Anal. Chem. 1992, 6 4 , 428R-442R (L30) Konklari, C. N.; Karayannls, M. I . Telenta 1991, 38, 1019-1026. (L31) Chakrabarti. C. L.; Headrlck. K. L.; Hutton, J. C.; Blcheng, 2.; Bertels, P. C.; Beck, M. H.. A M I . them. 1990, 62, 574-586. (L32) Martinerlozano, C.; P&er-RuIz, T.; Tomes, V.; Val. 0. Analyst (London) 1991, 116, 857-865. (L33) Poston, P. E.; Harris. J. M. Anal. Chem. 1991, 63,668-692. (L34) Harak, D. W.; Mottola. H. A. Blosensors Bloelecbw,. 1901, 6 , 589-594.
(L26) Drexler, C.; Ellas, H.; Fecher, B.; Wannowius, K. J. Fresenlus' J . Anal. Chem. 1991, 340, 605-615. (L27) Gund, G.; Wlen, F.; Weisweiler, W. Fresenius' J . Anal. Chem. 1991, 340. 616-620. (L28) Wlen, F.; Gund, G.; Welsweiler. W. Fresenius' J . Anal. Chem. 1991, 340, 621-628. M.; Wenbell, P. D.; Crouch, S. Anal. Chem. 1990, 62, (L29) L-z-Nleves, 304-308.
Size Exclusion Chromatographys Howard G. Barth* and Barry E. Boyest Du Pont Company, Central Research and Development, Experimental Station, P.O. Box 80228, Wilmington, Delaware 19880-0228, and Rockland Technologies, Inc., 538 First State Boulevard, Newport, Delaware 19804
INTRODUCTION Unlike other modes of chromatography, size exclusion chromatography (SEC) is an entropically controlled separation technique that depends on the relative size or hydrodynamic volume of a macromolecule with respect to the size and shape of the pores of the packing. With high-performance SEC, one can determine rapidly the relative molecular weight distribution of a polymer. If the column is calibrated with known molecular weight standards, average molecular weight values also can be obtained. Since the elution order is a function of molecular size, SEC is often used to aid in the identification of complex samples, especially low-molecular-weightcomponents, and also for sample cleanup for the removal of hi hmolecular-weight components. In addition, it is also possi le to determine molecular conformation and long-chain branching with the use of molecular-weight-sensitivedetectors. SEC has become a mature, and well-accepted technique for both synthetic polymers and bio olymers. Although highperformance SEC has lagged beknd conventional soft-gel packings for biochemical applications, the life science community is depending more on high-performance columns because of their speed and high resolution. For aqueous SEC (gel fitration chromatography),either silica- or organic-based packings can be employed. By and e, silica-based packings are used more often for quality contro and quality assurance, because of their higher efficiency (smaller particle size),shorter analysis time, and more robust nature. However, solutepacking interactions can be a concern. Hydrophilic polymeric-based packings are generally more inert and are often used for characterization studies. For organic-soluble polymers, polystyrene packings are still the media of choice. During the past 2 years, there has been less emphasis on SEC theory and band-broadening studies and more focus on applications. The use of on-line light-scatterin and viscometric detectors has greatly extended the usefufness of SEC. With these molecular-weight-sensitivedetectors, one can now determine branching, molecular size, and conformation as a function of molecular weight. Information-rich detectors, most notably UV diode array, are being used to determine compositional heterogeneity of copolymers and other complex pol eric systems. Research activities involving on-line N M R an% detectors suggest that interest in these types of systems will continue. In terms of sample types, we see increased applications regarding the characterization of block copolymer micelles, as well as the study of associated and aggregated states of biopolymers. Long-chain branching measurements of polymers are becoming more common as a result of commercially available light-scattering and viscosity detectors. Althou h most types of macromolecules can be characterized now y! SEC using the proper column acking and mobile-phase composition, amphipathic biopoPy"rs, that is, compounds containing both highly hydrophobic and highly ionic groups,
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Technologies, Inc. * Rockland Du Pont Contribution No. CON6218. +
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are still difficult to chromatogra h. Examples of this class of compounds are detergent-solu%ilizedintegral membrane protein, such as receptors and transport proteins. This review covers fundamental developments in SEC from December 1989 to the end of 1991, and is a continuation of our previous review (Bl). The database was Chemical Abstracts and Biosis. Our main focus was on hi h-performance SEC; however, studies utilizing convention8 packings were included if they were of fundamental significance or dealt with an unusual se aration. Areas that are included in this review are listed in 8able I. We have made every attempt to include only those articles that contained fundamental studies or described selected applications that we considered informative. The writing of this article was a long and laborious process that involved developing search strate ies, reading all abstracts and often times tracking down ful! articles, and categorizing this information. To this end, we are grateful to the thorough search routines enerated by Neil Feltham and the careful typing and proogeading by Rebecca Pennington of the Du Pont Co. We also wish to thank our respective companies for their support and encouragement in the preparation of this manuscript.
A. BOOKS AND SYMPOSIA In the last 2 years, there have been no books dedicated to
SEC that have been published. However, several texta on both
separation science and polymer characterization have appeared that provide useful information on SEC and related methods. Gltkker ( A l ) has written on gradient HPLC on Copolymers that includes comprehensive chapters on copolymer chemical heterogeneity, SEC of copolymers, chromatography cross fractionation, and a useful discussion on experimental problems associated with HPLC of polymers. This is a companion monogram to his previously published work, Polymer Characterization by Liquid Chromatography (&). Barth and Mays (A3) have coedited Modern Methods of Polymer Churacterization that includes contributed cha ters on most major techni ues, including several on SE8. Although somewhat oudated, the text Polymer Characterization (A4) covers fundamental principles of techniques related to solution roperties and dilute solution characterization of pol geveral other books iven in refs A5 and A6, which mention SEC, may %e of interest to those involved with synthetic polymer analysis and characterization. Giddings recent book on separation science (A7) reviews entropic effecta in SEC. Ahuja (AB)also briefly discussea the theory of SEC in his book on HPLC. There have also been a number of books published on HPLC of biologicals, which include chapters on various aspecta of SEC (see the following section on reviews), that are given in refs A9-A12. The International Symposium on Polymer Anal sis and Characterization (ISPAC) published proceedings of IgPAC-2, held in Austin, Texas, 1989 (A13). The roceedings of ISPAC-3, which took place in Bmo, CzechosEvakia, 1990, were also published (A14). In addition to a number of studies on
00Q3-27QQ/92/Q36e428R$10.00/0 0 1992 Amerlcan Chemical Society
gt?y
SIZE EXCLUSION CHROMATOGRAPHY Howard G. Barth is a member of the research staff of the Corporate Analytical Science Center at the Du Pont Experimental Station, Wilmington, DE. Before joining the Du Pont Co. in 1988, he was a research scientist and group leader at Hercules Research Center. He received his B.A. (1969) and Ph.D. (1973) in analytical chemistry from Northeastern University. His specialties Include polymer characterization, size exclusion chromatography, and high-performance liquid chromatography. He has published over 50 papers in these and related areas. Barth has also edited the book Modern Methods of Particle Size Analysis (Wiley, 1984) and coedited Modern Methods of Potymer Characterization (Wiley, 1991). He has also ediied four symposium volumes on polymer characterization published in the Journal of Applied Polymer Science. Barth was on the Instrumentation Advisory Panel of Analytical Chemktry and was Associate Editor of the Journal of Applied Polymsr Science. He is cofounder and Chairman of the InternationalSymposium on Polymer Analysis and Characterization. Barth is past Chairman of the Delaware Section of the American Chemical Society where he presently serves as councilor. Dr. Barth is a member of the American Chemical Society divisions of Analytical Chemistry, Polymer Chemistry, and Polymeric Materials Science and Engineering, Society of Plastics Engineers, and the Delaware Valley Chromatography Forum. He is also a Fellow of the American Institute of Chemists and a member of Sigma Xi. Barry E. b y e s is a research and development scientist with Rockland Technologies Inc., Newport, DE. He received his B.S. in biochemistry in 1982 at the University of Alberta (Edmonton, Alberta). Barry carried out his graduate work at the University of British Columbia (Vancouver, BC). where he received the M.S. in neurological research (1985), working with Professor S.C. Sung, and the Ph.D. in neurosciences (1991) under Professor Edith G. McGeer. Before joining Rockland Technologies, he spent 1’/* years as a visiting scientist in the Analytical Division of the Central Research and Development Department at the Du Pont Experimental Station. The research activiiies of Dr. b y e s include the development of analytical techniques for molecular neurobiology, the study of biopolymer structure by hydrodynamic and chromatographic methods. and the investigation of chromatographic materials for biochemical separations.
