a gel or a porous inorganic solid. The degree of retention is dependant on the size of the solvated solute molecule relative to the size of the pore. Smaller molecules will permeate the smaller pores, intermediate-sized molecules will permeate some pores and 

Size exclusion chromatography (SEC) is a method by which molecules can be separated according to their size in solution, thus relating indirectly to their molecular masses. To achieve this, stationary phases contain pores through which compounds are able to diffuse to a certain extent. Although the efficiency of separation can never attain that observed with HPLC, SEC has become an irreplaceable tool to separate natural macromolecules in order to study the distribution of synthetic polymer masses. Though the separation of compounds according to their sizes is not the most efficient process for small and medium molecules, this approach remains very useful in industry where the products are most often mixtures of compounds of very different masses. The instrumentation is comparable to that used in HPLC. 

Size exclusion chromatography (SEC), though an established technique for the separation of macromolecules using open column systems, met with limited success when applied to modern LC, as many of the commercially available packings did not meet the constraints and instrumental demands 

The principles causing retention behaviour, separation variables, molecular weight calibration and associated terminology such as interparticle and intraparticle volume, selective permeation, fractionation range and molecular hydrodynamic radius are as for open column size exclusion (Chapter 4). 

Medium-sized molecules can only utilize part of the available pore volume. A small coiled molecule with a statistical mean radius of r can remain in a larger pore volume than a larger molecule with a mean radius V2. The available pore volume is represented by the narrowly shaded area in Fig. 15.1. The 

Practical High-Performance Liquid Chromatography, Fourth edition Veronika R. Meyer 2004 John Wiley Sons, Ltd ISBN 0-470-09377-3 (Hardback) 0-470-09378-1 (Paperback) 

Size-exclusion chromatography, also called by its older designation gel-permeation chromatography, permits the separation of different sizes of molecules by retarding the smaller ones longer than the larger ones while passing over a specially prepared 

Size exclusion chromatography (SEC) separates macromolecules according to their hydrodynamic volume and has become the dominant method for the determination of molecular weight or mass distribution 

Crystalline polyolefins as polypropylene (PP) with high tacticity and copolymers of propylene with ethylene and/or higher a-olefins containing sufficiently long isotactic PP blocks can only be dissolved under conditions (solvent/temperature) which cause complete melting of the crystalline domains. Therefore, SEC of PP and crystalline copolymers of propylene must be carried out at elevated temperatures which requires the special equipment of high-temperature SEC (HT-SEC) which is commercially available from several sources, e.g. Millipore-Waters Corp. (Milford, MA, USA) and Polymer Laboratories Ltd (Church Stretton, Shropshire, UK). 

HT-SEC of PP is usually carried out at a temperature ranging from 135°C up to 150 C. This temperature must be maintained in the SEC apparatus from the point of injection of the sample solution throughout the chromatographic columns and the concentration detector or the multidetection system at the end of the SEC line. 1,2,4-trichlorobenzene (TCB) is preferably used as solvent and eluent. The concentration of the sample solution is usually in the range of 0.1-0.3 g/1. SEC columns based on 

As PP is sensitive to thermooxidative degradation, an antioxidant such as 2,6-di-tert.butyl-4-methylphenol is usually added to the solvent in a concentration of about 0.5 g/1. The complete dissolution of PP pellets usually requires at 150°C about 4h, and at 170°C about 1 h, respectively, and should be carried out in a nitrogen atmosphere with occasional stirring too intensive stirring may cause molecular degradation of the high molar mass fraction. 

In the case of propylene homopolymer, the universal calibration procedure established by Grubisic et al. [2] can be applied using anion-ically polymerized polystyrene molar mass standards with narrow MMD for TCB at 135°C, the following values of the parameters K and a in the equation  

In this approach, the size of the protein is taken into consideration. The size of the protein depends on the number of amino acids it contains. This property can be used in protein purification. The column material consists of a porous matrix for proteins to diffuse Into (Fig. A.3). The smaller proteins get entangled Inside the porous material and hence their mobility Is restricted. In contrast, the larger proteins do not get entangled and could just pass through. Hence, In the elution profile, the larger molecules would be the first ones to elute, while the smallest ones will be last to elute. 

Handbook of Size Exclusion Chromatography, Academic Press, New York, NY (1999). 

Mori and H.G. Barth, Size Exclusion Chromatography, Springer-Verlag, Berlin (1999). 

