Handbook of Size Exclusion Chromatography and Related ...

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Figure 6 Elution profile of PA6. Eluent: HFIP þ 0.05 M potassium-trifluoroacetate. Flow rate: 1 mL/min. Columns: 2 PSS PFG LinXl, 7 mm, 8 300 mm. Temperature: 258C. Detection: RI. Standards: 12 PSS PMMA calibration standards. Biopolymers such as silk and the very versatile group, proteins, can also be regarded as polyamides. For protein GPC analysis we have observed that PSS- NOVEMA columns driven in aqueous solvents became the most successful (19). Owing to the complex structure of proteins, complex GPC methods are often used. The large theoretical background that is needed for protein analysis makes it necessary to describe it in a separate article (19). Figure 7 Result of the PA6 analysis: molecular weight distribution relative to PMMA calibration. © 2004 by Marcel Dekker, Inc.
Figure 8 Result of natural spider silk analysis. Eluent: HFIP þ 0.1 M sodiumtrifluoroacetate. Flow rate: 1 mL/min. Columns: PSS PFG 100 A ˚ , 7 mm, 8 300 mm þ PFG 1000 A ˚ , 7mm, 8 300 mm. Temperature: 258C. Detection RI. Standards: 12 PSS PMMA calibration standards. 5 CONCLUSION The previous section described the most frequently used PSS methods for analysing polyesters and polyamides. We know from our long experience that the methods presented are the most reliable and reproducible. In routine analysis in the laboratories of PSS and of our customers it normally takes many years before the presented systems lose any efficiency. Molecular weight calibrations can be carried out very easily with the readily available PMMA standards; of course, polyester or polyamide standards also can be used. Owing to the unusually poor solubility of these polymers it is very important for customers to be in contact with a column supplier which knows how to overcome the difficulties that result and which is able to assist its customers. This assistance will become more and more important for customers because of the many modifications that will be made to high-performance plastics in the future. 6 ACKNOWLEDGEMENTS The author thanks the editor for his support and all the colleagues at PSS who contributed their work to this chapter. The author also thanks his wife Susanne and his son Jan-Luca for the care they took of him while writing. © 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
Page 129 and 130: sensitivity for many samples compar
Page 131 and 132: that is, log[h] vs. logM,generated
Page 133 and 134: Figure 8 Effect of band broadening
Page 135 and 136: Table 1 Measurement of Molecular We
Page 137 and 138: Figure 10 Differential refractomete
Page 139 and 140: Figure 11 (A) Mark-Houwink plot of
Page 141 and 142: Table 3 Measurement of Molecular We
Page 143 and 144: size information is derived from un
Page 145 and 146: Table 5 Molecular Weight Distributi
Page 147 and 148: By measuring the scattered intensit
Page 149 and 150: 8 APPENDIX: INSTRUMENT COMPANIES 8.
Page 151 and 152: 53. G Marot, J Lesec. J Liq Chromat
Page 153 and 154: 125. D Slootmaekers, JAPP Van Dijk,
Page 155 and 156: 200. JG Bindels, GJJ Bessems, BM De
Page 157 and 158: possibly with hydrodynamic volume (
Page 159 and 160: An insurmountable limitation of SEC
Page 161 and 162: to sDR(V) when either nPS ¼nPB ¼c
Page 163 and 164: 1,4-trans;and8%1,2-vynil.TheglobalC
Page 165 and 166: The goal is to find the MWD and CCD
Page 167 and 168: As expected, pS is closer to the no
Page 169 and 170: Figure 3 Example 3: MWD and BD of a
Page 171 and 172: 9. BH Zimm, WH Stockmayer. The dime
Page 173 and 174: 45. RO Bielsa, GR Meira. Linear cop
Page 175 and 176: solubility properties. They are use
Page 177 and 178: determination of the absolute molec
Page 179: Figure 4 Result of the PBT analysis
Page 183 and 184: 7 Size Exclusion Chromatography of
Page 185 and 186: As pointed out earlier, rubber must
Page 187 and 188: Table 2 Various Solvents and Operat
Page 189 and 190: Figure 2 Typical GPC calibrations w
Page 191 and 192: © 2004 by Marcel Dekker, Inc. Figu
Page 193 and 194: Natural and Synthetic Rubbe177 vari
Page 195 and 196: Another example, shown in Fig. 5 (3
Page 197 and 198: APPENDIX: SEC CONDITIONS FOR RUBBER
Page 199 and 200: Appendix (Continued) Polymer Column
Page 205 and 206: 5. J West, E Rodriguez. Rubber Chem
Page 207 and 208: 8 Size Exclusion Chromatography of
Page 209 and 210: associate. Girdler (67) and Speight
Page 211 and 212: Table 1 Fractions Obtained Using Co
Page 213 and 214: Figure 1 SEC analyses of an aged as
Page 215 and 216: Apparently more polar compounds are
Page 217 and 218: 40% was fractionated into four frac
Page 219 and 220: Figure 7 SEC analyses of an unaged
Page 221 and 222: Figure 9 Comparison of percentage L
Page 223 and 224: Figure 11 Comparison of SEC chromat
Page 225 and 226: Figure 12 Comparisons of SEC chroma
Page 227 and 228: percentage LMS at the other locatio
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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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9 Size Exclusion Chromatography of
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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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Appendix (Continued) Polymer Column
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Appendix (Continued) Polymer Column
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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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© 2004 by Marcel Dekker, Inc. Figu
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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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Page 549 and 550:
Page 551 and 552:
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
Page 569 and 570:
1 Segmental interaction energy para
Page 571 and 572:
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
Page 579 and 580:
Figure 3 Conventional SEC chromatog
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chromatogram at 4mL/min, which is f
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Sincethereductionoftheinternaldiame
Page 585 and 586:
Table 3 Summary of Column Performan
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Figure 11 Dependence of column perf
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. Two-dimensional chromatography ru
Page 591 and 592:
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
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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© 2004 by Marcel Dekker, Inc. Figu
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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
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Finally, the use of a light-scatter
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Figure 8 Linear and cubic increases
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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
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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
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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
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Figure 2 Configuration of an SEC se
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5 THE IMPORTANCE OF SOFTWARE Like a
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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
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Figure 13 Fitstodatacollectedatasli
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Table 1 Errors (in %) Characteristi
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homogeneous copolymer, that is, tak
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process may be used to identify the
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fractionated sample. They represent
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Figure 16 An example of poor SEC ch
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on) methods, whereby the average di
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23. WH Stockmayer, LD Moore Jr, M F
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condition, as is explained in this
Page 671 and 672:
Figure 2 Partition coefficients KL
Page 673 and 674:
4 COLUMNS FOR HOPC Detailsaregiveni
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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
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Figure 10 SEC chromatograms for som
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appropriate for the early fractions
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23 Size Exclusion/ Hydrodynamic Chr
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Figure 2 Calibration curve for the
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Figure 5 Elutionbehaviorofpolystyre
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Table 1 Comparison Between SEC and
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egular SEC column in which the pore
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Such an ideal separation can be obt
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