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CHROMATOGRAPHY, HPLC
Introduction
High performance liquid chromatography (hplc) is a powerful tool for characterization of synthetic and natural polymers by separating individual fractions by molecular weight, chemical composition, functional groups, etc. It is also used for isolation and purication of biopolymers and analysis of additives in complex polymer formulations. As in any chromatographic technique, separation occurs due to thermodynamic partitioning between the sample components in the mobile and stationary phases. In hplc, this process takes place in solution inside a chromatographic column packed with inorganic (usually, silica-based) or organic (synthetic resin, such as polystyrenedivinyl benzene and acrylic-based) porous particles, capable of resisting the high pressure created by a moving liquid (mobile phase) mechanically pumped through the column. Depending on application, isocratic (constant mobile phase composition) or gradient (variable composition) modes of separation can be employed, which signicantly extends the capabilities of the technique. Column liquid chromatography has been used for more than 60 years for analytical and preparative separations of polymers. BakerWilliams Fractionation, introduced in the mid-1950s, employed both temperature and solvent gradients to separate polymer fractions according to solubility through multiple precipitation redissolution steps. This technique has been signicantly improved in the last two decades and is still used for fractionation of synthetic copolymers and polymer blends under the term high performance precipitation liquid chromatography (hpplc). Dramatic changes in instrumentation and column preparation for liquid chromatography in 1970s, as well as better understanding of the mechanisms of separation, had a dramatic impact on polymer separation approaches.
Encyclopedia of Polymer Science and Technology. Copyright John Wiley & Sons, Inc. All rights reserved.

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Thus, a new technique Chromatography, SEC (a.k.a. gel-permeation chromatography for organic solutions or gel-ltration chromatography for aqueous mobile phases) emerged at that time as a premier method for molecular weight distribution (MWD) determination of synthetic and natural polymers. Since the early 1970s, sec as well as traditional types of hplc (reversed-phase, ion-exchange, hydrophobic-interaction, and afnity chromatography) have been widely used for analysis of proteins and other biopolymers. For two decades, normal- and reversedphase chromatography have attracted signicant interest as methods of separation of synthetic copolymers and polymer blends by chemical composition. Twodimensional hplc techniques (also called chromatographic cross-fractionation) have been used for most complete polymer characterization in recent years. Thus, consequently coupling two different modes of chromatographic separation can result in determination of both molecular weight and chemical composition distributions of synthetic copolymers.

Instrumentation for HPLC

Each contemporary hplc instrument consists of a pump for solvent delivery, an injector device for sample introduction, column(s) for sample separation, detector(s) for visualization of the separated components (solutes), and a computer for system control and data acquisition and reduction, as depicted in Figure 1. Precise temperature control of columns and some other parts of a chromatographic system becomes an important prerequisite for a successful separation. Solvent Delivery. The function of the solvent delivery system is to deliver the mobile phase (eluent) through the chromatograph, accurately and reproducibly. The most popular are reciprocating-piston pumps, usually with two

Eluent Sample A+B

Pump Injector

Column

Detector Detector Signal B A Computer

Time (Volume)

Fig. 1. Schematic presentation of separation of two components, A and B. Reprinted from Ref. 1 with permission.

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pump heads containing two sets of moving parts: the check valves and seal-piston assembly. For analytical chromatography, such pumps can deliver liquid at a ow rate from 100 L/min up to 10 mL/min. Microscale capillary chromatography needs syringe (positive displacement) pumps with capability to produce a ow rate as low as 1 L/min, while preparative chromatography utilizes ow rates up to 50 mL/min. Gradient pumping system is capable of delivering more than one solvent during an analysis with variable mobile phase composition. The blending of the solvents can occur in one of two ways: high pressure mixing or low pressure mixing. In the former case the solvents are mixing on the injector side, while in the latter case the mixing takes place at atmospheric pressure and a single high pressure solvent delivery system is used to pump the mixture. Accurate and reproducible eluent ow rate and composition are important prerequisites for successful separation and detection. This is achieved in contemporary intelligent pumps through completely independent, digitally controlled piston drives. The sophisticated, software-driven mechanism produces pulse-free eluent delivery, compensates for changes in eluent viscosity, and automatically purges any gaseous mobile phase, thereby enhancing performance for both isocratic and gradient applications (2). The quality of solvents affects the accuracy of ow and baseline noise. On-line solvent ltering and degassing become the necessary elements of any commercial hplc system. Sample Introduction. To minimize the dispersion and broadening of peaks, sample solution must be injected as a sharp plug with minimal interruption of ow. Typically, two-position six-port valves are used for sample injection, which may be operated manually or automatically (such as in an autosampler, where the sample is introduced from a sample vial held in an injection station). While the general intention is to keep the injection volume as small as possible, the limits are imposed by column dimensions, the sensitivity of detectors, and the nature of separation. An additional factor is the concentration of the analyte, which for high molecular weight species may often be selected very low to reduce solution viscosity and avoid intermolecular interaction. Stationary and Mobile Phases. Chromatographic columns provide the means for polymer fractionation or separating mixtures into components. The selectivity, capacity, and efciency of the columns are all affected by the nature of the packing material. Most modern hplc packings are porous spherical microparticles with 3- to 10-m size. The efciency of a column increases with decreasing particle size, but so does the backpressure, which usually cannot exceed 27.2 MPa (4000 psi) on traditional instruments. The use of new generation of ultra-high pressure chromatographs with backpressure up to 690 MPa (100,000 psi) can potentially reduce the particle size up to 1 m or lower. In general, silica-based columns can withstand higher pressure as compared with resin-based columns. Pores (mean diameter usually from 6 to 100 nm) play a crucial role in hplc, as they provide the surface where retention and hence separation occur. The chemical structure of the surface determines the thermodynamic environment for the analyte in thin surface layer (stationary phase) and in such a way determines the mechanism of retention. Flexible macromolecules approaching the internal surface of a solid particle change their spatial conformations because of steric interaction, which plays an important role in any mode of polymer chromatography. This interaction restricts the uctuation motion of a macromolecule, decreases its

