Multiple Detection in Size-Exclusion Chromatography - American

Chapter 1

Separation Science of Macromolecules: What Is the Role of Multidetector Size-Exclusion Chromatography?

Downloaded by LINKOPINGS UNIV on October 29, 2013 | http://pubs.acs.org Publication Date: November 4, 2004 | doi: 10.1021/bk-2005-0893.ch001

André M. Striegel Department of Chemistry and Biochemistry, The Florida State University, Tallahassee, F L 32306-4390

The importance of separation science in the study of natural and synthetic macromolecules is described, highlighting the role of size-exclusion chromatography (SEC) and emphasizing that of multiple detection in this analytical technique. Particular attention is paid to the ability of separation science in general, and of multi-detector SEC in particular, to determine the distributions of key macromolecular parameters and the end-use properties these affect. The historical development of SEC is reviewed and mention is made of how non-separation techniques can complement the type of information provided by SEC and other macromolecular separation method.

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© 2005 American Chemical Society

In Multiple Detection in Size-Exclusion Chromatography; Striegel, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.


3 The end-use application of most macromolecules is determined not only by their chemical identity but also, and sometimes more importantly, by the distributions and sequences of key physical and physicochemical parameters. The distribution of molar mass is known to affect a large number of properties, as for example with elastomers, where narrowing the molar mass distribution (MMD) results in superior mechanical properties but relatively poor processing characteristics. The distribution of properties such as long-chain branching (LCB) and three-dimensional structure and/or conformation (referred to herein collectively as "architecture") can affect areas as dissimilar as the abilities to form inclusion complexes and to modify flow through drilling pipes. Chemical composition distribution can affect the miscibility and morphology of polymer blends, particle size distribution affects the packing and rheological behavior of resins and melts, while charge distribution influences the drug-delivering ability of polymeric sequestering/transfer agents. It is well known that children who inherit sickle cell anemia from both parents rarely live beyond the age of two. This genetic disease is known to arise from a change in -0.3% of the amino acid sequence of hemoglobin, a change of merely two amino acids out of 574. Figure I shows examples of various polymeric distributions; a more comprehensive list is given in Table I along with the types of end-use properties affected. The ability to determine these and many other distributions is gained through separation science. As seen in Table I, the use of chromatographic and other methods permits the study of a large and varied array of macromolecular property distributions and sequences. Some methods, such as temperature rising elutionfractionation(TREF), are intended for use with crystalline polymers such as polyethylene. Rheology, enzymology, and matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been included as well; while not separation methods per se, the information they yield is often complementary to that obtained by techniques such as size-exclusion chromatography (SEC). The preeminent role of SEC, in particular of multidetector SEC techniques, is seen by its abundant representation in the table, which has been highlighted using boldface type. The role of the complementary, non-separation methods will be discussed briefly at the end of this chapter. It has been said that "[t]he only completely satisfactory description of the molecular weight (i.e., the degree of polymerization) of a macromolecular compound is the distribution curve...as determined through fractionation." Analogous truisms apply to all the other properties mentioned in Table I. As seen, to achieve the "completely satisfactory description[s]" requires enlisting the aid of a large number of separation methods (many still in their infancy), usually in combination with spectroscopic, hydrodynamic, etc. methods of detection. Though the separation techniques each possess their own individual thermodynamic and kinetic (and instrumental) identities, they are also fundamentally united in ways that allow for their complementarity, study, and improvement. The polymer molecules (including natural and synthetic polymers as well as oligomers) thus play a dual role, that of analyte and that of probe. In the former, separation science is used to enhance our knowledge of the

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Molar mass

Branching

AABBA ABBBB AABBB AAAAB Chemical Composition —

Potyelectrolyte charge

_ Crosslinking

AB ABB ABBB BBABB Chemical heterogeneity

Differential weight fraction

Figure 1. Generic examples of (top) various macromolecular heterogeneities and (bottom) their distributions as a continuous function ofmolar mass. MMD: molar mass distribution, CCD: chemical composition distribution, CCD-MMD: CCD as a function of MM (this is normally represented as a contour plot), CH: chemical heterogeneity, LCB: long-chain branching, SCB: short-chain branching, M : molar mass between crosslink +/-: polyelectrolytic charge. c