il
Table I. Review Contents A. B. C. D. E.
F.
G.
H. I.
J.
K.
L. M. N.
SEC, these proceedings also covered other major technique areas. The proceedings of ISPAC-4 (Baltimore, MD) is in press. The proceedings of the International GPC Symposium ’89, which took place in Newton, MA, also was published (A15). The 1991 International GPC Symposium (San Francisco, CA) proceedings are scheduled for publication in 1992. The First International GPC Viscometry Symposium was held in 1991 in Houston, T , but only abstracts of this meeting are available (A16). The 1991National ACS Meeting in New York City also sponsored a symposium on SEC and related techniques, and an ACS Symposium Edition on this meeting is due out in 1992.
s
B. REVIEWS Reviews of specific topics in SEC are listed in the appropriate sections. Barth and Boyes (B1) presented a comprehensive coverage of the literature on SEC from 1988 to the end of 1989, this present review being a continuation of that format. Patton (B2)has written an excellent review on the fundamentals of SEC that includes theory, calibration, experimental considerations, aqueous SEC, determination of long-chain branching, and copolymer analysis. The characterization of complex synthetic polymers by SEC and other separation techniques was surveyed by Balke (B3). This chapter also includes extensive discussion on the quantitative interpretation of data. A chapter by Meira (B4)emphasized data treatment associated with instrumental band-broadening corrections as applied to determining the molecular weight distribution and branching frequency of polymers. The measurement of long-chain branching in polymers by SEC was covered by Rudin (B5). Lindner and Huang (B6)presented the theory and practice of low-angle laser-light scattering
Books and Symposia Reviews Theory Band Broadening Calibration General Information Universal Calibration Data Processing Non-Size-ExclusionEffects Shear Degradation/Hydrodynamic Effects Sample Concentration Effects Adsorption Effects/Mobile-Phase Selection Detectors General Information Molecular-Weight-Sensitive Detectors Light Scattering Detectors Viscometers Miscellaneous Detectors Evaporative Light Scattering Detector Inductively Coupled Mass Spectrometer Infrared Spectrometer NMR Spectrometer Optical Activity Packings Silica-Based Packings Organic-Based Packings Compositional Heterogeneity SEC with Selective Detectors Interactive Liquid Chromatography Physiochemical Studies Biopolymers Structure/Conformation Studies Association/Ligand Binding Synthetic Polymers Inverse SEC Branching/Mark-Houwink Constants/Conformation Partition Coefficients/Association Polymer Degradation/Kinetic Studies Microcolumn SEC Preparative SEC Coupled Columns/Column Switching Selected Applications Biopolymers Proteins and Peptides Nucleic Acids Particles and Assemblies Synthetic Polymers Asphalt, Bitumens, and Related Products Starch, Cellulosics, Lignin, and Other Polysaccharides Humic and Related Substances Small Molecules Inorganic Compounds Sample Cleanup/Pretreatment Miscellaneous Applications
including its use as an online SEC detector. There has been a significant number of studies of biochemical interest (see Selected Applications section), including a number of chapters on the use of SEC for the characterization of peptides (B7)and proteins (B8-BII). Sebille (BIZ) discussed the use of SEC to measure interactions involving proteins. Characterization of protein conformation and folding by SEC is reviewed in refs B13-B15. SEC reviews of membrane proteins (B16),antibodies (B17), and protein purification (B18)also have been published.
C. THEORY Dubin’s group ( C l ) used a phenomenological approach to identify a polymer dimension that controls peak elution in SEC. Polymer dimensions that were examined included viscosity radius, radius of gyration, and contour length. Since none of these size parameters appeared to control retention uniquely, a new dimension was proposed which progressively deviates from the hydrodynamic radius with increasing macromolecular asymmetry. Assuming a stagnantipore liquid phase, Poppe and colleagues (C2) developed a theoretical ANALYTICAL CHEMISTRY, VOL. 64, NO. 12, JUNE 15, 1992
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model to describe the molecular wei ht calibration curve that includes both SEC as well as hydro$ynamic chromatographic processes. Dubin et al. (C3)evaluated a theoretical model for the free energy of partitioning of charged spheres (anionic polyelectrolytes) into cylindrical pores of like charge. Hoagland (C4) examined the interaction of a rodlike polyelectrolyte with nearby surfaces of like charge. A combination of electrostatic and entropic effects was predicted to produce both chain orientation and segmental depletion, the latter of which was incorporated into a model for SEC of polyelectrolytes. Using a sucrose polymer, Dubin et al. (C5) measured the dependence of the SEC distribution coefficient on molecular size. The results closely agreed with a model in which the stationary phase is considered to be a Gaussian distribution of cylindrical cavities. McGeavy et al. (C6)proposed a network model to describe the intraparticle porous structure on an SEC column. Buergy and Calzaferri (C7) proposed that oligomeric silsesquioxanes could serve as ideal hard-sphere solute models to test out SEC retention because of their nearly spherical structures. Gorbunov and Skvortsov (C8) compared the SEC resolution of random-coil polymers to that of globular proteins. These authors found that optimum resolution for both types of macromolecules could be achieved at an SEC partition coefficient of 0.3 and that the resolution for proteins was 1.5-1.8 times worse than for random-coil polymers. Moussaouj et al. (C9)measured the diffusion of globular proteins in gels and concluded that diffusion retardation of the proteins was caused by the obstruction effect of the gel matrix. Fukuda et al. (ClO)described the separation mechanism of simple ions by SEC using a liquid-liquid partition process. The main mechanism was proposed to be related to the differences in thermodynamic stabilities of the ions in the mobile phase and stationary gel phase rather than involving size exclusion and adsorption effects.