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conformation entropy, and effectively reduces the retention. In sec, where particles with inert surface are used and the size of macromolecules is comparable with the internal pore diameter, steric interaction is dominant and separation occurs according to size of macromolecules. All other modes of polymer chromatography also exploit enthalpic (attraction) specic interactions between macromolecules and the functional groups on the surface. For this reason they are often lumped together as interaction polymer chromatography (ipc). The name adsorption polymer chromatography is also used because the size of macromolecules usually far exceeds the size of the surface functional groups participating in interaction, and retention has the adsorption mechanism. The nonsteric interactions in ipc depend on the chemical structure of the analyte, and also on nature of stationary and mobile phases. In normal- or reversedphase hplc, neutral solutes are separated on the basis of their polarity. In the former case, polar stationary phases are employed (eg, bare silica with polar silanol groups) and less polar mobile phases based on nonpolar hydrocarbons are used for elution of the analytes. Solvent selectivity is controlled by adding a small amount of a more polar solvent, such as 2-propanol or acetonitrile or other additives with large dipole moments (methylene chloride and 1,2-dichloroethane), proton donors (chloroform, ethyl acetate, and water), or proton acceptors (alcohols, ethers, and amines). Correspondingly, the more polar the solute, the greater is its retention on the column, yet increasing the polarity of the mobile phase results in decreased solute retention. The opposite situation takes place for reversed-phase separation, when nonpolar stationary phase is accompanied by a polar mobile phase, typically water, to which a less polar solvent such as acetonitrile or methanol is added, and solvent selectivity is controlled by the nature and amount of the added solvent. As a result, a decrease in the polarity of the mobile phase leads to a decrease in solute retention. Many organic polymers are not soluble in water, and other polar solvents can be used. Thus, the combination of acetonitrile as an initial solvent with addition of tetrahydrofuran (THF) can be used in reversed-phase separation of many styrenebased copolymers and acrylic polymers. Modern reversed-phase chromatography typically refers to the use of chemically bonded stationary phases, where functional groups, such as alkyl ( CH3 , C4 H9 , C8 H17 , and C18 H37 ) groups, phenyl ( C6 H5 ) groups, cyano [ (CH2 )3 CN] groups, and amino [ (CH2 )3 NH2 )] groups, are bonded to the silica. Note that some of these bonded packings, such as those with cyano- and amino-groups, as well as diol groups [ (CH2 )3 OCH2 CH(OH)CH2 OH], can also be used in normal-phase separations, as they contain moderately polar moieties. The same stationary phases are also used for separation of biomolecules in hydrophobic-interaction chromatography. Weak nonionic interactions between hydrophobic portions of proteins and nonpolar moieties of the stationary phase have an entropic nature and are highly sensitive to salt concentration in a totally aqueous, buffered mobile phase of high ionic strength. In ion-exchange chromatography, the ionic species, such as proteins or synthetic polyelectrolytes, are separated on the basis of differences in electric charge. The stationary phases with ionic groups (xed ions) are employed, and the mechanism of retention is the electrostatic attraction of ionic solutes in solution to opposite charged ions (anions or cations) on the stationary phase support. The stationary phase for ion-exchange chromatography is called ion exchanger and