In older references SEC is sometimes referred to as gel permeation chromatography. Applications of SEC are many and include  

A linear detection response to amoimts of water derived fulvic acids separated on the gas chromatography 100 column was obtained enabling direct measurement of amounts of fulvic acid in water samples. The detection limit was 40ng for copper EDTA and 12pg of fulvic acid. 

Gardner ef al. [9] recognised that because component separations on size exclusion columns with distilled water are affected by chemical physical interactions as well as component molecular size, distilled water size exclusion chromatography will also fractionate dissolved metal forms. These workers interfaced distilled water size exclusion chromatography with inductively coupled argon plasma detection to fractionate and detect dissolved forms of calcium and magnesium in lake and river waters. 

1 Object and principle of size-exclusion chromatography, nature of the gel 

The chromatography which interests us in this book is a liquid state chromatography applied to polymer solutions. In practice, the available sample is polydisperse, and the question is the separation of polymers with different masses. The aim of this separation is either an analysis of the polydispersion of the sample, or a fractionation of the sample for further applications. In the first case, the required equipment is much lighter than in the second one. 

The principle of the method is as follows the polymer sample, dissolved in a proper solvent, is injected at time zero at the entrance of a column which, in general, is metallic and which is filled with a porous gel then it is swept along the column by an eluent, i.e. pure solvent, which an adequate applied pressure forces to circulate through the column (see Fig. 1.13). The experiment shows that polymers with higher masses come out first. 

After a time t, a certain amount of liquid has flowed through the column this volume of liquid is measured and it is called the elution volume. Simultaneously, the instantaneous mass concentration of the solution issuing from the column is continuously measured. This measurement is made either by differential refraction or by light absorption. 

If a polymer having a well-defined molecular mass is injected at the entrance of the column, a certain elution volume flows out and then a sharp signal indicates the passage of the polymer, provided that the presence of polymer in the solvent modifies the optical properties of the solution in an appreciable way. Thus, elution volume and molecular mass correspond to each other (see Figs 1.14 and 1.15) 

At present the resolution of peptides and proteins smaller than 10 kDa using SEC is not adequate and this mode of chromatography is 

Another appUcation is in the recovery of peptides after chemical modification, e.g. to remove the alkylating agent after alkylation (Fig. 11.2.5). Accurate molecular weight determination, even with a narrow range of standards, is not reliable unless carried out in the presence of strong denaturants. 

The entire molar mass distribution, including the higher-order average molar masses, such as A/, etc. can be measured by either size- 

As an analogy describing the SEC process, consider an art museum with an entry on one side and an exit on the other side of the building. On a 

For strictly linear chains, universal calibration is extremely useful because the Mark-Houwink coefficients have been tabulated for all common linear polymers. The calibration curve allows [q M to be deter-mined from the elution volume. The Mark-Houwink equation [Eq. (1.100)] then allows the SEC measure of [r]]M to determine the molar mass of the polymer  

In practice, SEC columns are often calibrated using linear monodisperse polystyrene standards, generating a calibration curve like Fig. 1.26. Then any linear polymer that is soluble in the same solvent, for which a Mark-Houwink equation is known, can have its molar mass determined by this calibrated SEC experiment. The elution volume of the polymer determines [q M from the calibration curve and the Mark-Houwink 

This is an effective and relatively simple method for characterizing silica sols and other colloids [75]. It has also been used to determine the particle size distributions of polymer lattices [76,77]. Separations are performed in a column packed with particles having pores substantially of the same size. A carrier liquid is passed through the column as a mixture of colloidal particles passes through the bed, the larger ones exit first since they are too large to sample the pore volume. Intermediate sized colloids enter the pores and are retained according to the volume that can be 

A variety of materials are used as stationary phases for size-exclusion chromatography, including cross-linked dex-tran, polyacrylamide, agarose, polystyrene-divinylbenzene, porous glass, and combinations of the above. Beads of these materials are porous with pore sizes that allow small molecules to be temporarily entrapped. Molecules too large to enter the pores remain entirely in the mobile phase and are rapidly eluted from the column. Molecules that are intermediate in size have access to various fractions of the pore volume and elute between the large and small molecules according to the relation  

Vr(A) - retention volume for solute A Vr(B) = retention volume for solute B w(A) bandwidth (units of volume) measured at base for solute A 

Resolution also is expressed in terms of time, with V lA) and V,.(B) being replaced with retention times t A) and and w(A) and w(B) being expressed in units of time. 

In this variation of high performance liquid chromatography (HPLC), the columns are packed with material that will hold back molecules dependent on the molecular size. Using the correct solvent system and column types, the molecular weight and distribution of the polyols can be determined. Standard samples are needed to calibrate the system. The molecular weight distribution of prepolymers can also be determined. This will enable an estimation to be made of the number of soft segment chains there are. 