D. BAND BROADENING Feng and Fan (01) reported on a multi-Gaussian model.for deconvoluting T 's integral equation for band broadening. These authors us?a Fourier-transform technique to reduce Tung's equation to a simple expression for the calculation of the true molecular weight distribution. Gugliotta et al. ( 0 2 ) compared several deconvolution techniques and a novel heuristic method for their ability to correct for band broadening. On the basis of a proposed band-broadening shape function, Omorodion and Hamielec ( 0 3 ) discussed contributions from pore-size distribution (04),column arran ement (04), flow rate (05,D6), and applicationto a twc+broa!-standard method of column calibration (07). Lederer and co-workers (08) studied the concentration dependency of band broadening using an on-line light-scattering detector. This group also investigated the influence of the delay volume between a concentration and light-scattering detector on band broadening and tested the applicability of universal calibration (09).
E. CALIBRATION General Information Martire and colleagues (El) prepared and evaluated a bimodal SEC column using a mixture of 80- and 50(FA poresize silica. Keller and Kolycheck (E2)obtained molecular weight distributions using two log-normal distributions. Tennikov et al. (E3) proposed a procedure for estimating the polydispersity (M,M,) of an unknown sample without the use of standards. ori (E4)derived conversion factors for relating polystyrene molecular weights to molecular weights of polymethacrylates. Peng and Liu (E5) evaluated the use linear and nonlinear least-squares fits of calibration curves constructed from samples having broad distributions. Balke and co-workers (E6) used plots of residuals to compute the deviation of calibration curves fitted with a polynomial. Calibration curves obtained from conventional polymer standards, intrinsic viscosity calibration, and universal calibration were evaluated. By the use of a LALLS, viscometer, and refractometer detection system, the authors concluded that calibration curve devia-
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tions were caused by the packings themselves rather than uncertainties associated with the polymer standards. This group also applied two numerical optimization methods for column calibration (E7) and discussed error analysis in SEC (E@. Also, the evaluation of a thermal pulse flowmeter was described by these authors (E9).Papazian and Murphy (ElO) reported on long- and short-range precision obtained with narrow- and broad-molecular-weight standards using an internal standard for flow rate corrections. McNair's group (Ell) evaluated a series of alkanes, aromatics, alcohols, and phthalates to construct calibration curves for small molecules. Tsitsilianis and Dondos (E121 described the calibration of low-molecular-wei ht poly(methy1 methacrylate). Chouman et al. (EI3) triefvarious approaches to establish a calibration curve for phenolic resin intermediates including the use of alkanes, phenols, aromatic compounds, and polystyrenes. Cabre et al. ( E l 4 ) used inverse regression for treating protein calibration data to obtain hydrodynamic arameters of lobular proteins. Fishman and co-workers (El5~preaent.d a &tail study of the use of a five-component fit to Characterize the molecular distribution of pectins. These data were used to calculate the radius of gyration as well as average molecular weights, From this investigation, the authors proposed that pectins possess quaternary structures that may vary with the pectin source. Berth et al. ( E l 6 ) used a series of well-characterized pectin standards for SEC column calibration. Soubelet et al. (El7)prepared and evaluated a series of cellulose nitrate standards for SEC.
Universal Callbratlon With increased use of SEC viscometric detectors, there has been a great deal of interest regarding the validity and application of universal calibration. For further treatment of this area, please consult section F on non-size-exclusion effecta as well as section G on viscometers. Sanayei and ODriscoll (E18)established universal calibration for polystyrene and poly(methy1 methacrylate) standards. Intrinsic viscosities of the oligomers of these polymers were estimated by extrapolation of the MarkHouwink equation, and these data were used to extend the universal calibration to the low-molecular-weight region. Timpa (El9,E20) applied universal calibration to characterize cotton and starch using dimethylacetamide containing lithium chloride as the mobile phase and an on-line viscometer. Vidal et al. (E21) evaluated the use of universal calibration for cellulose tricarbanilate using known Mark-Houwink constanta for the cellulose derivatives. Himmel et al. (E22) used universal calibration obtained from an on-line viscometer to characterize acetylated lignins. Universal calibration was used for the characterization of polyoxymethylene (E23), carbonic acid polymers (E24), poly(viny1 chloride) (E%), poly(tert-butyl acrylate) (E%),poly(3-hexylthiophene) (E27), and polymer latexes (E28). In the area of aqueous SEC, universal calibration using known Mark-Houwink constants was applied to 6-glucans, pullulan, and alginate (E29),and aurintricarbo lic acid (EN). Dawkins and co-workers (E31) evaluated x e validity of universal calibration using water/methanol mobile phases with a cross-linked polyacrylamide gel. Poly(ethy1ene glycol) and poly(ethy1ene oxide) obeyed universal calibration. Highmolecular-weight pullulan standards also fell on the universal calibration curve; however, lower-molecular-weight pullulans were displaced to higher elution volumes. These authors also found that universal calibration was obiserved for polystyrene, poly(ethy1ene glycol), and poly(ethy1ene oxide) usin dimethylformamide as the mobile phase. Chikazumi and bhta (E32) investi ated the universal calibration behavior of globular and ienatured proteins. Proteins denatured with guanidine hydrochloride followed universal calibration, while proteins dissolved in sodium dodecyl sulfate did not. and Terwilliger (E33) used universal calibration obtaine with an on-line viscometer to characterize cationic polymers including poly(ally1amine) in terms of molecular weight and Mark-Houwink constants. Nagy (E341 also showed that universal calibration was valid for poly(ethy1ene oxide) and polysaccharides. Universal calibration was employed by Wu and Senak (E35) to characterize quaternized poly(viny1pyrrolidone-co-(dimethylamin0)ethylmethacrylate)
N"6"
SIZE EXCLUSION CHROMATOGRAPHY
copolymers. These results were in good agreement with data obtained from an on-line low-angle laser light scattering (LALLS)detector. Lesec and Volet (E36)studied the validity of universal calibration of poly(ethy1ene oxides) and pullulans as a function of column type, mobile phase composition, and injected polymer concentration. Universal calibration curves were obtained directly from an online viscometer and LALLS detector. Data Processing
Kiltz (E37) discussed the importance of elution volume precision and calibration curve fitting in terms of obtaining reliable molecular weight data. ALSO described was a software package for analytical automation and data processing. Huber and Billiani (E38)reported on two rsonal computer-based software pack e for processing SE data. Another software package for S C was briefly reviewed by Beinert (E39).
ee
3
F. NON-SIZE-EXCLUSION EFFECTS Shear Degradatlon/Hydrodynamlc Effects
Chubarova and Nesterov ( F I ) reported on the shear degradation of polystyrene and poly(methy1methacrylate) as a function of flow rate and the size, shape, and pore size of the packing. Slalom chromatography, a term used to describe the mechanism in which high-molecular-weight DNA is eluted after smaller fragments, was studied by Hirabayashi et d (F2). By examining the effects of packing and pore size, flow rate, temperature, and the nature of the mobile phase, these authors concluded that slalom chromatography is not caused by adsorption or an equilibrium phenomenon but by a hydrodynamic effect. Sample Concentration Effects
Hu and co-workers published a series of papers on the concentration effects for polydisperse polymers and applied their theory to universal calibration (F3),to radius of gyration calculations (F4), and to polyisobutylene rubber characterization (F5).Cheng and Yan (F6) studied the effect of polystyrene concentration on elution volume and were able to determine the critical concentration for coil shrinking. Their observationswere in agreement with Lohse’s mean-field theory regarding the variation of polymer chain dimensions in solution. Czok and Guichon (8’7)reported that viscous fingering is more pronounced with large-diameter columns than with microbore columns. Methods proposed to reduce this effect included increasing the viscosi of the mobile phase to match that of the injected sample an following the in’ected sample with a large plug of eluent having a viscosity skghtly higher than the sample. Ye et al. (F8)studied the effect of polymer concentration in vacancy SEC, a seldom used technique, in which the polymer solution is employed as the mobile phase with pure solvent injected as the sample.