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is classied as an anion-exchange material when the xed ion carries a positive charge, and as a cation exchanger in the opposite case. There are strong and weak ion exchangers depending on the pH range over which the stationary phase can retain the charge on the xed ion. Acrylic-based copolymers treated with an appropriate reagent to produce the desired functional group are the most used ion exchanger because of wider pH range (pH 113) and higher ion-exchange capacity as compared with silica-based columns (pH 28). Eluents for ion-exchange chromatography are aqueous solutions of salts with small addition of organic solvent. Selectivity in the separation of polyelectrolytes may be varied by changing either the pH of the mobile phase or the nature or concentration of the displacing (competing) ions. Chromatography, SEC is used for the separation of biomolecules on the basis of the lock-and-key mechanism prevalent in biological systems. Detectors. When separated macromolecules leave the column, they are detected by one or more on-line electrical devices with signals proportional to the concentration of the analyte. In all hplc detectors, the eluent ows through a measuring cell, where the change of a physical or chemical property with elution time is detected. Each monitored trace represents a chromatogram: a set of chromatographic peaks separated by baseline regions. The sensitivity (the ratio of peak height to the sample concentration), signal-to-noise ratio (the amount of signal visible above the baseline noise), as well as linear dynamic range (maximum linear response divided by the detector noise) are the most important characteristics of any detector. Ultraviolet (uv) absorption photometers capable of absorbing light at a selected wavelength (in the range 190350 nm for uv detectors, or 190700 nm for uv/Visible detectors) are the most popular detectors in hplc of both synthetic and biological polymers. In case of proteins, wavelengths that are typically monitored include the regions of absorbance of peptide bonds (210220 nm), and tyrosine and tryptophan side chains (ca 280 nm). Many synthetic polymers also contain chromophoric groups. Variable-wavelength and photodiode array detectors have the advantage that more than one wavelength can be monitored during a single run. Photodiode array detectors allow for simultaneous measurement of an entire uv spectrum at any point of the chromatogram, which can aid in peak identication. Among other selective (solute property) detectors used in hplc of polymers are uorescence and IR photometers. For example, uorescence detectors can provide higher sensitivity, as well as greater selectivity, in the detection of proteins containing tyrosine and tryptophan residues that have intrinsic uorescence. Both types of detectors are limited to certain mobile phases. A good alternative is offline coupling of ftir to hplc system using an evaporative interface (3). In such a system, the eluent is sprayed onto a Germanium disc, which can be transferred to any ftir spectrometer to yield the full spectrum information over any peak of the chromatogram. The same principle is applied also to the matrix-assisted laser desorption/ionization mass spectrometer, which otherwise is difcult to couple to the hplc system (4). A few universal (bulk property) detectors are of frequent use in hplc of polymers. Refractive index detector is limited to isocratic elution and is utilized primarily in sec. The same is true for the recently introduced density detector based on mechanical oscillator principle (B. Trathnigg in Ref. 4). Evaporative light

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scattering detector (ELSD) becomes very popular in nonaqueous gradient elution of synthetic copolymers and polymer blends (3). The eluent is nebulized, and the solvent is evaporated from the droplets. Each droplet containing nonvolatile material will form a particle, which scatters the light while crossing a light beam. This detector has signicant advantages over the uv detector for ability to work with aliphatic polymers without chromophore groups, and also for its applicability to practically any mobile phase which does not contain a nonvolatile buffer. Despite these obvious merits, ELSD cannot be considered as an ultimate choice for quantitation because of nonlinear concentration response, which depends on numerous factors such as chemical nature of the sample including its molar mass and chemical composition, the eluent composition, viscosity and surface tension, and operating conditions. Coupling ELSD to another detector can help to solve these problems with quantitation. The quadrupole mass spectrometers, single-quadrupole (MS) or triplequadrupole (MSMS), have proved to be the detectors of choice for interfacing with chromatographs. The resulting combinations LCMS or LCMSMS have become very popular among protein scientists. Soft ionization methods, such as electrospray ionization and matrix-assisted laser desorption, are used extensively for biopolymers and some ionic synthetic polymers (5). The interface usually requires the use of very low ow rates (below 10 L/min) and therefore necessitates capillary chromatographic equipment, or a sample splitter. Miniaturization of separation with laboratory-on-a-chip technology and microuidics with nanoscale columns can additionally improve the compatibility of column chromatography with mass spectrometry detection. In addition to the widespread use of mass spectrometry as a detection technique, scientists report investigations of other spectroscopic techniques such as nuclear magnetic resonance (nmr) and inductively coupled plasma mass spectrometry. These emerging technologies in a few years may have a major impact as online detection methods for characterizing both synthetic and biological polymers. Miniaturization of separation makes these detectors more feasible for on-line coupling. Using internal reectance spectroscopy to study molecular interactions at phase surfaces has helped researchers to understand retention mechanisms and to conrm or expand chromatographic data. Data Treatment. The traditional hplc method involves two types of data analysis: qualitative (peak identication) and quantitative (determining the analyte concentration). Both analyses require specic data manipulations, such as matching retention times, peak integration and peak calibration. The use of spectroscopic detectors (photodiode array, ftir, and mass spectrometry) suggests additional mathematical procedures because of the necessity to manipulate with uv-, ftir-, and MS-spectra and corresponding library matching (6). Most commercial chromatographic software packages contain necessary functionalities and can be applied to hplc of proteins, peptides, and DNAs. The characterization of other natural and all synthetic polymers is a much more difcult problem, which includes calculating the distributions of different macromolecular parameters, such as molecular mass, chemical composition, branching frequency and end group functionality, from raw chromatograms. This involves additional calibration procedures (column calibration), calculation of statistical moments of distributions, etc. Two-dimensional chromatographic cross-fractionation of synthetic copolymers