5 Table I. Macromolecular distributions: Their measurement and end-use effects

Macromolecular Property


Molar mass

Representative end-use properties affected

Elongation, tensile strength, adhesion Long-chain Shear strength, tack, peel, branching crystallinity Short-chain Haze, stress-crack resistance, branching crystallinity Crosslinking Gelation, vulcanization, surface roughness Architecture Flow modification, diffusion encapsulation Tacticity Crystallinity, anisotropy, solubility Chemical Morphology, miscibility, composition solubility Chemical Toughness, brittleness, heterogeneity biodegradability Chemical comp. Mechanical properties, vs. molar mass blending, plascticization Block sequence Dielectric properties, reactivity, miscibility Base pair sequence Genetic code, heredity, mutations Flocculation, transport, Polyelectrolytic charge binding of metals Particle size Packing, drag, friction, mixing

Separation method usedfor determination* SEC, FFF, HDC, TGIC, CEC SFC, MALDI-MS, rheology SEC-MALS, SEC-VISC, rheology, enzymology SEC-IR, SEC-NMR, TREF , CRYSTAF , enzymology SEC-MALS, SEC-VISC, rheology b

b

SEC-MALS-QELS-VISC SEC-NMR, TGIC, LCCC, GPEC, TGIC SEC-spectroscopy/spectrometry, LCCC, PFC 2D-LC {e.g., SEC-GPEC) SEC-spectroscopy, 2D-LC (e.g., PFC-SEC) Automated DN A sequencing, MALDI-MS SEC-conductivity FFF, HDC, PSD A, sieving

Many techniques require a concentration-sensitive detector (e.g., a differential refractometer), not included here for simplicity. *SEC: size-exclusion chromatography, FFF: field-flow fractionation, HDC: hydrodynamic chromatography, TGIC: temperature-gradient interaction chromatography, CEC: capillary electrokinetic chromatography, SFC: supercritical fluid chromatography, MALDI-MS: matrix-assisted laser desorption/ionization mass spectrometry, MALS: multi-angle light scattering, VISC: viscometry, IR: infrared spectroscopy, NMR: nuclear magnetic resonance spectroscopy, TREF: temperature rising elution fractionation, CRYSTAF: crystallization fractionation, QELS: quasi-elastic (dynamic) light scattering, LCCC: liquid chromatography at the critical condition, GPEC: gradient polymer elution chromatography, PFC: phase fluctuation chromatography, 2D-LC: two-dimensional liquid chromatography, PSDA: particle size distribution analyzer. *¥οτ crystalline polymers only.


6 macromolecules and to improve, tailor, and predict their end-use properties. In the latter, the analytes themselves help to develop a clearer picture of the fundamental processes, of the commonalities and distinction lying below the alphabet soup that is the "Separation Method" column of Table I. As our focus is principally on size-exclusion chromatography, we now take a moment to remember how we got to where are, i.e., by briefly reviewing the development of SEC.