Y
Adsorptlon Effects/Moblle-Phase Selectlon
Garcia et al. (8’9) presented a thermodynamic treatment of non-size-exclusion effects that are observed with mixed eluents. These authors used a modified Flory-Hug to calculate the SEC distribution coefficients for meric-based and silica-based packings. By assuming cylindrical pore shapes and taking into account the influence of the preferential solvation of the polymer in the mobile and stationary phases, a good correlation was obtained between experimental and predicted distribution coefficients. This oup also studied the elution behavior of sodium polystyrenesulfonate) and dextran on a silica-based support as a function of polymer concentration, ionic strength, and pH (FIO). On the basis of the above thermodynamic formalism, the authors developed a semiempirical equation to relate the effective pore volume to the molecular weight of the polyion. A general diecussion on non-sizeexclusion effeds in aqueous SEC, including concentration and flow rate effects, was presented by Rinaudo and Tinland (FII).Bolte and Troquet (FI2) used a parameter design to o timize the experimental conditions for the SEC of water-solufhe polymers. Perez-Paya et al. (F13)evaluated the elution behavior of sodium poly-
f%$zT
F
(styrenesdfonate)on both silica and polymeric-based pacas a function of pH and ionic strength. An empirical correlation was proposed to take into account deviations observed
with this anionic polymer as compared to nonionic polymer standards. The applicability of this correlation was tested for peptides and proteins. Inouye (FI4) studied the chromatogra hic behavior of proteins on TSK-gel Toyopearl. It was fountfthat electrostatic interactions could be eliminated by the addition of 0.3-0.5 M NaCl and hydrophobic interadions reduced by the addition of 30% ethanol to the mobile phase. The retention behavior of proteins on TSK-G3000SW as a function of mobile- hase ionic strength and pH also was studied by Shi and Hu b15). These authors reported that when the pH of the mobile phase was below the PIof the protein, adsorption occurred; when the pH was above the I, the protein was excluded. With this approach it was possibe to separate proteins of similar molecular weight, but different PI values. Hagarova et al. (F16) investigated the chromatographic properties of proteins on Perlose cellulose packings. A concentration of 0.1% sodium dodecyl sulfate was required to prevent adsorption. Hayes et al. (FI7) studied the SEC behavior of C-terminal peptide amides using a dihydroxyalkyl-bonded silica support. These peptides were adsorbed via hydrophobic interactions as compared to C-terminal acid analogues which were not retained. This property was used for the purification of insect neuropeptides. Hu et al. (FI8) found that the ion pair of cephalosporins with phosphate increased the SEC distribution coefficient significantly so that high-molecular-weight impurities could be separated. Chuan e (FI9)studied the influence of the ionic strength of DMSd on the separation of starch using a cross-linked polystyrene packin . A sodium hydroxide mobile phase was used by Suortti andPessa (F20) with Ultrahydrogel columns for SEC of natural and modified starches. To reduce ion exclusion exhibited by carrageenan oligosaccharides on Bio-Gel P-6, ionic strength of the mobile phase was increased (8’21). Eremeeva et al. (F22) studied non-size-exclusion effects of nitrocellulose usin THF as the mobile phase and silanized silica packings. Sufxtitution heterogeneit and the presence of ionic grou on nitrocellulose influencdthe SEC behavior of nitrocellxe. Addition of acetic acid to the mobile phase suppressed nonexclusion effects and led to the validity of universal calibration between nitrocellulose and polystyrene. Wu et al. (F23) evaluated the SEC of nonionic and anionic m p o pers of vinylpyrrolidoneusing aqueous and nonaqueous mob’ e phases. Mori (F24,F25) studied the elution behavior of sodium pol (styrenesdfonate)and pdulan on a silica-based packing and &3K-GMPW as a function of phos hate buffer concentration. Malfait et al. (F26) evaluated & h y d r o g e l columns and tested the validity of universal calibration with sodium poly(styrenesulfonate). The influence of adsorption, polymer concentration, and flow rate was discussed. A salt concentration of 0.1 M at 60 “C was required to prevent adsorption, and i n j d concentrations below 1mg mL were required for high-molecular-weight standards. niversal calibration was applied to determine the molecular weight distributions of K-carrageenans. Kim and Cotta (F27)discussed the difficulties of performing SEC of polyimide precursors (amic acids and esters). These problems were caused by strong polyme-olvent interactions, electrostatic interactions, and the possibility of partial imidization in solution. For the esters, anomalous peaks and unexpectedly large apparent molecular weights were caused by artial imidization catalyzed by basic impurities in the m o b e phase. Papazian (F28) studied the elution behavior of polyacrylamides and partially hydrolyzed polyacrylamides as a function of mobile-phase pH. Strege and Dubin (F29) optimized mobile-phase conditions for the SEC of poly(dimethyldiallylammonium chloride), poly(((methacry1amido)pro y1)trimethyla”onium chloride), polyethylenimine, and pufulan using Superose 6 packing. Pasch et al. (F30, F31) developed a mobile-phase system for the SEC of sultonated urea-formaldehyde resins. Electrostatic interactions including ion exclusion and association phenomena were eliminated by adding sodium sulfate or ion-pair reagents to the mobile phase. Reidle et al. (F32) investi ated the effect of mobile- hase com osition on the elution?behaviorof phenol-formal8ehyde resoE. A propriate Mark-Houwink constants were determined antf used for
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universal calibration. Sellers and Prewitt (F33) evaluated various SEC systems for the characterization of phenolformaldehyde resins and phenolic compounds. Spychaj (F.34) studied adsorption behavior of low-molecular-weight styrene-acrylic acid copolymers on Styragel columns in THF. Nguyen et al. (F35)studied the chromatographic behavior of magnesium salts using Sephadex G10 and Bio-Gel P2 packings as a function of mobile-phase composition. Polman and Quigley (8'36)investi ated a number of SEC systems for the analysis of alkali-solu%ilizedcoals. Emphasis was placed on eliminating adsorption effeds. Mobile-phase optimization of SEC of humic acids and related components was reported by Min'ko et al. (F37) and Krasyukov and Lapin (F38). Kononov et al. (F39) were able to separate siloxanes with polar end groups (-OH, -OLi, -ONa, -OK) from regular siloxanes of similar molecular weights based on adsorption on Styragel columns. The thermodynamics of adsorption of dioxins, furans, and polychlorinated bi henyls on an SEC polystyrene packing with chloroform and t$chloromethane was studied by Bicking and Wilson (F40). Hewfort (F41)showed that the size of phenolic inhibitors is increased by solvation with THF. These inhibitors could then be size-separated from common monomers that lack hydroxyl functionality necessary for hydrogen-bond formation with THF. Johnson et al. (F42)discussed mobile-phase optimization of lignins. Hvilsted et al. (F43) re orted that aliphatic diphenyl esters in THF, toluene, and!chloroform result in esteraolvent interactions which obscure SEC analysis. Isotactic polypropylene and ethylene copolymers were analyzed using a binary solvent consisting of cyclohexane and decalin (F44).A mixture of dichloroacetic acid and a chlorohydrocarbon was used for the SEC of aromatic polyesters (F45).Fluoroacetylatd derivatives of Nylon 66 were analyzed by SEC (F46). Ono (F47) reported the analysis of a number of engineering plastics at 40 "C using Shim-pak GPC-80 columns. Polymers and associated mobile phases were as follows: polysulfone-dimethylformamide; poly(thioether sulfone)-chloroform;polyimide-dichloroacetic acid/chloroform (1:9); poly(ether imide)-dimethylformamide;poly(parabanic acid)-dimethylformamide; poly(meth 1pentene)chloroform/ l,l,l-trichlorotrifluoroethane(8/2); e dyleneviny1 alcohol copolymedimethylformamide. A patent was issued that described the SEC of heat-resistant polymers in nonaromatic aprotic dipolar mobile phases at 165-260 "C with silica packings, e.g., poly(thieppheny1ene) in N-methylcaprolactam
(Fa).