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requires the construction of two-dimensional distributions to characterize the copolymer heterogeneity (7).

Retention Mechanism and Modes of Separation Isocratic Elution. Contrary to sec, where any nonsteric effects are undesirable and suppressed, week nonsteric adsorption interactions between a solute and a stationary phase play a dominant role in ipc. The best way to demonstrate the difference between size-exclusion and interaction (adsorption) modes of polymer separation is to use the so-called molecular weight calibration curves (log M vs V, where M is the molecular weight, and V is the elution volume) depicted in Figure 2. The set of narrow polydispersity polystyrene samples with molecular weights from several hundred up to million were subjected to isocratic elution on the reversed-phase column (silica bonded with octadecyl chains) with different acetonitrile/tetrahydrofuran (ACN/THF) mixtures as eluents. THF is less polar than ACN (polarity parameters 4.0 and 5.8, respectively). Pure THF completely suppresses nonpolar interactions between polystyrene and alkyl chains on silica, and conventional sec behavior is obtained, that is, elution time increases with decreasing molar mass of the sample. The separation occurs within pore volume V P = V T V 0 , where V T is the liquid volume inside the column and V 0 is the interstitial volume between particles. The steric interactions prevail until the amount of ACN in the mobile phase reaches ca. 50%. After that, a completely reversed retention behavior is obtained, which is typical for the adsorption mode of separation, when retention volume V R exceeds V T . At 45 and lower % THF, retention

6.0 5.5 5.0 log M 4.5 4.0 3.5 3.0 70 80 40 100 50 48 45

0.0

1.0

2.0

3.0

4.0

5.0

6.0

V0

VT V, mL

Fig. 2. Transition from size-exclusion to adsorption mode for isocratic elution of polystyrene samples with low polydispersity. Column: Nova-Pak C18 (Waters Corporation, USA), mobile phase: THF/ACN mixtures (numbers on the graph represent vol% THF), 1 mL/min, detector: ELSD Model 500 (Alltech Corp.).

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dramatically increases with molecular weight and becomes extremely sensitive to the eluent composition, so that high molecular weight fractions are practically irreversibly retained by the column. For this reason, the IPC separation of high molecular weight polymers is usually performed in gradient mode. It is important to note the elution at 48% THF: Retention becomes completely independent of molecular mass, and all fractions elute with the columns liquid volume V T . At this transition point, which is termed critical point of adsorption (CPA), entropy driven size-exclusion effects are completely compensated by enthalpic adsorption interactions. Although both types of interaction strongly depend on molecular weight, this mutual compensation phenomenon leads to molecular weight independent retention at CPA. This phenomenon allows for practical implementation of isocratic polymer separation other than sec. Thus, because of chromatographic invisibility of a main polymer chain, effective separation of telechelic polymers based on types of end-groups can be performed (Ref. 7, p. 125). The same principle is applied for isocratic separation of some block copolymers (Ref. 7, p. 151). The isocratic separation at CPA requires very careful control of chromatographic conditions, such as the quality of stationary phase, mobile phase composition, and temperature, because of high sensitivity of retention to these parameters, especially for polymers with high molecular weight. Another limitation of the techniques is associated with the dynamic effects. The success of separation depends on the ability of all macromolecules to penetrate all pores (the same way as the solvent molecules). But small diffusion coefcients and steric restrictions make the equilibration time for large macromolecules and narrow pores unreasonably long. Because of this, the use of isocratic separation at CPA is practicable only when the internal pore diameter exceeds the size of macromolecules in solution. Gradient Elution. In gradient mode, the elution strength of the mobile phase is gradually increased within the chromatographic run to facilitate the elution of strongly retained compounds. This is especially important for high molecular weight polymers that otherwise are practically irreversibly retained by the column in the adsorption mode. Such gradient can be performed by changing the chemical composition, ionic strength or pH of the eluent, or even temperature. In protein separation, the salt gradient is used in both hydrophobic-interaction and ion-exchange chromatographies. During the chromatographic run, the salt concentration is decreased in the former case and increased in the latter one. Typical reversed-phase gradient elution of synthetic polymers is demonstrated in Figure 3 for the same set of polystyrene samples as in Figure 2. The increase in eluent strength is achieved by linear increase of the concentration of less polar THF in the mobile phase that allows the elution of all polystyrenes within 8 min. The elution of lower molecular weight samples (below 10,000 Da) occurs in the adsorption mode at low content of THF in the mobile phase, and strongly depends on molecular weight. Excellent resolution for polystyrene oligomers in this mode allows one to distinguish between fractions with different end groups (double peaks on the chromatograms). In contrast, all higher molecular weight polystyrenes have practically the same elution volume, which coincides with the volume where eluent reaches its CPA (48% THF). The principle that gradient elution of high molecular weight polymers occurs at CPA has been introduced

