Handbook of Size Exclusion Chromatography and Related ...

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22 High Osmotic Pressure Chromatography Iwao Teraoka and Dean Lee Polytechnic University Brooklyn, New York, U.S.A. 1 INTRODUCTION High osmotic pressure chromatography (HOPC) was developed in 1995 as a tool for preparative separation of polydisperse polymers by molecular weight (MW) using analytical-size columns (1). Since then, HOPC has been applied to separation of various polymers, demonstrating a high resolution and a large processing capacity (2–10). In HOPC, a concentrated, viscous solution of polymer is injected into a column packed with porous materials. The concentration is much higher than the overlap concentration; the solution is in a semidilute range. The pore diameter must be sufficiently small to exclude most of the polymer at low concentration but not too small to exclude low-MW components at high concentrations. The injection continues typically until the whole column is filled with the solution. Upon detecting the first polymer in the eluent, solvent is injected to wash the column, and the eluent is collected by a fraction collector. The collection continues until the eluent concentration drops to a low level. Any soluble polymer can be separated by HOPC. Advantages of HOPC over conventional preparative-scale chromatography include a high processing capacity and a high resolution. The latter requires fine-tuning of the separation © 2004 by Marcel Dekker, Inc.
condition, as is explained in this chapter. The injected solution is concentrated, as is the eluent. Therefore, it is easy to recover solid polymer in each of the fractions. With minimal consumption of often hazardous organic solvents, HOPC is an environmentally friendly separation method for a wide variety of polymers. The first half of this chapter explains the separation principle of HOPC in a good solvent condition. A couple of examples of separation are given. The second half focuses on recent extension of HOPC into theta solvent condition. The latter solvent allows a superior resolution and a greater processing capacity compared with the good solvent. In particular, use of weakly adsorbing porous packing makes it possible to produce narrow-distribution fractions from early to late eluent, yet rejuvenating the column at the end of each batch. 2 SEPARATION PRINCIPLE: HOPC IN A GOOD SOLVENT The separation in HOPC is based on partitioning of a concentrated solution of polymer between a pore space (stationary phase) and a surrounding unconfined space (mobile phase). When the polymer is monodisperse and the solution is dilute (much lower than the overlap concentration c*), the partition coefficient K is a sharply decreasing function of MW of the polymer (dashed line in Fig. 1). This principle is widely used in size exclusion chromatography (SEC). As in Figure 1 Partition coefficient K is schematically drawn as a function of MW in a logarithmic scale. Dashed lines, low concentrations; dash-dotted lines, higher concentrations; monodisperse polymer, solid lines; higher concentrations, polydisperse polymer. © 2004 by Marcel Dekker, Inc.
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MARCEL DEKKER Handbook of Size Excl
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CHROMATOGRAPHIC SCIENCE SERIES A Se
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60. Modern Chromatographic Analysis
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Preface to the Second Edition Gel p
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Preface to the First Edition Molecu
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Contents Preface to the Second Edit
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17. Size Exclusion Chromatography o
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James F. Curry Analytical Departmen
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Iwao Teraoka, Ph.D. Othmer Departme
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The graphical data display typicall
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polymer molecule can elute. The tot
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individual states) allowed to them.
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unsaturation while the IR detector
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Sampleconcentrationshouldbeminimize
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averagesdefinedintermsofthemolecula
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information regarding appropriate M
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Analytical. Two models, the KMX-6 a
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Figure 6 Overlay of time-sliced pea
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accurately for very large and very
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4 GENERAL REFERENCES The interested
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46. TA Chamberlin, HE Tuinstra. US
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materials are commercially availabl
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Table 1 Commercial Column Packing M
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necessarily comparable. For this re
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adjusted such that the pore size di
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narrowmolecular weight range, indiv
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Figure 8 Effect of particle size on
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Figure 9 SEC calibration using poly
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Table 4 Typical Eluent Systems for
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Figure 12 Analysis of poly-2-vinyl
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24. E Meehan, S O’Donohue. The ro
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into accepted laboratory techniques
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Table 1 Trace Metal Impurities in C
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mass and biological activity, for m
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Experimental efficiency vs. velocit
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Table 3 Properties of SEC Silica Ge
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operate at aflow rate that can easi
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Figure 3 Efficiency of amide-bonded
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mechanically stable packing materia
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Figure 5 Pore size distributions of
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Figure 6 Separation of polystyrenes
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60-120 A ˚ are large enough to be
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Table 6 Selected Silica-Based Colum
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Table 7 Separation Ranges for Polym
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the decline in permeability is perm
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sample components can be varied by
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Figure 10 Diol bonding reactions. I
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ecause of the higher relative molec
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valuesfork1,k2,anda,theunknownmolec
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water-soluble and detergent-soluble
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the importance of secondary retenti
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discussed in Table 8(33). Based on
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individual dispersion processes ins
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The detector time constant can dist
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5.3 Mobile Phase A mobile phase is
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less steep and still linear at high
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35. P Bristow, J Knox. Chromatograp
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112. K Gooding, K Lu, G Vanecek, F
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for branched polymers or copolymers
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Figure 1 “Bridge design” flow-r
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Figure 3 A light-scattering photome
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Figure 5 SEC universal calibration
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where hn is the hydrodynamic volume
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It should be noted that dn=dc also
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sensitivity for many samples compar
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that is, log[h] vs. logM,generated
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Figure 8 Effect of band broadening
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Table 1 Measurement of Molecular We
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Figure 10 Differential refractomete
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Figure 11 (A) Mark-Houwink plot of
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Table 3 Measurement of Molecular We
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size information is derived from un
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Table 5 Molecular Weight Distributi
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By measuring the scattered intensit
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8 APPENDIX: INSTRUMENT COMPANIES 8.