Arnold, A.C., and I.M. Hutchings. The Mechanisms of Erosion of Unfilled Elastomers by Solid Particle Impact. Wear 138 (1990) 33-46. 

Blickensderfer, R., J.H. Tylczak, and B.W. Madsen. Lab Wear Testing Capabilities of the Bureau of Mines. Bureau of Mines information circular 9001, U.S. Dept, of the Interior, 1987. 

Names such as gel filtration chromatography (mobile phase is water), used by biochemists, and gel permeation chromatography (mobile phase is an organic solvent), used by polymer chemists, describe this technique. Size exclusion chromatography, however, is the recommended term. Molecular weight distribution of polymers can be obtained by this technique, and proteins, enzymes, peptides, nucleic acids, hormones, polysaccharides, and so on can be separated. 

Sephadex is a popular molecular-sieve material for the separation of proteins. It is a polymeric carbohydrate material that, because of hydroxyl groups along the polymer chain, is fairly polar and so will adsorb water. The amount of crosslinking 

Sephadex Type Fractionation Range for Peptides and Globular Proteins (MW) Bio-Gel Type Fractionation Range (MW) 

Bio-Gel is a more chemically inert, series of molecular-sieve gels, consisting of polyacrylamides. These are insoluble in water and common organic solvents and can be used in the pH range of 2 to 11. The inert gel decreases the possibility of adsorption of polar substances adsorption can be a variable with Sephadex, causing changes in the chromatographic behavior of these substances. Table 21.1 lists the different Bio-Gel preparations and their separation properties. 

Styragel is a polystyrene gel that is useful for purely nonaqueous separations in methylene chloride, toluene, trichlorobenzene, tetrahydrofuran, cresol, dimethyl-sulfoxide, and so on. It cannot be used with water, acetone, or alcohols. Gels of this can be prepared with exclusion limits for molecular weights of from 1600 to 40 million. 

5 Human Insulin For proteins of higher molecular weight 

The size-exclusion chromatography (or gel-chromatography) is a means of separation which is exclusively dependent on the exchange of solute molecules between the solvent of the mobile-phase and the same solvent within the pores of the column-packing material. In reality, it is the pore-size-range of the packing material that solely determines the molecular-size-range within which a particular separation can take place effectively. 

The timely adoption of the cross-linked dextran gels (i.e., Sephadex) in late-fifties as a packing material for column chromatography opened an altogether new horizon of chromatographic separation whereby substances, in general, undergo separation more or less as per their molecular size. 

In actual practice, the inert gels of dextran (I)-a polyglucose or other types of polymers, for instance agarose and polyacrylamides, wherein the macromolecules invariably are cross-linked to afford a reasonably porous 3D-structure, served as the stationary phases in size-exclusion chromatography. 

Sephadex Grade Water Regain (g/g) Mol.Wt. Bed Vol. (ml g1) Swelling Time (h) 

1 Preparation of [ED A]-dendri-PAMAM -(C02H)m Shell Reagents 

Amine-terminated, G3 (PAMAM) dendrimer, (0.316 g 45.7 moles) was dissolved in anhydrous methyl sulfoxide (5 ml) in a 100 ml round-bottom flask flushed with dry nitrogen. After dendrimer had completely dissolved, succinic anhydride (Aldrich) (0.363 g 3.6 mmol) was added to the reaction mixture with vigorous stirring, and the mixture was allowed to react for 24 h at room temperature. The product solution was diluted with deionized water, transferred to 3500 MWCO dialysis tubing (Spectrum) and dialyzed against deionized water (18 Mil) for 3 d. The retentate solution was clarified by filtration through Whatman No. 1 filter paper, concentrated with a rotary evaporator, and lyophilized to yield a colorless powder (0.435 g, 94%). The product was analyzed by 13C-NMR, FT-IR, SEC and MALDI-MS. The analytical data were consistent with the expected carboxylic acid-terminated product. 

Let us examine a silica-based cationic (sulfonate) ion exchange separation (Fig. 4.9). The column is equilibrated in 50mM sodium acetate. An injection of amines and an alcohol in the mobile phase is made. The same mobile phase, or one containing increased amounts of sodium acetate, is used to elute fractions. 

The alcohol will come off in the void volume of the column since it has no attraction to the column. The amines will be retained, because at the pH of the acetate solution they are protonated and have a positive charge. As more mobile phase passes the through the column, its sodium ions begin to compete for the sulfonate sites with the bound amines. Through a mass effect, the amines are displaced down the column until, finally, they elute into the detector. The amine that has the strongest charge and binds the tightest is eluted last. 