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Styrene Oligomers 2,890 9,100

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50 40 30 20 10 0

43,900 190,000 355,000 706,000 1,090,000

THF, %

5 V, mL

Fig. 3. Gradient elution of polystyrene samples from Figure 1. The same chromatographic system and conditions as in Figure 1, except eluent gradient: 100% ACN to 100% THF over 10 min (dashed line). Numbers on the graph: molecular weights of individual polystyrenes. Broken line: critical point of adsorption (48% THF).

recently (8). It provides the basis for gradient separation of copolymers and polymer blends by chemical composition. Each compositionally homogeneous fraction of a copolymer or a polymer blend elutes at its own CPA independently of molecular weight. By this means, gradient elution produces the chemical composition distribution of copolymers and polymer blends. In general, gradient elution is a much more versatile technique for polymer separation as compared with isocratic elution at CPA Pore and particle sizes and hence dynamic effects do not impose such signicant restrictions. Macromolecules do not need to penetrate the entire pore volume, and elution at CPA is achieved when their size exceeds the pore diameter. Gradient run allows for a sample precipitation on the frit or inlet of the column, when starting eluent is a nonsolvent for the polymer. This is a rather common situation because the solubility window for many polymers is relatively narrow, and the eluent with a poor elution strength often has a poor thermodynamic quality. This is true even for some proteins (membrane proteins or collagen), which can precipitate at the intermediate stage of the gradient separation. However, the elution of different components of a polymer sample still follows the rules of gradient separation described in the previous paragraph as soon as all polymer fractions are redissolved and adsorbed by the column during the chromatographic run. Only if macromolecules elute without any adsorption interactions immediately after redissolution, as in hpplc, the separation based solely on polymer solubility occurs. In this case, retention always strongly depends on molecular weight and concentration of the sample (9). Multidimensional Separation. In some cases, a single separation step is not enough to complete the analysis of biological or synthetic polymers. In this case, coupling two or more different modes of separation involving column-switching mechanism is widely used in multidimensional polymer

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chromatography. This approach provides the opportunity to simplify complex mixtures before their characterization by spectroscopic techniques, especially LCMS and LCMSMS. A typical example is the combination of ion-exchange or sizeexclusion separation with reversed-phase chromatography in protein purication (10). Gradient or isocratic elution at CPA followed or preceded by size-exclusion separation is often used in chromatographic cross-fractionation of synthetic polymers (11). The use of ipc as a rst step is a more feasible way to achieve the true two-dimensional characterization of copolymers or polymers with functional groups, because it allows for molecular weight independent separation by chemical composition or by other structural differences in macromolecules. Size of macromolecules in each compositionally homogeneous fraction obtained after the rst separation, is directly related to molecular weight thereof, and the following sizeexclusion separation of all these fractions can complete the cross-fractionation. The coupling of two modes of polymer separation can be achieved off-line or on-line. In the former case fractions from one chromatographic system are transferred to another system manually, in the second one, automatically, when the eluent from the rst instrument ows through the injection valve of the second instrument. The most sophisticated automatic two-dimensional chromatographic systems employ two six-port valves or one eight- or even ten-port injection valve (10). Two storage loops make it possible to collect fractions continuously without losses. The proper coordination of the ow rates in consecutive chromatographic steps is an important feature of any automated system. Thus, in chromatographic cross-fractionation, the collection time of one hplc fraction should coincide with the analysis time in the following sec step (Ref. 7, p. 198).