G. DETECTORS One of the most rapidly advancing areas in SEC has been the use of molecular-weight-sensitive detectors for absolute molecular weight measurement of eluting components. These detectors can be used not only for absolute mass measurements but also for the study of polymer branching, determination of Mark-Houwink constants, and obtaining molecular size information. There has been also increasing interest in the use of UV, NMR, and IR detection systems for the characterizationof chemically heterogeneous polymers and blends. For related papers in these areas, see also section E under Universal Calibration, and sections I and K. General Information
Balke et al. (Gl)described a systematic approach for interpreting data obtained from multidetector systems. Yau ((32)reviewed new capabilities that are now possible with molecular-weight-sensitive detectors with emphasis on viscometric detectors. A new detection system involving an on-line osmometer was also introduced. Lesec and Volet (G3) used a triple detection system consisting of a low-angle laser-light-scattering photometer (LALLS), viscometer, and refractometer for the characterization of acrylate copolymers, dextran, pullulan, and polyacrylamide. The LALLS detector gave absolute molecular weights and the viscometer gave information on long-chain branching. Grubisic-Gallot et al. (G4) described an SEC detection system comprised of a viscometer, LALLS, UV spectrophotometer, and refractometer. This instrument was used to characterize ethyl methacrylate-deuterated methyl methacrylate block copolymer, styrene-methyl methacrylate block copolymer, and polystyrendimethyliloxane diblock 432R
ANALYTICAL CHEMISTRY, VOL. 64, NO. 12, JUNE 15, 1992
co olymers. Alain (G5) reported on the characterization of cchmides using on-line LALLS,viscometer, polarimeter, Egefractometer .
f
Mohcular-Welght-Senrive Detectors
Light-Scattering Detectors. Mourey and Miller (CS) discussed the importance of obtaining an accurate interdetector volume between a LALLS photometer and a concentration-sensitive detector. Several methods of measur' the interdetector volume using narrow-molecular-we' ht sa% are 'ven. Best results were obtained when p e f onsets were usefrather than peak maximum. Furthermore, the authors presented an approach that uses the LALLS as an absorption spectrophotometer. Jackson et al. (G7)used an multiangle laser-li ht-scattering (MALLS) photometer as an SEC detector to Jetermine the radius of gyration of polystyrene. For nearly monodisperse standards, measurements >10 nm were possible. For polydisperse samples, the radius of gyration had to be >18 nm. Johann (G8)used an online MALLS detector to determine the radius of gyration and the degree of branching of polystyrene and cross-linked polystyrene. Krull's group reviewed the use of LALLS for the characterization of biopolymers (G9) and used SEC and gradient HPLC with an on-line LALLS detector for protein analysis (GI(FC12). SEC-LALLS has been used for the characterization of pectins (C131,cereal 8-glucans (G14),starch (CIS), and K- and A-carrageenans (CIS). In the area of SEC-LALLS of synthetic polymers, ap lications include polyphosphazines (GI7), poly(viny1butyraly (CIS), polypropylene (GI9),irradiated polystyrene (C20),urea-formaldehyde resins (C21), and poly(macromers) (C22). Viscometers. New developments in the area of on-line SEC viscometers were presented by Yau and -workers (G23, G24). Included was a discussion of intrinsic viscosity distributions and calculating radius of gyration values from intrinsic viscosity data A commercially available single-ca iuary viscometer was described and evaluated by Kuo et al. rGW), Ekamis and Skinner (G26),and Lesec et al. (G27). A laboratory-constructed single-capillary viscometer was used b Safieddine and Heater (G28)as a detector for preparative of water-soluble polymers.
Sd
Miscellaneous Detectors
Evaporative Light-ScatteringDetector. This detector has also been referred to as a mass detector, light-scattering detector, or an evaporative analyzer. The principle of operation is that eluent from a column is nebulized into a heated chiminey, and the mobile phase evaporates and leaves behind solute particles. The stream of solute particles then pass through an incident beam,and scattered light is detected. The intensity of scattered light is a function of solute particle concentration as well as the size of the particles. Burkow and Henderson (G29, G30) used this type of detector for the analysis of polymers from autoxidized fish oils. Karlsson ((331) described the application of an evaporative light-scattering detector for peat-derived liquefication products. Inductively Coupled Mass Spectrometer. Mason et al. (G32) reported on the feasability of coupling SEC to an ICP-MS instrument for the separation and elemental analysis of metalloproteins in biological samples. ICP-MS was used as an SEC detector for the analysis of iron-con (G33), lead and other trace elements in blood ( 34), proteins and metal-containing compounds in feedstock tower bottoms (C35). Infrared Spectrometer. Dekmezian et al. ((236)described an interface to collect solvent-free lymer fractions for ETIR analysis. Eluent is nebulized anEprayed onto KBr plates. This technique was used to determine the com ositional heterogeneity of ethylene in ethylene-propylene ruibers and the analysis of block copolymer reaction roducts. Jansen (C37) developed a thermospray moving-ielt interface for FTIR detection. Nishikida et aL (C38)reported on the des' of a high-temperature SEC-FTIR system and used it to termine short-chain branching in high-density and low-density polyethylenes. Markovich et al. (G39) also used an on-line FTIR detector for characterizing polyethylenes and acrylic
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SIZE EXCLUSION CHROMATOGRAPHY
acid-ethylene copolymer. Other applications of SEC-FTIR have been reported for the characterization of pro ellants (G40),cellulose esters (G41),and poly(viny1 chloride7 (G42). NMR Spectrometer. Hatado’s oup has been active in the use of NMR as an on-line SEFdetector. The instrumentation was described in refs G43-G44. Applications included determining the number-average molecular weight of poly(methy1 methacrylate) which contained one terminal tert-butyl group per chain ((343, G44). In addition, the instrument was used for characterizing butyl methacrylatemethyl methacrylate block copolymer and chloral oligomers (G45,C46). Optical Activity. An SEC on-line optical activity detector was used by Heyraud and Rinaudo (G15, G47) for the characterization of cyclodextrins and starches. Swadesh ( G M ) described a commercial on-line laser polarimeter for HPLC.
ti. PACKINGS The most popular packing for organosoluble polymers continues to be cross-linked polystyrene. Advances in this area have focused on improving polymerization processes for increased particle stability toward different mobile phases. Although most high-performance packings are in the range of 10 pm, there has been some development re arding smaller size packings for higher column efficiency. ith respect to aqueous SEC, hydrophilic organic-based packings appear to be the packin type of choice, and we see more effort in the synthesis of ties, materials. For a general review of SEC packings, consult H1.