The first commercial HPLC system was sold to do gel permeation (GPC) or size separation chromatography. It is the simplest type of chromatography, theoretically involving a pure mechanical separation based on molecular size. 

The column packing material surface is visualized as beads containing tapered pits or pores. As the mobile phase sweeps the injection passed these pits, the dissolved compounds penetrate, if their largest diameter (Stokes radius) is small enough to fit (Fig. 4.10). If not, they wash down the column with the injection front and elute as a peak at the column void volume, which is called the exclusion volume. 

The first fraction of solute emerging from a gel column is eluted in v() that contains molecules too large to enter the gel micropores. v0 is the macroscopic pore space in the gel bed, not otherwise participate in the sieving mechanism. For a given column, v0 is constant. The solute interacting with the liquid stationary phase on the column surface elutes in order of the magnitude of a fraction s partition coefficient (Kp) between the elution volume (vel) and the volume of stationary solvent in the micropores (vs), fixed at 100 mL, because of difficulty in its measurement (Bio-Rad, 1971). At Ts = 100 mL, 

It is self-evident from Eq. (7.2) that the smallest sizes possess the largest Kp, because they are the last to emerge from the column (largest vel). Kp is relatively large also, if there is bonding between the solid support and the eluting solute, for the obvious reason that bonding extends the retention time and hence vel. Kp is variable with temperature. 

The difficulties presented by log Kp vs log of standard M are obviated by alternatively plotting log[r ]M vs log wel. For each fraction of a polysaccharide homologous series, log[r ]M vs log we, is superimposable on a so-called universal calibration curve (Grubisic et al., 1967) from which an unknown M2 may be estimated, after substitution in the equation 

Size exclusion chromatography did not differentiate lower molecular weight, extended coils from higher molecular weight, compact coils (Berth, 1988). 

For a polymer in dilute solution we have seen that / 2 0-5 is proportional to M°Ja, where a is the chain expansion factor (see Chapter 11 note that previously we related R2 °-S to the number of segments, but this is obviously equal to the molecular weight of the chain, M, divided by the molecular weight of segment M.  

Accordingly, if we assume that Vh is proportional to V, then the intrinsic viscosity should be related to the molecular weight by Equation 12-55  

We know that for a good solvent a is proportional to Af01, while in a 0 solvent a = 1, so this gives us our final result (Equation 12-56) where a varies between 0.5 and 0.8. 

This model is obviously flawed, we would not expect the solvent molecules within the volume defined by a polymer coil to move with exactly the same velocity as this coil, for example. But, in spite of its simplicity it gives a surprisingly good representation of the data. Also, it makes plain why the constants K and a vary with the polymer/solvent system and also the temperature. Finally, it is also a concept that is important in understanding SEC or GPC measurements, the final topic in our discussion of molecular weight determination. 

When we talk about chromatography we are referring to a wide range of techniques that are used to separate the components of a mixture. The field has its origin in the work 

If the sample is a single polymer, the chromatogram represents the MW distribntion. This method is very valuable for determining the MW distributions of polymers up to very high MWs. The actual calculation is nsnaUy based on a comparison with a standard polymer material 

Schematic representation of GFC Molecules of different size in the frame are separated according to size during migration through (b) the gel-filtration matrix as shown in the middle and right frames. 

Care must be taken in interpreting retention data to estimate MWs since the separation is based on the size of the molecule rather than on the actual MW. This means that the shape of molecule has a significant effect on the results. For example, two molecules of the same MW, one straight chained and the other highly branched, will be retained somewhat differently because each has a different penetrating power into the column particle pores. This must be taken into account when selecting standards for calibrating the MWs. 

An advantage of SEC is that it can be carried out at room temperature and the samples are not decomposed because of exposure to high temperature. This is especially important when dealing 

If multiangle light scattering detection is used with SEC, a distribution of Rq values can be obtained (Striegel et al. 2009). SEC is a powerful technique to characterize HS and has been applied by a number of researchers (Baigorri et al. 2007 Asakawa et al. 2008 Maia, Piccolo, and Mangrich 2008 Trubetskaya et al. 2008). 