Theory of Polymer Chromatography Thermodynamic Treatment. Each component of a polymer sample injected in the chromatographic system can be characterized by its retention volume V R , ie, the volume of solvent that passes through the column from the time of sample injection to the detection of the corresponding peak. To eliminate the variability of retention caused by operational variance of the ow rate, mobile phase, or other parameters, the normalized quantity k = (V R V T )/V T (capacity factor) is often used instead of V R . The resolution of the chromatographic system, ie, its ability to discriminate between individual macromolecules, depends on the selectivity of separation, which is described by the ratio of corresponding capacity factors. The basic assumption in any chromatographic theory is that retention is determined by the thermodynamic factors. In such a way, mobile and stationary phases are interpreted as true thermodynamic phases with volumes V M and V S , respectively, so that retention volume depends on the partition (distribution) equilibrium coefcient K of the solute in these two phases: V R = V M + KV S . By definition, all enthalpic and entropic interactions between the macromolecules and the chromatographic surface occur in the stationary phase. If the size of macromolecules in solution is comparable with the internal diameter of pores, the entire pore volume represents the stationary phase, V S = V P , yet the mobile phase is formed by the interstitial volume only, V M = V 0 . This is not always the case for

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gradient separation of peptide or proteins, when the pore diameter usually far exceeds the size of compact macromolecules, and practically the entire liquid volume, V T = V 0 + V P , represents the mobile phase. The role of thermodynamic theory is to relate the distribution coefcient K (or free energy change due to solute sorption on the stationary phase) to the parameters describing the congurational and conformational structure of a macromolecule (such as molecular weight, chemical composition and shape), as well as temperature, pore geometry, mobile phase composition, etc. These parameters determine the entropy of steric interaction and the energy of multipoint nonsteric interactions between the macromolecule and the surface, and in this way affect the retention. Construction of a universal thermodynamic model for chromatographic separation of proteins and other biological polymers is hardly probable because of the complexity of chemical and spatial structure of such macromolecules, which can undergo a diversity of conformational transitions during separation. Even for small peptides composed of 515 amino acids, which are assumed to exist in solution as a random coil, amino acid identity and position, as well as peptide sequence, all play an extremely important role in retention. Nevertheless, semiquantitative approaches applied to specic modes of separation often allow for understanding the mechanism of separation, as well as the effect of operating parameters such as salt type and concentration, temperature, nature of packing material, organic modiers, and pH. Examples are the solvophobic (12) and preferential interaction (13) theories applied to hydrophobic interaction and reversed-phase chromatography of proteins (14). Several useful retention models exist in ion-chromatography, where the equilibria, which govern ion-exchange separations, are generally well understood (1). In the case of synthetic polymers, a simple continuous random-ight model of a exible polymer chain allows for quantitative consideration of the chromatographic process in a rather general way (15). The theory provides a simple analytical expression for the distribution constant K of a macromolecule penetrating the slit-like or cylindrical pore and interacting with its walls, in terms of macromolecule and pore sizes, and a segment energy of nonsteric interactions between the macromolecule and stationary phase. Depending on the mobile phase composition, the competition between steric (repulsive) and nonsteric (attractive) interactions may produce either size-exclusion, K<1, or adsorption, K>1, modes of separations, yet the intermediate condition, K=1, represents the critical mode of adsorption, where both types of interaction counterbalance each other (see Fig. 2). Present models of gradient elution (16) assume that migration in gradient mode occurs in the same way as for isocratic elution. That is, given the dependence of K on eluent composition, which changes during the mobile phase gradient, one can calculate the gradient elution volume for gradients with various slopes and shapes using the well-known mass-balance integral equation (17). The results of such calculations applied to gradient separation of polymer homologous series can explain (8) the peculiarities depicted in Figure 3. Thus, the combination of molecularstatistical theory of dilute polymer solutions in conned (porous) media with conventional chromatographic theory produces a valuable tool in elucidating the polymer chromatography (8). Recent extension (18) of the concept

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on copolymers and stationary phases with heterogeneous surface (which actually is inherent in most if not all of available sorbents) signicantly improves the feasibility of the theory for separation of complex polymer formulations. Dynamic effects. Besides the thermodynamically driven selectivity, the resolution of the chromatographic system depends also on dynamic (kinetic) factors that determine the efciency of separation. These factors cause the broadening of the chromatographic peaks so that even peaks from individual compounds such as proteins have a nite width. For polydisperse polymers this chromatographic peak broadening (or bandspreading) is usually hidden by the overall polymer distribution, but still can produce erroneous results if no correction has been made to compensate for this effect. The major contributor to peak broadening in polymer chromatography is a stagnant mobile phase mass transfer, that is, kinetics hindrance due to penetration of macromolecules into the pores of the support packings. The molecular diffusion is the only way for macromolecules to reach the internal surface of pores, whereas the migration in interstitial volume is governed by the convection mechanism. The effect of bandspreading tends to diminish with the ratio of characteristic times of these two processes, tD = L2 /D (L is the average length of pores, D is a solute diffusion coefcient), and t0 = V 0 /F (F is the ow rate). The diffusion coefcient of macromolecules inside pores drops rapidly as molecular weight increases, and so does peak broadening. As the length of an individual pore is usually comparable with the size of a particle [exception is perfusive particles, which contain very broad ow-through pores with only shallow diffusive pores and are mostly used in preparative chromatography 19], the most popular solution to date for minimizing the problem in biological applications is the use of small porous particles of 25 m so that the depth of the stagnant pools is not excessive (20). It is signicant that in gradient separation of synthetic polymers the internal surface area is not so important, and separation usually takes place only at the inlet of very narrow pores, which signicantly reduces the dynamic effects.