Historical Development of SEC It has been over a half century since Wheaton and Bauman noted the fractionation of non-ionic substances in the passage through an ion exchange column, indicating that the separation of molecules based on size should be possible in aqueous solution. In 1959, Porath and Flodin demonstrated that columns packed with crosslinked polydextran gel, swollen in aqueous media, could be used to separate various water-soluble macromolecules by size. This became known as gel filtration chromatography (GFC). Soon other hydrophilic gels were developed for separation of compounds of biological interest. Being capable of swelling only in aqueous media, however, limited their use to watersoluble substances. By the early 1960s, the relationship between the molar mass distribution of synthetic polymers and their physical characteristics such as chemical resistance, toughness, melt viscosity, etc. (see Table I) was well known. Consequently, workers in the polymers and plastics fields were interested in a method to obtain not only molar mass averages but, more importantly, molar mass distributions of synthetic polymers. During this time, work had begun on making hydrophobic gels and columns were packed consisting mainly of crosslinked polystyrene, with the crosslinking performed in the absence of diluents. It was soon recognized that crosslinking in the presence of diluents that are solvents for the monomer altered the structure of the gel networks. When the diluent is a non-solvent for the resulting polymer a rugged, stable internal gel structure of good permeability may be obtained. In 1964, John Moore published the first paper on a technique that he termed gel permeation chromatography (GPC). In said paper, he described the preparation of polystyrene beads of sufficient crosslinking to impart a desired rigidity to the gel while still regulating the permeability by altering the amount and nature of the diluent. The composition of the gels consisted mainly of varying proportions of styrene/divinylbenzene/toluene. Large changes in the permeability of the gels were effected by changing the diluent. Columns packed with these gels were used to separate a series of polystyrenes and of poly(propylene glycoI)s over an extended range of molar mass. To monitor the composition of the eluent a differential refractometer was used. This last was a special design, by James Waters, with a smaller optical cell than was commercially available at the time and with provision for continuous flow in 3

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both sides of the cell. Moore recognized that the chromatographic separation appeared to be close to an equilibrium process in which the solute molecules diffused rapidly into all available parts of the gel network. The thermodynamic equilibrium of the separation process remains a topic of interest to this day for size-exclusion chromatography (SEC), the all-encompassing term that is used nowadays for both GPC and GFC. 6

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Separation Science and Biopolymers The field of biopolymers and copolymers is one rich with possibilities and areas of interest. Biodegradable copolymers, in particular, combine biological and synthetic, linear and branched, neutral and polyionic and, occasionally, lightly crosslinked materials for applications in drug delivery, "smart" sutures, and tissue regeneration, for example. Characterizing these copolymers' properties leads directly to improved capabilities and predictive behavior. For example, the biocompatibility and biodegradability of implantation materials made from poly(DL-lactic acid/glycine) copolymers are directly related to both molar mass and chemical composition. Determining the distributions of these and other key functional parameters cannot be accomplished without the use of SEC and related techniques» Equally interesting, and oftentimes more challenging, is the field of glycopolymers. At the oligomeric level, oligosaccharides perform a large number of biological roles,fromnutrition to being essential components of plant cell walls, to moderating biosynthesis, structure, and transport functions of glycoproteins. While there are not many distributions of properties at this level, oligosaccharide analysis is aided, many times critically, by separation techniques such as high-performance liquid chromatography (HPLC) and anionexchange chromatography (AEC), or by SEC for identification and measurement of key conformational properties. At the macromolecular level, many botanical glycopolymers possess limited solubility (cellulose); ultra-high molar masses and broad molar mass distributions (amylopectin); long- and short-chain branching (dextran) and, in some cases, hyperbranching (certain Type II arabinogalactans); a variety of anomeric configurations and glycosidic linkages (xanthan, which is also a polyelectrolyte); etc. Dextran sulphate, for example, is a glycopolymer known for its anti-coagulant and bioinhibitory properties (e.g., inhibiting enzyme release from macrophages). It is also a high molar mass, polydisperse, polyanionic, potentially branched polymer. Their use in biological processes and pharmaceutical formulations, adhesives and rheological modifiers, foods and feeds, textiles and non-wovens and, more recently, biodegradable products shows the criticality of applying separation science to the study of these macromolecules. Multi-detector SEC can determine virtually all of the 9

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8 properties mentioned in this section, exceptions being the chemical composition distribution, determined by e.g., gradient polymer elution chromatography (GPEC, though this technique has seen limited application in the study of biopolymers), and the various anomeric configurations and glycosidic linkages, for which we must rely on the help of enzymology and/or mass spectrometry.