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53. G Marot, J Lesec. J Liq Chromat
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125. D Slootmaekers, JAPP Van Dijk,
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200. JG Bindels, GJJ Bessems, BM De
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possibly with hydrodynamic volume (
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An insurmountable limitation of SEC
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to sDR(V) when either nPS ¼nPB ¼c
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1,4-trans;and8%1,2-vynil.TheglobalC
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The goal is to find the MWD and CCD
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As expected, pS is closer to the no
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Figure 3 Example 3: MWD and BD of a
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9. BH Zimm, WH Stockmayer. The dime
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45. RO Bielsa, GR Meira. Linear cop
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solubility properties. They are use
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determination of the absolute molec
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Figure 4 Result of the PBT analysis
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Figure 8 Result of natural spider s
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7 Size Exclusion Chromatography of
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As pointed out earlier, rubber must
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Table 2 Various Solvents and Operat
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Figure 2 Typical GPC calibrations w
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© 2004 by Marcel Dekker, Inc. Figu
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Natural and Synthetic Rubbe177 vari
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Another example, shown in Fig. 5 (3
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APPENDIX: SEC CONDITIONS FOR RUBBER
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Appendix (Continued) Polymer Column
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5. J West, E Rodriguez. Rubber Chem
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associate. Girdler (67) and Speight
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Table 1 Fractions Obtained Using Co
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Figure 1 SEC analyses of an aged as
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Apparently more polar compounds are
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40% was fractionated into four frac
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Figure 7 SEC analyses of an unaged
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Figure 9 Comparison of percentage L
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Figure 11 Comparison of SEC chromat
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Figure 12 Comparisons of SEC chroma
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percentage LMS at the other locatio
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the total area into up to 12 sectio
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attempted to develop an SEC method
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parameterisbasedontheinternalpressu
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5 MODIFIED ASPHALTS The addition of
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Oxidation of the rubber and asphalt
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Figure 20 SEC chromatogram for an a
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Figure 21 Comparisons of apparent m
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© 2004 by Marcel Dekker, Inc. S/
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PS/10 4 þ 10 3 þ 500 þ 100 THF/1
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Nevertheless, SEC of asphalts is es
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56. NW Garrick, RR Biskur. Trans Re
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Asphalts 235 122. M-S Lin, JM Chaff
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Table 1 Applications of Acrylamide
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(AM/AA), and acrylamide/dimethyldia
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polymer, a large size filter of 5,
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Figure 1 Size exclusion chromatogra
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Table3 MWandMWDofaBroad-MWDPAMStand
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Another commercially available MW d
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Table 5 Weight-Average Molecular We
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Figure 6 (a) Size exclusion chromat
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compared to anormal product. By com
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StudyingtheKineticsofaChemicalReact
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this information, the higher MW pea
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46. CJ Kim, A Hamielec, A Bendek. J
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A relatively new technique utilizin
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where ais the exponent of the Mark-
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Figure 2 TDS chromatograms for full
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Figure 5 Comparison of molecular we
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Table 3 Summary of TDS Molecular We
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thepartiallyhydrolyzedPVAwiththecol
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Table 5 Summary of Conformation and
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a¼0.627andlog(K) ¼23.249.Theseare
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The limits of detection of the PVA
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Figure 12 PVAc broad molecular weig
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4 SUMMARY Advances in SEC character
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2.1 Molecular Weight Grades of PVP
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exclusion chromatography with low-a
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weightandmolecularweightdistributio
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Figure 4 PEOcalibrationofUltrahydro
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Figure 6 Overlay of GPC chromatogra
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plus Ultrahydrogel 120 A ˚ ,Shodex
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Figure 10 SEC traces of PVP/AA copo
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Figure 11 Molecular weight distribu
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2 CHEMICAL, MACROMOLECULAR AND MORP
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Table 2 The Relative Composition of