Applications Separation of Biological Polymers. The development of wide-pore bonded-phase packings has dramatically affected the separation of biopolymers in the life sciences and biotechnology (21). Nevertheless, several factors should be taken into account in selecting the optimum mechanism of separation for a specic application. Reversed-phase chromatography is the most popular mode for separation of peptides because of exceptional selectivity and resolution. But in case of proteins, hydrophobic interaction is often the method of choice because of the much milder effect on solute conformation. Many proteins are denatured by acids or organic solvents, as well as by the alkyl chain of the bonded phase, the usual constituents of the reversed-phase separation (22). These conformational transitions may cause additional chromatographic problems, such as peak broadening, multiple peaks, poor sample recovery, and ghosting, if the conformational transition time is of the same order as the migration time (23). Hydrophobic interaction chromatography as well as ion-exchange chromatography have been

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predominantly used to analyze proteins, nucleic acids, and other biological macromolecules when maintenance of the three-dimensional structure is a primary concern (24). With the human genome now sequenced (primarily by capillary electrophoresis technology), scientists are turning their attention to proteomics. The term proteome has been introduced to dene the full complement of proteins in a cell. It also requires a description of the localization, concentration, and multisubunit association of each of these proteins (25). The multidimensional hplc with automated column switching technology can successfully complement the more conventional approaches to protein separation, such as two-dimensional gel electrophoresis, which usually involve many manual steps and are not conducive to high throughput applications. Separation of Synthetic Polymers. Interest in interaction polymer chromatography as a technique complementary to sec for characterization of synthetic polymers and polymer blends has quickened in recent years. The principle difference between these two methods is depicted in Figures 4 and 5, where three polymers with close average molecular weights, as well as the equimolar blend of these polymers were subjected to isocratic size-exclusion and gradient reversed-phase separations, respectively. In the rst case (Fig. 4), only molecular weight information is available from the chromatograms, the result of second separation (Fig. 5) reveals the individual components, ie, chemical composition of the blend. This way, many statistical, block and graft copolymers and blends were fractionated by chemical compositions in gradient mode on both

Refractive Index Response

15

20 V, mL

25

Fig. 4. Size-exclusion chromatograms of polystyrene (1), styrenebutadiene statistical copolymer (2), styreneacrylonitrile statistical copolymer (3), and equimolar mixture of them (4, dotted line). Columns: 3 HR Styragel (Waters Corp.), mobile phase: THF, 1 mL/min, detector: refractometer model 2410 (Waters Corp.).

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1

611

2 THF, % 60 40 20 0 0 10 V, mL 20 3

Fig. 5. Gradient reversed-phase elution of the same polymers as in Figure 3. Column: SymmetryShield C8 (Waters Corp.), mobile phase: 100% ACN to 100% THF over 20 min (dashed line), 1 mL/min, detector: ELSD Model 500 (Alltech Corp.).

normal- and reversed-phase columns, without any practical limitations on the molecular weight of polymers [see reviews in Refs. 7, 11 (chapt. 9), and 2629]. On the other hand, several reported (7) isocratic separations of block copolymers at CPA were limited by the oligomeric range of molecular weights. Many recent publications deal with two-dimensional characterization of copolymers, where the molecular weight independent separation is achieved by ipc as the rst step (7,30,31). To summarize briey, in spite of solid age, hplc of polymers remains the workhorse of the biotechnology and chemical industries, and at the same time presents a fruitful eld for future development and improvement.

BIBLIOGRAPHY
Chromatography in EPSE 1st ed., Vol. 3, pp. 731745, by P. M. Kamath and L. Wild, U.S. Industrial Chemicals Co.; Chromatography in EPSE 2nd ed., Vol. 3, pp. 491531, by J. F. Johnson, The University of Connecticut at Storrs. 1. P. R. Haddad and P. E. Jackson, Ion Chromatography: Principles and Applications, Elsevier, New York, 1990, Chapt. 5. Journal of Chromatography Library, Vol. 46. 2. J. B. Li, J. Morawski, Liq. Chromatogr./Gas Chromatogr. 16, 468 (1998). 3. P. C. Cheung, S. T. Balke, and T. C. Schunk, in T. Provder, H. G. Barth, and M. W. Urban, eds., Chromatographic Characterization of Polymers: Hyphenated and Multidimensional Techniques, American Chemical Society, Washington, D.C., 1995. 4. J. L. Dwyer, in T. Provder, ed., Chromatography of Polymers: Hyphenated and Multidimensional Techniques, American Chemical Society, Washington, D.C., 1999. ACS Symposium Series 731. 5. S. A. Carr and co-workers, Anal. Chem. 63, 2802 (1991).