+
Slllca-Based Packlngs
Czok and Guiochon (H2) introduced columns packed with bundles of aligned porous silica fibers that are 18 pm in diameter and have an average pore size of 270 A. Since these columns have low interstitial volume, the pore volume per column is higher than conventional packings. An in situ silaniziation procedure was used to modify the packing with a diol stationary phase. Berek and Novak (H3) described the properties of a surface-crazedsilica packing. Lee and Jarrett (H4) prepared silica packings consisting of a covalently bond glucose stationary phase, and evaluated these columns for protein separation. A patent was issued for the synthesis of porous glass ceramic beads, which were evaluated for use as an SEC packing (H5). Organic-Based Packlngs
Meehan et al. (H6) evaluated mixed-bed columns, which contain mixtures of individual pore-size cross-linked polystyrene packings. The authors suggest that for the analysis of polymers above 1 x 106 g/mol, 10-pm particles, rather that 5 pm, should be used to avoid polymer shear degradation. In addition, the larger size packing should be employed at high temperatures (100-150 “C) for increased column life. A comparison was made between narrow- and broad-standard calibration approaches, and both were found to give similar results. Kulin et al. (H7) described the preparation of macroporous poly(styrene-divinylbenzene) particles using a multistep swelling process. Packings of 5,10, and 20 pm were evaluated in terms of column efficiency and resolution. Finally, the theoretical prediction that HETP is pro ortional to the square of the particle diameter was confiimefby these authors. The review of hydrophilic polymeric packings for biopolymers was written by Makino and Hatano (H8). Hirayama and Ihara (H9) discussed the polymerization chemistry involved in the production of hydrophilic packin s. Meehan et al. (HIO)evaluated Aquagel packings, whic! consist of polyhydroxy and polyacrylamide surfaces. Universal calibration, which was found to hold with these packings, was used for the analysis of poly(viny1 alcohols). A detailed study of Superdex 75 was presented by Drevin et al. ( H I I ) . These authors studied the effect of mobile-phase pH and ionic strength on the elution behavior of protein with different pZ values. The evaluation of macroporous glycidyl methacrylateethylene dimethacrylate copolymers was reported by Tennikova et d. (H12). Hirayama and ceworkers (H13)described the preparation of poly(y-methyl L-glutamate) packings for
SEC. A glucomannan packing was prepared and evaluated by Morita and colleagues (H14, H15).Patents were awarded for a cellulose packing (H16) and for the preparation of an SEC su port by suspension polymerization of acrylonitrile with a $vinyl monomer (H17).Finally, cross-linked hemicellulose (xylan), called xylogel, was evaluated as an SEC packing (H18).
I. COMPOSITIONAL HETEROGENEITY This section deals first with the use of SEC and selective detectors to determine the chemical composition distribution in copolymers,blends, and other complex polymeric materials. The second part covers the application of interactive liquid chromatographic modes of separation for measuring compositional heterogeneity in these types of polymers. In this approach, the polymer is usually fractionated first according to molecular wei ht, either by SEC or solvent-nonsolvent fractionation, an8 then characterized by an LC method. SEC wRh Selective Detectors
Garcia-Rubio and co-workers (11,12) used simulation studies to investigate joint molecular weight-chemical comosition distribution and secondary fractionation effects of inear copolymers. Kilz (13,14) used a four-detector system consisting of a multiangle laser-light-scattering photometer, refractometer, UV detedor, and an on-line viscometer for the characterization of block and statistical copolymers as well as for star- and comb-shaped polymers. Grubisic-Gallot et al. (15) also used a four-detector system for the complete characterization of several different types of block copolymers. In their system, a low-angle laser-li ht-scattering photometer was employed. Trathnigg (16,18 used an on-line density detector in series with a refractometer to characterize the composition of poly(ethy1ene 1ycol)-poly(propy1ene glycol) blends and copolymers of ettylene oxide and tetrahydrofuran. UV spectro hotometric detection is the most widely used approach for fetermining chemical heterogeneity of complex polymers. Mork and Priddy (18) determined the extent of phenolic end-capping in bisphenol A polycarbonates using SEC-UV detection. Warner et al. (19) used SEC-UV to determine the amount and location of functional groups in styrenic polymers. Pasch and co-workers (110) relied on W detection for the analysis of polymers containing UV stabilizer units. UV-SEC was used for the characterization of polystyrene-poly(2,4-dimethyl-l,Cphenyleneether) blends (111) and polyester-polycarbonate blends (112). Podzimek et al. (113)used SEC-UV for determining the composition of polyesters. The compositional heterogeneity of nonstoichiometric polyelectrolyte complexes between polymethacrylate and poly(N-ethyl-4-vinylpyridiniumbromide) was investigated by Efremov et al. (114) usin SEC-UV. Styrene-propylene oxide block copolymers (1157 and linear and cyclic siloxane cowlwners (116) also were characterized - usingthis technique. Palladino and &hen (117)used a photodiode array detector with SEC to measure second-order derivative spectra of DOlypeptides. By comparison of these s ctra to t h b e of known amino acids and heterodipe tides, igntification of the composition of unknown pepti es was possible.