Separation Science and Synthetic Polymers There are a great many synthetic polymers that also possess distributions in a number of parameters critical to their end-use applications. Even a cursory review of the literature, or of this book, will reveal a multitude of examples. In contrast to natural polymers, some control may be exerted over properties such as molar mass and its distribution, branching, copolymerization, etc. in synthetic polymers. Nonetheless, polydispersity may exist not only in molar mass, but also in properties such as tacticity, crystallinity, branching, chemical composition, functionality type, etc. The molar mass distribution becomes but a minimum datum that needs to be measured for many "real world" polymers. It is interesting to note that certain properties may possess both a polydispersity as well as a non-uniform distribution across the MMD. One example, in the case of random copolymers or terpolymers, e.g., a random AB copolymer, is that the ratio of A to Β may not be distributed uniformly across the MMD of AB (assuming AB is polydisperse with respect to molar mass). This is referred to as chemical heterogeneity and may be measured by SEC using spectroscopic (IR, NMR) or spectrometry (MS) methods of detection. The spectroscopic detection methods are discussed in the chapters by DesLauriers and by Montaudo, while mass spectrometry detection methods are discussed in the chapters by Montaudo, by Sadi et al. by Lecchi and Abramson, and by Prokai et al. The chemical heterogeneity may also be measured using other separation techniques such as liquid chromatography at the critical condition (LCCC, discussed in the chapter by Pasch) or phase fluctuation chromatography (PFC, discussed in the chapter by Teraoka). Regardless of whether the A:B ratio remains constant or not across the MMD of AB, however, there may also be a polydispersity in the amount (mole or weight percent) of either A or Β (or both). This is referred to as the chemical composition distribution (CCD) and, as mentioned above, must be determined using non-SEC methods such as GPEC or temperature-gradient interaction chromatography (TGIC). Generic examples of chemical heterogeneity and chemical composition distribution are shown in Figure 1. As seen in Table 1, the chemical heterogeneity and the chemical composition distribution can each influence different end-use properties of materials; thus, both parameters need to be controlled and measured accurately for copolymers. 9

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9 Another example is that of short-chain branching (SCB). As described in the chapter by DesLauriers, SEC-FTIR may be used to measure the distribution of short-chain branches across the MMD of polyolefins. Theoretically, at least, this should also be possible by SEC with C-NMR detection, though this author has been unable to find any reference to this in the literature; however, SEC-NMR is still in its infancy though, as described in the chapter by Montaudo, it is quickly coming of age. Additionally, the SCB may also have a distribution and a polydispersity, and recently this has been measured using analytical temperaturerisingelution fractionation (A-TREF). One advantage in studying synthetic polymers is the ability to make materials such that individual parameters can be isolated. Polyethylene, for example, has been a benchmark polymer in the study of long-chain branching (LCB), as it fulfills all the requirements that are necessary for accurate, quantitative calculation of LCB via the classic Zimm-Stockmayer theory: Linear standards exists with the same chemistry as the branched material; the standards cover the MMD region of interest of the branched material; the branching functionality is usually known a priori due to a refined understanding of free-radical, Ziegler-Natta, etc. polymerization mechanisms; and there are even a modest amount of relatively narrow molar mass polydispersity standards commercially available for this polymer. The ability to isolate parameters to study their individual effect(s) is also seen in polystyrene, where narrow molar mass polydispersity standards exist over several orders of magnitude in molar mass, and where linear, broad molar mass polydispersity PS may be compared to /-functional stars with varying, but controlled, number of arms. Many synthetic polymers do not possess these conveniences, however, and in these cases (in the absence of superior synthetic strategies) assumptions and extrapolations need to be made in order to advance our knowledge of the field. These "real world" polymers are abundant and many possess a number of the distributions given in Table I. An example, studied by this author and others, is poly(vinyl butyral) or PVB, the main component of the polymeric interlayer in laminated safety glass. This macromolecule is actually a random terpolymer, with polydispersities in molar mass (as shown by SEC-MALS) and chemical composition (determined by GPEC), perhaps possessing chemical heterogeneity (though one study has shown otherwise, using SEC-IR), with long- (shown by SEC-MALS-VISC) and possibly short-chain branching and, occasionally, crosslink- or graft-induced branching as well (SEC-MALSVISC). Additionally, PVB lacks narrow molar mass polydispersity standards or adequate (i.e., same chemical heterogeneity and CCD) linear standards for branching calculations, and possesses intra- and inter-molecular hydrogenbonding the affects solubility and dissolution. As mentioned, in order to further our knowledge of this and other "real world" polymers assumptions, compromises, and extrapolations must be made, and we should always remember that any of these could be wrong. For more information on MALS 13


