Handbook of Size Exclusion Chromatography and Related ...

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endered useful in the separation of polymers according to their stereoregularity (61,62) and for discrimination of linear and cyclic macromolecules with similar molar masses (63–65). Numerous critical systems are listed in Refs. 4, 5, and 66. LC CC was also included in two-dimensional HPLC (4,5,62,67). During application of LC CC, its numerous important experimental limitations were revealed (68,69). Most of them are mentioned at the end of this section. Unfortunately,the drawbacks of LC CC were ignored in some recent books (4,5). Cifra and Bleha (70) also showed by Monte Carlo modelling that the molar mass region in which perfect entropy–enthalpy compensation takes place may be limited (Fig. 4, curve 9). Still, LC CC remains attractive for characterization of many complex polymers both in direct (one separation system) and twodimensional arrangements. LC CC proved especially successful in molecular characterization of oligomers (4) where, for example, the problems connected with strong interaction of large polymer species with walls of narrow pores (Sec. 3.2.1) are less important and also relatively high concentration of analytes are experimentally feasible. The latter feature of oligomer HPLC, including LC CC, allows application of a less sensitive flow-through densitometer as an additional detection system (4). A situation similar to entropy/enthalpy compensation may also appear in systems where two enthalpic retention mechanisms, for example, adsorption and enthalpic partition, affect retention volumes in an opposite way (71). The following practical hints may be useful for LC CC users: 1. Whenever possible, the enthalpic partition retention mechanism is preferential. It is difficult to maintain the HPLC system at the critical adsorption point. Minute variation in eluent composition due to preferential evaporation and moisture absorption may strongly affect adsorption of polymer analytes. Consequently, the LC CC system must be frequently controlled and critical conditions adjusted for example by temperature variations. If, however, the adsorption mechanism has to be applied, try to identify both adsorli and desorli, which are as good solvents for the analysed polymer as are possible. Critical composition of the eluent, which is situated in the vicinity of the theta point or even near the precipitation threshold, may further increase instability of the critical adsorption point and increasingly deteriorate repeatability of measurements. 2. Avoid using very narrow pore column packings. The danger of backward curvature of critical calibration curves, as well as problems connected with peak broadening and decreased sample recovery decrease with rising pore diameter (47). Unfortunately, the selectivity of SEC separation for lower molar mass polymer species is at least partially sacrificed when narrow pore column packing is removed. © 2004 by Marcel Dekker, Inc.
ThisisimportantwhenLCCCisappliedtocharacterizationofpolymer blendsandblock-,graft-,orstar-copolymerswhereonekindofpolymer chains elutes under critical conditions, irrespective of its molar mass (chromatographic invisibility). Another kind of polymer chain is eluted under SEC conditions and its MMM/MMD is to be determined in the conventional way.Reduced separation selectivity of the latter chains affects the accuracy of the results obtained. 3. Single-component critical eluents are rather rare (30). Therefore, one is forcedtousemixedmobilephasesinLCCC.Ifpossible,binaryeluents are preferred over multicomponent eluents. Usually, experimental problems increase with the increasing number of eluent components. Temperature and pressure dependence of preferential sorption and preferentialsolvation(Sec.4.2)aswellaspreferentialevaporationfrom themobilephaseincreasinglycomplicatenotonlycriticalpointstability butalsosampledetection.Evaporativelightscatteringdetectors(ELSD) may be used to suppress detection problems, although the response of ELSD is often rather nonlinear and depends on polymer composition, molar mass, as well as on eluent composition (72) (Sec. 10). 