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considered to be 3. After the DS ha
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een extensively studied by methods
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The silylation was performed in LiC
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observed using column materials hav
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Table 6 (Continued) Packing materia
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4.5 Other Cellulose Derivatives Bes
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Table 8 SEC Conditions for Characte
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dextrans swell too much in cadoxen
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stirring at room temperature for an
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Figure 2 MMDofunbleached(HP)andblea
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Figure 4 MMDofbirchkraftpulpdegrade
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adequate when evaluating the influe
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differential refractive index (DRI)
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30. HA Krässig. Effects of structu
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67. E Sjöholm, K Gustafsson, A Col
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104. B Fleury, J Dubois, C Léonard
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136. D Miller, D Senior, R Sutcliff
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166. SS Cutié, CG Smith. Determina
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200. R Berggren, F Berthold, E Sjö
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13 Molar Mass and Size Distribution
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measurements, and vapor pressure os
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The calibration of SEC columns by c
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The division according to eluent ty
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Apolymer produced by UV irradiation
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Because DMAC/LiCl is also agood sol
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compoundsandwasexplainedbyadsorptio
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isolated lignins because they are e
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The Separon HEMA and Separon HEMA B
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Figure 4 GPC elution curves of the
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liquor is highest throughout the co
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topological anisotropy in the polym
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29. M Ristolainen, R Alen, J Knuuti
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eds. Proc Int Symp 1998. Canton, Pe
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92. L Jurasek. Towards a three dime
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Table 1 Selected Industries Connect
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into C3-metabolites, which then may
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Table 4 Key Enzymes in the Biosynth
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Table 5 Cloned Mutants of Starch En
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Figure 3 Modeled x-ray diffraction
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In the native environment (plant ce
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Figure 6 (a) Large starch granules
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number of branching points); crossl
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Both absolute approaches are extrao
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Table 7 (Continued) Approach Experi
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structures, excluded volumes, and t
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Figure 10 (a) SEC elution profile o
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Figure 12 Wheat glucans: DRI elutio
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Figure 13 Wheat glucans. Normalized
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Figure 16 Wheat glucan. Normalized
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Figure 18 Wheat glucan.Elution prof
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Figure 20 Wheat glucans. Absolute m
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Figure 22 Wheat glucan. (a) Intrins
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Figure 24 Wheat PA-glucan. Elution
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an Ubbelohde-viscometer for concent
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Rheological investigations of stabi
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shifts to applied laser light, whic
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Table 8 Characteristic Parameters f
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Table 8 (Continued) and H2O. A part
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10. SG Ball, MHBJ van de Wal, RGF V
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52. W Praznik, R Beck. J Chromatogr
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3 PROTEIN PARTITIONING IN SEC 3.1 G
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Figure 1 Plot of Kav vs. log M for
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yconsideringtheproteinsolutestobesp
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globular proteins and selected flex
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silanol groups (52-54); even capped
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most proteins to be maximally stabl
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Figure 4 Chromatograms of BSA and P
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concentrated) steps of protein puri
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Table 3 Characteristics of Dextran-
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preparative SEC. The capabilities o
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37. GR Noll, N Nagle, JO Baker, DJ
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Figure 2 Separation of total E. col
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Figure 3 Separation of HaeIII-cleav
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The recovery of DNA fragments has b
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Endotoxin should also be removed fr
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Table 3 Best Columns for the Separa
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Figure 10 Dependence of HETP on flo
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Appendix (Continued ) Polymer Colum
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REFERENCES 1. CT Wehr, SR Abbott. J
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MALDI-MS has been applied to determ
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Figure 1 Calibration curvesof SEC c