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6. A. Weston and P. R. Brown, hplc and CE: Principles and Practice, Academic Press, San Diego, Calif., 1997, p. 220. 7. H. Pasch and B. Trathnigg, hplc of Polymers, Springer, Berlin, 1998, Chapt. 7. 8. Y. Brun, J. Liq. Chromatogr. Relat. Technol. 22, 30673090 (1999). 9. D. W. Armstrong and R. E. Boehm, J. Chromatogr. Sci. 22, 378 (1984). 10. H. J. Cortes and L. D. Rothman, in H. J. Cortes, ed., Multidimensional Chromatography: Techniques and Applications, Marcel Dekker, Inc., New York, 1990. Chromatographic Science Series 50. 11. G. Gl ckner, Gradient hplc of Copolymers and Chromatographic Cross-Fractionation, o Springer, Berlin, 1991, p. 148. 12. C. F. Poole and S. K. Poole, Chromatography Today, Elsevier, New York, 1991. 13. S. L. Wu, K. Benedek, and B. L. Karger, J. Chromatogr. 359, 3 (1986). 14. M. I. Aguilar and M. T. W. Hearn, in M. T. W. Hearn, ed., hplc of Proteins, Peptides and Polynucleotides, VCH Publishers, New York, 1991. 15. A. A. Gorbunov and A. M. Skvortsov, Adv. Colloid Interface Sci. 62, 31108 (1995). 16. J. Jandera and J. Churacek, Gradient Elution in Column Liquid Chromatography Theory and Practice, Elsevier, Amsterdam, 1985. 17. L. R. Snyder and M. A. Stadalius, in C. Horvath, ed., High-Performance Liquid Chromatography: Advances and Perspective, Vol. 4, Academic Press, Inc., Orlando, Fla., 1986, p. 195. 18. Y. Brun, J. Liq. Chromatogr. Relat. Technol. 22, 30273065 (1999). 19. N. B. Afeyan and co-workers, J. Chromatogr. 519, 1 (1990). 20. R. L. Cunico, K. M. Gooding, and T. Wehr, Basic hplc and CE of Biomolecules, Bay Bioanalytical Laboratory, Richmond, Calif., 1998, p. 23. 21. K. M. Gooding and F. E. Regnier, hplc of Biological Macromolecules: Methods and Applications, Marcel Dekker, Inc., New York, 1990. 22. F. E. Regnier, Science 238, 319 (1987). 23. A. Sadana, Bioseparation of Proteins: Unfolding/Folding and Validations, Academic Press, Inc., San Diego, Calif., 1998, Chapt. 5. 24. M. T. W. Hearn, in S. Ahuja, ed., Handbook of Bioseparations, Vol. 2, Academic Press, Inc., San Diego, 2000. 25. M. R. Wilkins, K. I. Williams, R. D. Appel, and D. F. Hochstrasser, eds., Proteome Research: New Frontier in Functional Genomics, Springer-Verlag, New York, 1997. 26. S. Mori, in Ref. 3. 27. T. C. Chunk, J. Chromatogr. A 656, 591615 (1993). 28. S. Teramachi, in D. Berek, ed., Chromatography of Polymers (special issue), Macromol. Symp. 110, 217 (1996). 29. H. Sato, K. Ogino, T. Darwint, and I. Kiyokawa, in Ref. 28. 30. P. Kilz, R.-P. Kruger, H. Much, and G. Schulz, in Ref. 3. 31. B. Trathnigg, M. Kollroser, M. Parth, and S. Roblreiter, in T. Provder, ed., Chromatography of Polymers: Hyphenated and Multidimensional Techniques, American Chemical Society, Washington, D.C., 1999.

GENERAL REFERENCES
Refs. 6 and 19 are recommended as introductory material to hplc of biopolymers. Refs. 14 and 20 deal critically with all aspects of hplc of biological molecules.

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Refs. 23 and M. Kastner, ed., Protein Liquid Chromatography, Elsevier, Amsterdam, 2000, J. Chrom. Library, Vol 61, reect the current state of the art in protein chromatography. U. D, Neue, hplc Columns: Theory, Technology, and Practice, Wiley-VCH, Inc., New York, 1997, in-depth review of column technology. Refs. 7 and 11 reect the current state of hplc of synthetic polymers. Refs. 1, 8, and 17 deal with theoretical aspects of polymer chromatography.

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