P
B
Interactive Llquld Chromatography
Gl6ckner’s two books on liquid chromatography of polymers are excellent and comprehensive texts on interactive HPLC of polymers as well as SEC (AI, A2). This author also presented a brief review on the use of HPLC to elucidate end-group effects, chain configuration of oligomers, and copolymer com ition distribution (118). Glijckner and Mueller (119) studieEhe elution behavior of statistical and block copolymers of styrene and tert-butyl methacrylate on silica with isooctane-THF gradients and on a phenyl-bonded packing using methanol-THF gradients. Glockner and Barth (120) presented an overview of normal-phase and reversedphase HPLC for copolymer characterization. During this review period, Mori published a series of studies dealing with gradient liquid adsorption chromatography of styrene copolymers of methac lates and acrylates (121-127) and poly(styrene-vinyl acetate7 block copolymers (128). For ANALYTICAL CHEMISTRY, VOL. 64, NO. 12, JUNE 15, 1992
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SIZE EXCLUSION CHROMATOGRAPHY
these separations, the mobile-phase composition consisted of chloroform-ethanol or 1,2-dichloroethane-ethanol. Shultz and Engelhardt (129) discussed the advantages and limitations of high-performance recipitation chromatography (HPPLC). This study was un&rtaken with polystyrene on normal-phase and reversed-phase packings. This group (130) also used HPPLC for the characterization of statistical acrylonitrile-styrene copolymers. Martire and co-workers (131) investigated the elution behavior of polystyrene using isocratic and gradient reversed-phase HPLC with methylene chloridemethanol mobile phases. Chiantore (132) reported on reversed-phase and normal-phase HPLC of polystyrene. Litvinova et al. (133) studied various experimental parameters of TLC of polystyrene utilizing silica-coated plates of different pore sizes. Products from the grafting of methyl methacrylate onto ethylenepropenediene rubber were characterized by Augenstein and Stickler (i34). SEC and gradient HPLC cross fractionation were used in which an evaporative light-scattering detector was employed for the latter separations. This group (135)also used the cross-fractionationapproach for the characterization of statistical and block copolymers of decyl methacrylate and methyl methacrylate. These authors demonstrated that the block copolymers were more highly retained than the statistical copolymers of similar composition using normal-phase HPLC. They concluded that the elution of the block copolymers seemed to be controlled by the length of the methyl methacrylate block. Hunkeler et al. (136) and Gorshkov et al. (137) investigated the concept of critical conditions in SEC. In short, critical conditions describe the point a t which the enthalpic and entropic energies are in balance. As a result, the polymer elutes at the total permeation volume of the column. Thus any deviation from the total elution volume signifies an imbalance between these thermodynamic parameters, suggesting a change in molecular weight or chemical composition. Hunkeler et al. (136) reviewed the concept of critical condition chromatography and studied this mechanism with respect to polystyrene and poly(methy1 methacrylate). Gorshkov et al. (137) used critical chromatography to determine the structural inhomogeneity and functional group distribution of copolymers of ethylene oxide and propylene oxide. In addition to the studies by Mori (121-127), the compositional heterogeneity of styrenemethacrylate copolymers was also reported by others us’ gradient reversed-phase (138-140) and normal-phase HPL?f140,141). It is of interest to note that Sat0 et al. (140) utilized an acrylonitrile copolymer packing with a nonpolar mobile phase (normal phase) and a cross-linked polystyrene packing with a polar mobile phase (reversed-phase) for the characterization of methyl methacrylate-styrene copolymers. Asada et al. (142) determined the chemical composition distribution of acrylonitrilebutadiene copolymers using a cross-linked 2-chloroacrylonitrile packing with gradient elution of chloroform-hexane. Schimmel et al. (143) used adsorption chromatography to separate hydroxy-terminated dimethylsiloxanes from linear and cyclic dimethylsiloxanes. Huang (144)used ion-exchange HPLC with a sodium chloride gradient elution to study the chemical heterogeneity of cationic acrylamide copolymers.
J. PHYSIOCHEMICAL STUDIES For a survey of other modes of liquid chromatography to measure physiochemical parameters, please consult the Physiochemical Studies section of the Column Liquid Chromatography review article in this issue. Biopolymers
Structure/Conformation Studies. SEC remains a popular method for estimation of protein molecular weight and/or hydrated volume under native conditions, and representative examples are presented in the Selected Applications section of this review. Stuting and Krull ( J I )describe the use of SEC-LALLS for the determination of growth hormone molecular weights under native eluent conditions. The use of second-derivative UV absorption spectroscopy with SEC for the characterization of interleukin-2 (E-2) and mutants was described by Ackland and colleagues (J2). The sensitivity of intrinsic tyrosine and 434R
ANALYTICAL CHEMISTRY, VOL. 64, NO. 12, JUNE 15, 1992
trypto han residues to the protein microenvironment permittexdetection of altered conformational states of certain IL-2 mutants. The use of denaturants for the investigation of protein subunit associations,protein folding, and protein stability by SEC methods was reported by many investigators. Valuable reviews of the application of SEC to the analysis of protein folding can be found in refs B13-Bl5. This ex erimental ap roach uses SEC to determine the apparent hy8odynamic vokkne of a protein across conditions that lead to progressive protein dissociation and/or folding and unfolding. Examples of such conditions include the presence of urea, guanidinium salts, and low pH. The reversible fold’ /unfolding reactions of monomeric proteins studied using %C methods included chicken cystatin (J3), human stefin A (J3, J4), and eye y11-crystallin (J5).The folding pathways of growth hormones from several species, as well as their chemically modified derivatives, were investigated in detail (J6,J7), yielding critical information on folding intermediates and on the stabilizing effects of intrachain disulfides. The effeda of freezing low-pH solutions of ovalbumin on the conformation and ag egation state of the protein were described by Koseki et al. 88).The conformational states and associations of rabbit skeletal tropomyosins were studied under low-pH conditions by Crimmins and Holtzer (J9). The association states and folding pathways of several multisubunit proteins were analyzed by SEC. The unfolding of the dimeric enz e aspartate aminotransferase and a truncated mutant G v e d from the enzyme was studied by Herold and colleagues (J10,J11). The SEC resolution was sufficient in this case to distinguish the hydrodynamic changes accom anying subunit dissociation, formation of folding intermeiates, and the final unfolded monomer. The foldin athway of bacterioph e P22 tailspike protein was studie! gy Fuchs et al. (JI2). %n renaturation, the tailspike polypeptide was observed to first form a structured monomer, which then associated to form a trimer, and in turn, slowly folded to the native trimeric protein. It should be noted that in many of the cited studies of protein folding and unfolding, various spectroscopic techniques (i.e.’, fluorometry, spectrophotometry, circular dichroism) were used in conjunction with SEC analyses. In most cases excellent agreement between SEC changes and spectral changes were observed; however, it is prudent to use SEC methods in conjunction with additional measures of protein conformation. The complementarity of SEC and circular dichroism for the investigation of protein folding is well illustrated in a review by Becktel and Lindorfer (B15). The structure of the eptidoglycan of Staphylococcus aureus was investigated y! selective hydrolysis, followed by analysis of the fragment distribution by SEC (J13)to investigate the mechanism of cross-link formation. In order to study the mechanism of gelatin hydrolysis by hydrochloric acid, Sumita et al. (J14) examined the molecular wei ht distribution of liberated products. Wang and ceworkers (&SI describe the coexistence of double-helical and monomeric xanthan conformers at low ionic strength. Association/Ligand Binding. An overview of SEC methods for the study of protein-ligand interactions and protein association was presented by Sebille (BI2). Casey and Reithmeyer (J16)described the oligomerization state and cytoskeletal associations of deter ent-solubilized Band 3 (anion transport) protein of human cell membrane. Analysis of the aggregation state of another isolated human red cell membrane protein, the glucose transporter, was carried out using SEC-LALLS (JI 7). Detergent-solubilized calcium-ATPase monomer-dimer equilibrium was studied using SEC-LALLS (J18), in order to characterize the state of the enzyme during the catalytic cycle. Sone and Takagi (JI9) described the use of SEC-LALLS in detergent-containing eluents for measuring detergent-solubilized- rotein relative molecular mass and mass of protein-bound fetergent. This approach was illustrated by the characterization of the structures of cytochrome c oxidase and the cytochrome b-cl complex. By a combination of SEC and small-angle X-ray scattering, Harada et al. (J20)studied the TF1 ATPase, concluding that the minimal a6 heterodimer reversibly associates to an a3j3 catal is. Martel and colleagues (J21)described ZELdo%c angff-line small-angle neutron scattering for
ret!