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10 and VISC as detection methods in SEC, as well as on QELS and refractometry, the reader is referred to the chapters by Reed and by Cotts. Data handling for refractometry, static light scattering, and viscometry is discussed in the chapter by Brun, and a number of applications of SEC-MALS are given by Podzimek in his chapter.


Polymers as Analytical Probes While obviously an area rich with possibilities, we will deal here only briefly with the use of polymers as analytical probes, i.e., as used to shed light on the separation processes and methods. Polymers may be used to determine reduced column factors, selfsimilarity features of separation media, etc. They may be used to study chromatographic band broadening, as seen in the chapter by Netopilik, as well as in the study of local polydispersity effects that can plague hydrodynamicvolume-dependent separations such as SEC. More generally, polymers can serve to demonstrate the limitations and biases of current techniques, such as the apparent limits of SEC, as compared to TGIC, toward characterizing narrow polydispersity polymers. Macromolecules can also serve to showcase the advantages of new methodologies, such as phase fluctuation chromatography (PFC) for determination of the chemical composition distribution of copolymers. This technique, and its coupling to SEC, is explained in depth in the chapter by Teraoka. 25

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Conclusions Multi-detector SEC plays a pivotal role in the study of natural and synthetic macromolecules. This technique (or these techniques) has the ability to measure an abundance of parameters and, more importantly, their distributions. It is not, however, the end-all/be-all of analytical methods and is oftentimes aided by other members of the large family of separation methods to which it belongs. Incestuously, it tends to couple with some of these other methods in the form of two-dimensional liquid chromatographic separations. Multi-detector SEC can also be intimately liked to other, nonseparation methods. Liaisons with enzymic and mass spectrometric methods have been hinted at above. The generally tepid relationship between SEC and rheology is slowly warming, as separations scientists recognize the ability of rheology to measure LCB, as well as supra-molecular properties, of materials that may be difficult to analyze chromatographically due to problems with dissolution, non-size-exclusion effects during separation, etc. and rheologists recognize the ability of SEC to measure the distribution of parameters such as


11 LCB and SCB across the MMD of a polymer (oftentimes with orders of magnitude less sample than necessary for rheology studies), data unavailable from rheological measurements. More often that not these two techniques have the ability to complement each other, and this ability is slowly being exploited by researchers. ' In the meantime, seemingly unrelated techniques are entering the fray, as with the case of atomic force microscopy (AFM), where a recent report has demonstrated the measurement of the number-average molar mass (M ) as well as the MMD by combining AFM with the Langmuir-Blodget technique. This is also the case with dynamic surface tension which, as described in the chapter by Synovec et ai, is now being used as a detection method in SEC. Witness also the growing number of applications for fluorescence detection in SEC, covered in this book by Maliakal et al and by Yokoyama and Knuckles, where each group applies the technique to completely different ends than the other. Finally, the determination of polymer size, conformation, etc. can now be aided intimately by the variety of computer modeling methods available, including ah initio, semi-empirical, Monte Carlo, molecular mechanics, and molecular dynamics techniques, among others. " 29 31

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Multiple Detection in Size-Exclusion Chromatography - American

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