5.2 Barrier Coupled Procedures When compared with HLPC of low molar mass substances, polymer HPLC exhibits several specific features. For example, in the case of porous particulate or monolithic column fillings, we are confronted with generally large differences between mobilities of macromolecular analytes and eluent molecules. With the exception of LC CC, macromolecules that are partially or fully excluded from the filling pores tend to move along the HPLC column much faster than molecules of eluent that penetrate most filling pores. This allows the creation of “barriers” of small molecules, which are impermeable for macromolecules possessing certain enthalpic interactivity. Such barriers force macromolecules to decelerate their progression along the column and to elute at the barrier edge. In other words, macromolecules accumulate on the corresponding barrier of small molecules and may elute irrespectively of their molar mass. This is a specific approach to the mutual compensation of entropic and enthalpic effects within HPLC columns. Adsorption-promoting barriers, that is, zones of liquids with low solvent strength, are utilized for adsorbing macromolecules. Enthalpic partition and phase separation barriers are created from thermodynamically poor solvents or nonsolvents for polymer analytes. Barriers can be . Continuous, created by the mobile phase, or . Local, formed by pulses of appropriate liquids. The local barriers can be single or multiple. © 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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Page 479 and 480:
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
Page 493 and 494: REFERENCES 1. CT Wehr, SR Abbott. J
Page 495 and 496: MALDI-MS has been applied to determ
Page 497 and 498: Figure 1 Calibration curvesof SEC c
Page 499 and 500: Figure 3 Chromatograms of epoxy res
Page 501 and 502: wherenandn0 aretheRIsofthesample an
Page 503 and 504: Figure 5 SEC chromatogram of n-alka
Page 505 and 506: Figure 7 Refractive indexes of comm
Page 507 and 508: Another detector recently applied i
Page 509 and 510: Figure 11 Absolute MW calibration p
Page 511 and 512: Figure 12 Chromatogram of Acrawax C
Page 513 and 514: 18 Two-Dimensional Liquid Chromatog
Page 515 and 516: largerispressuredropandresultingexp
Page 517 and 518: elations between molar masses and s
Page 519 and 520: Figure 2 (Continued) chapter, one o
Page 521 and 522: where Nis the column efficiency exp
Page 523 and 524: composition/architecture/molar mass
Page 525 and 526: solvent. Here again, solvent-solven
Page 527 and 528: or repulsive interactions between m
Page 529 and 530: macromolecules may strongly differ
Page 531 and 532: temperature. The efficacy of an ads
Page 533 and 534: however, aliphatic bonded groups ma
Page 535 and 536: Figure 10 Schematic representation
Page 537 and 538: packing materials for SEC of synthe
Page 539 and 540: Quantitative relations between sila
Page 541 and 542: Both strength and thermodynamic qua
Page 543: where Cand Dare constants for apart
Page 551 and 552: However, strong adsorption of macro
Page 553 and 554: elution step may be sufficient for
Page 555 and 556: Figure 16 Block scheme of a full ad
Page 557 and 558: processing of 2D-HPLC data. Knowled
Page 559 and 560: Figure 20 Set of full retention-elu
Page 561 and 562: distributions of complex polymers a
Page 563 and 564: constituents were not discriminated
Page 565 and 566: (Sec. 7) and therefore they can be
Page 567 and 568: 2. Identify sample solvents. First
Page 569 and 570: 1 Segmental interaction energy para
Page 571 and 572: 58. BG Belenkii. Pure Appl Chem 51:
Page 573 and 574: 19 Methods and Columns for High-Spe
Page 575 and 576: Table 1 Summary of Methods for Incr
Page 577 and 578: not savealot of time and solvent, d
Page 579 and 580: Figure 3 Conventional SEC chromatog
Page 581 and 582: chromatogram at 4mL/min, which is f
Page 583 and 584: Sincethereductionoftheinternaldiame
Page 585 and 586: Table 3 Summary of Column Performan
Page 587 and 588: Figure 11 Dependence of column perf
Page 589 and 590: . Two-dimensional chromatography ru
Page 591 and 592: The standard deviations for the mol
Page 593 and 594: Figure 15 Comparison of time requir
Page 595 and 596:
Figure 17 Accuracy and precision of
Page 597 and 598:
polymer analysis. Samples are “ru
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just ignoring the fractionation of
Page 601 and 602:
20 Automatic Continuous Mixing Tech
Page 603 and 604:
out at low enough concentration tha
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educed viscosity, h r, to be comput
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Page 609 and 610:
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)
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Figure 11 Light scattering from sol
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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
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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
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These include the differential weig
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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
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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 and 668:
23. WH Stockmayer, LD Moore Jr, M F
Page 669 and 670:
condition, as is explained in this
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
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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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