SIZE EXCLUSION CHROMATOGRAPHY
the hydrodynamic characterization of smaller polypeptides, as exemplified by a cobra neurotoxin and analogues. The cyanide-linked monomeldimer equilibrium of Chromatium uinosum ferricytochrome c’ was studied, and the relative pH independence of the equilibrium was observed (522).Preliminary SEC studies on the dimerization of HIV-1 aspartic proteaae showed an ionic strength dependence (J23). Applications of size exclusion methods for the analysis of immunochemical reactions and refined techniques for the kinetic analysis thereof were described in detail by Stevens (J24).In a further study, using similar kinetic analyses (J25), the subunit association equilibria of two dimeric venom phospholipases A2were investigated as a function of pH and calcium concentration. The self-association of Rep helicase and its interaction with DNA was detailed by Chao and Lohman (J26).The association state of the helicase was determined at various concentrations, in the presence of nucleotide cofactors, and on binding to both short and long single- and double-stranded DNAs. Protein cross-linking studies of the DNA-bound protein were conducted. On the basis of these experiments, the authors demonstrate that the active helicase results from DNA-induced dimerization of the Rep protein. A highly sensitive method for the analysis of peptide antigen complex formation with a major histocompatibility complex protein, HLA-DR 1, was reported (J27). In this assay, the fluorescent signal generated by intrinsic tryptophan residues of the HLA-DR 1increased on complex formation. Binding of a specific peptide was demonstrated for both the intact heterodimer and its dissociated subunits. A technique for the determination of calcium ions ti htly bound to proteins was described by Nitta and Watanabe b28). The protein-calcium complex was dissociated in a postcolumn reador following SEC separation, allowing free calcium to be detected by fluorometry using the specific indicator Quin-2. Dihydrotestosterone binding to the androgen binding protein of rat tissues was analyzed by Fremont et al. (529). Binding site kinetics for the association of the acidic cephalosporin, ceftriaxone, to human serum albumin and to albumin in diluted serum samples was determined by the HummelDreyer method (J30). The binding of warfarin to human serum albumin was used to demonstrate the application of a specialized SEC-interactive column, the internal surface reversed-phase (ISRP) column, by Shibukawa and colleagues (J31)using frontal analysis and by Pinkerton and Koeplinger (J32)using a modified Hummel-Dreyer method. The latter report includes a useful discussion of analytical conditionsand appropriate techniques of data reduction for drug-protein interaction studies. Synthetlc Polymers
Inverse SEC. With inverse SEC, the material to be characterized is packed into a column, and standards are injected to probe the pore structure of the packed material. Yan et al. (J33)presented a review of this technique for the pore structure of wet polymeric porous ma%=%?eke and Brueckner (J34)used SEC to determine the porous structure of activated carbon samples and compared these results to those obtained from other techniques. Jerabek and Setinek (J35)studied the structures of stron acidic ion-exchange resins using deuterium oxide, sugars, an dextrans as probes. Jerabek (J36)also characterized the porous structure of regenerated pearl cellulose swollen in water or tetrahydrofuran. Branching Mark-Houwink Constants(Conformation. Vilenchik et (537)used a rather interestmg approach for characterizinga single sample of an unknown polymer. From an intrinsic viscosity measurement, the Huggins constant is f i t determined from which the Mark-Houwink exponent can be calculated from a proposed relationship. From a @venial calibration experiment, the Mark-Houwink K value IScomputed. From these data, the molecular wei ht distribution of the unknown polymer m be generated. &e authors claim that this approach can be applied to any homogeneous linear flexible-chain nondraining macromolecule. Gonzalez et al. (J38)derived a relationship for determining the flexibility parameter of a polymer, defined as the ratio of persistent length to monomeric unit projection length, as a function of molecular weight using universal calibration. In
d
$.
addition, the projection len h also was determined. Siochi et al. (539)used SE with a viscometer and low-angle laser-light-scattering detector (LALLS) to obtain branching parameters and unperturbed chain dimensions of model grafted oly(methy1 methacrylates). Kuo et al. (J40)determined t1e degree of branching in poly(viny1 acetate) using a viscometric detector. Johann and Kilz (J41)reported on the characterization of the structural heterogeneity of a star-shaped block polymer and polystyrene microgels by examining the dependence of the radius of gyration on molecular weight. Long-chain branching in polyethylene using SEC-LALLS or viscometry was discussed by Pang and Rudin (J42). SEC-LALLS was also used by Dickie and Koopmans (J43) and Zhang et al. (J44)for determining long-chain branching in irradiated polyethylene. Huber (J45)used a combination of SEC-LALLS with universal calibration to determine Mark-Houwink constants, olymer-particle-equivalentradii, and mass density distrigution of p u l l h , short-chain branched dextrans, and highly branched starch derivatives. This researcher also employed this technique to examine aggregation in hydrolyzed starch solutions (546). Partition Coefficients/Association. Tji et al. (J47) presented a method for determining the charge of ions from SEC partition coefficients. In SEC, the gel particles and eluent represent two phases of different polarity. If a solute ion Pz is added to an eluent containing electrolyte NX, the transfer of an ion Pz+from the eluent into the gel phase must be accompanied by a simultaneous transfer of z ions X- from the eluent to the gel phase. Thus the partition coefficient of Pz depends on z. This method was verified with inorganic ions using two eluents containing different electrolytes over a range of ionic strengths and Bio-Gel P-4 as the packing. Prochazka et al. (J48,J49)developed a theoretical model to describe the SEC behavior of reversibly associating block copolymers (micellizingsystems). The model takes into account fast e uilibration of the unimer (molecularly dissolved copolymer) ?Ietween mobile and stationary phases, the continuous perturbation and reestablishment of unimel-micelle equilibrium, and axial dispersion. Winnik’s group (J50)used SEC to study the adsorption of styrene-poly(ethy1ene oxide) diblock copolymer onto polystyrene latices. In the presence of excess block copolymer, latex, micelle, and unimer peaks were observed. This work was also extended to triblock copolymers (J51). Dejardins and Eisenberg (J52)studied the colloidal pro erties of styrene-metal (sodium or cesium) methacrylate block copolymers using SEC and found no apparent micelle dissociation-association equilibrium taking place in good solvents for the polystyrene blocks. Funasaki et al. (J53) determined the aggregation properties of poly(ethy1ene glycol) dodecyl ethers by frontal SEC. Simulation studies based on plate theory were used to support their findings re arding the aggre ation number of micelles that were observecf Jennings et al. $554) used SEC to characterize asphalts and to determine their relative ability to self-assemble. Polymer Degradation and Kinetic Studies. Chiantore (J55) resented a review on the use of HPLC to study polymer degraxation. Enamples given included the use of SEC to track chain scission of polystyrene and the thermal degradation of styrene-acrylonitrile copolymers. Mohammadi et al. (J56) used SEC to follow chain scission during fracture of lassy polystyrene. Yamada and Larsen (J57)utilized Sic to measure the kinetics of coal conversion to soluble products and analyzed these results using kinetic schemes based on the behavior of cross-linked macromolecular networks. SEC was used to study cure kinetics of epoxy amine formulations (J58),and polymerization kinetics o l,l’-bi-lHindene (J59),phenol-formaldehyde resol resin (J60),and polyurethane prepolymer (J61).
8
8-
!
K. MICROCOLUMN SEC Cortes et al. (K1)coupled an SEC microcolumn to a ca-
illary GC column for on-line MS of polymer additives. An gEC microcolumn was evaluated for the analysis of triglycerides and steroid esters by Ghijs and co-workers (K2). Ruban (K3)reported on a suspension procedure for packing