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Figure 3 Chromatograms of epoxy res
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wherenandn0 aretheRIsofthesample an
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Figure 5 SEC chromatogram of n-alka
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Figure 7 Refractive indexes of comm
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Another detector recently applied i
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Figure 11 Absolute MW calibration p
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Figure 12 Chromatogram of Acrawax C
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18 Two-Dimensional Liquid Chromatog
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largerispressuredropandresultingexp
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elations between molar masses and s
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Figure 2 (Continued) chapter, one o
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where Nis the column efficiency exp
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composition/architecture/molar mass
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solvent. Here again, solvent-solven
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or repulsive interactions between m
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macromolecules may strongly differ
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temperature. The efficacy of an ads
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however, aliphatic bonded groups ma
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Figure 10 Schematic representation
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packing materials for SEC of synthe
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Quantitative relations between sila
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Both strength and thermodynamic qua
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where Cand Dare constants for apart
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ThisisimportantwhenLCCCisappliedtoc
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However, strong adsorption of macro
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elution step may be sufficient for
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Figure 16 Block scheme of a full ad
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processing of 2D-HPLC data. Knowled
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Figure 20 Set of full retention-elu
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distributions of complex polymers a
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constituents were not discriminated
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(Sec. 7) and therefore they can be
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2. Identify sample solvents. First
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1 Segmental interaction energy para
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58. BG Belenkii. Pure Appl Chem 51:
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19 Methods and Columns for High-Spe
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Table 1 Summary of Methods for Incr
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not savealot of time and solvent, d
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Figure 3 Conventional SEC chromatog
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chromatogram at 4mL/min, which is f
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Sincethereductionoftheinternaldiame
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Table 3 Summary of Column Performan
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Figure 11 Dependence of column perf
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. Two-dimensional chromatography ru
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The standard deviations for the mol
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Figure 15 Comparison of time requir
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Figure 17 Accuracy and precision of
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polymer analysis. Samples are “ru
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just ignoring the fractionation of
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20 Automatic Continuous Mixing Tech
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out at low enough concentration tha
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educed viscosity, h r, to be comput
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Figure 2 Raw ACOMP signals from mul
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Figure 4 Mw vs. monomer conversion,
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decreases smoothly and monotonicall
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Figure 7 Effects of fluctuating rea
Page 617 and 618: Finally, the use of a light-scatter
Page 619 and 620: Figure 8 Linear and cubic increases
Page 621 and 622: where r(t) is now given by 2 6 r(t)
Page 623 and 624: Figure 11 Light scattering from sol
Page 625 and 626: 3.1 ASimple System: Equilibrium Cha
Page 627 and 628: Figure 13 (a) ACM applied to the ch
Page 629 and 630: Also of note in Fig. 13b is how hig
Page 631 and 632: 15. JM Starita, CL Rohn. Rheologica
Page 633 and 634: 52. M Hulden. Hydrophobically modif
Page 635 and 636: 21 Light Scattering and the Solutio
Page 637 and 638: therefore, is to remove any lingeri
Page 639 and 640: is often found in the literature wh
Page 641 and 642: Figure 2 Configuration of an SEC se
Page 643 and 644: 5 THE IMPORTANCE OF SOFTWARE Like a
Page 645 and 646: standard deviations of the measured
Page 647 and 648: These include the differential weig
Page 649 and 650: Figure 8 Data of Fig. 7corrected fo
Page 651 and 652: Figure 11 Data of Fig. 10 with spik
Page 653 and 654: Figure 13 Fitstodatacollectedatasli
Page 655 and 656: Table 1 Errors (in %) Characteristi
Page 657 and 658: homogeneous copolymer, that is, tak
Page 659 and 660: process may be used to identify the
Page 661 and 662: fractionated sample. They represent
Page 663 and 664: Figure 16 An example of poor SEC ch
Page 665 and 666: on) methods, whereby the average di
Page 667: 23. WH Stockmayer, LD Moore Jr, M F
Page 671 and 672: Figure 2 Partition coefficients KL
Page 673 and 674: 4 COLUMNS FOR HOPC Detailsaregiveni
Page 675 and 676: and concomitant clogging of columns
Page 677 and 678: Figure 5 Concentration dependence o
Page 679 and 680: Table 1 Solution Volume, Polymer Ma
Page 681 and 682: in Fig. 4a. The middle fractions (8
Page 683 and 684: Figure 10 SEC chromatograms for som
Page 685 and 686: appropriate for the early fractions
Page 687 and 688: 23 Size Exclusion/ Hydrodynamic Chr
Page 689 and 690: Figure 2 Calibration curve for the
Page 691 and 692: Figure 5 Elutionbehaviorofpolystyre
Page 693 and 694: Table 1 Comparison Between SEC and
Page 695 and 696: egular SEC column in which the pore
Page 697: Such an ideal separation can be obt
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