To determine the number MWDs of the samples, the obtained dry polymers were dissolved in stabilized tetrahydrofuran (THF, Biosolve, A.R.) at 1 w/v% and filtrated using 0.2 pm filters. SEC analyses were carried out on a Waters 2690 Separation Module, using 4 PL-gel mix C columns (300 x 7.5 mm. Polymer Laboratories) at 40°C with THF as eluent at a flowrate of 1.0 mL min. A Waters 410 differential refractometer was used for detection. Calibration was performed using narrow-distribution polystyrene standards (Polymer Laboratories) ranging from 370 to 7.5 10 g moF. All SEC analyses were performed in duplicate. Only those traces for which comparable duplicate analyses were obtained, and the chromatogram baseline before and after elution of the polymer exhibited no non-linear variations were used for data analyses. Some discussion on the quality of the SEC set-up can 

SEC chromatograms were converted into number MWDs according to the definitions by Shortt [6]. Universal calibration based on the Mark-Houwink equation [7, 8], was used to determine the MWDs of the produced polymers. 

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Wei G T, Liu F K and Wang C R C 1999 Shape separation of nanometre gold particles by size-exclusion chromatography Anal. Chem. in press... 

Two classes of micron-sized stationary phases have been encountered in this section silica particles and cross-linked polymer resin beads. Both materials are porous, with pore sizes ranging from approximately 50 to 4000 A for silica particles and from 50 to 1,000,000 A for divinylbenzene cross-linked polystyrene resins. In size-exclusion chromatography, also called molecular-exclusion or gel-permeation chromatography, separation is based on the solute s ability to enter into the pores of the column packing. Smaller solutes spend proportionally more time within the pores and, consequently, take longer to elute from the column. 

In size-exclusion chromatography, the smallest solute that can be separated from other solutes all smaller solutes elute together. 

Examples of the application of size-exclusion chromatography to the analysis of proteins. The separation in (a) uses a single column that in (b) uses three columns, providing a wider range of size selectivity. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

Calibration curve for the determination of formula weight by size-exclusion chromatography. 

Size-exclusion chromatography can be carried out using conventional HPLC instrumentation, replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is the most common means for obtaining the chromatogram. 

A series of polyvinylpyridine standards of different molecular weight were analyzed by size-exclusion chromatography, yielding the following results. 

SI units stands for Systeme International d Unites. These are the internationally agreed on units for measurements, (p. 12) size-exclusion chromatography a separation method in which a mixture passes through a bed of porous particles, with smaller particles taking longer to pass through the bed due to their ability to move into the porous structure, (p. 206)... 

Fig. 3. Overview of puriftcation sequence for the nonrecombinant tissue plasminogen activator (t-PA) which also contains urokinase plasminogen activator (u-PA). Serum-free culture conditional media is from normal human ceU line. The temperature for aU. steps, except for size-exclusion chromatography...

Column Si. Size-exclusion chromatography columns are generally the largest column on a process scale. Separation is based strictly on diffusion rates of the molecules inside the gel particles. No proteins or other solutes are adsorbed or otherwise retained owing to adsorption, thus, significant dilution of the sample of volume can occur, particularly for small sample volumes. The volumetric capacity of this type of chromatography is determined by the concentration of the proteins for a given volume of the feed placed on the column. 

The total stationary-phase volume required to process a given feed stream is proportional to the inlet concentration and volume of the feed. For example, for a typical inlet concentration of protein of 10 g/L, in a 100 L volume of feed, a column volume of at least 100 L is needed for size-exclusion chromatography. In comparison, an ion-exchange column having an adsorption capacity of 50 g/L would only require 20 L of column volume for the same feed. 

This reversed-phase chromatography method was successfully used in a production-scale system to purify recombinant insulin. The insulin purified by reversed-phase chromatography has a biological potency equal to that obtained from a conventional system employing ion-exchange and size-exclusion chromatographies (14). The reversed-phase separation was, however, followed by a size-exclusion step to remove the acetonitrile eluent from the final product (12,14). 

The molecular weight of SAN can be easily determined by either intrinsic viscosity or size-exclusion chromatography (sec). Relationships for both multipoint and single point viscosity methods are available (18,19). Two intrinsic viscosity and molecular weight relationships for azeotropic copolymers have been given (20,21) ... 

Poly(ethylene oxide). The synthesis and subsequent hydrolysis and condensation of alkoxysilane-terniinated macromonomers have been studied (39,40). Using Si-nmr and size-exclusion chromatography (sec) the evolution of the siUcate stmctures on the alkoxysilane-terniinated poly(ethylene oxide) (PEO) macromonomers of controlled functionahty was observed. Also, the effect of vitrification upon the network cross-link density of the developing inorganic—organic hybrid using percolation and mean-field theory was considered. 

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