Handbook of Size Exclusion Chromatography and Related ...

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procedurewasusedtodetermineahydrolysisdistribution,similarinmannertothe measurement of amolecular weight distribution. The fractionation of PVAby composition rather than molecular weight was carriedoutusingagradient liquid chromatographysystem comprisingtwoModel 64 pumps and a Model 50 programmer (Knauer, Germany), a Model 7125 injection valve (Rheodyne, USA), and aModel 950 evaporative mass detector (Polymer Laboratories, UK). The HPLC column was a polystyrene– divinylbenzene-based type with a particle size of 8m and a pore size of 4000 A ˚ , 50 7.5 mm. A linear gradient of water:THF (98:2%, v/v) to water:THF (30:70%, v/v) over a9-min period was employed. These conditions yielded aseparation where the first components to elute are the high hydrolysis PVA fractions followed by lower hydrolysis PVA components. An average degree of hydrolysis of 70% or greater produces satisfactory results using this methodology. Figure 11 is an overlay of three chromatograms from the reversed phase HPLC of PVAfractions with degrees of hydrolysis (determined by 1 H-NMR spectroscopy) of 88.0, 84.3, and 81.8 mol%. Also included is the parent PVAsample, from which the three fractions were collected using preparative HPLC. The different elution times of the three fractionsiseasilyobservedandthewidehydrolysisdistributionoftheparentPVA is revealed by the broad chromatogram. Plots of retention time for fractions of knownhydrolysiswereusedtoconstructcalibrationcurvesfromwhichhydrolysis distributions were computed (7). 3 RECENTADVANCES IN THE CHARACTERIZATION OF PVAC Since 1995, the published material on SEC of PVAc has been somewhat limited. Thissectionwillbrieflyreviewsomeofthepublishedworkswhichhaveappeared in the literature dealing with PVAc. PVAc is an amorphous, atactic polymer that is soluble in many organic solvents.THFisprobablythemostwidelyusedsolventforSECofPVAc(18).As an example, the SEC chromatogram and corresponding molecular weight distributionofacommerciallyavailable,PVAcbroadstandard(AmericanPolymer Standards Corporation) is shown in Fig. 12. There have been published several different values for the Mark–Houwink constants of PVAc over the years. These values fall in the range K ¼ 0.51 10 24 to 3.50 10 24 and a ¼ 0.63–0.79. It is interesting to note that Lawrey (18) points out the intrinsic viscosity behavior of PVAc is very close to that of polystyrene. Polystyrene and linear PVAc elute at nearly the same retention time for the same molecular weight in THF (18). The calculated molecular weight vs. polystyrene for the PVAc broad standard in Fig. 12 are very close to the © 2004 by Marcel Dekker, Inc.
Figure 12 PVAc broad molecular weight standard, manufacturer’s values: Mw ¼ 275,000, M n ¼ 65,700. manufacturer’s values. The same is true for the molecular weights calculated using universal calibration, with K ¼ 3.50 10 24 and a ¼ 0.630 for PVAc and K ¼ 1.28 10 24 and a ¼ 0.712 for polystyrene (18). A study on the use of a single capillary viscometer detector, utilizing a pulse-free pump on a Waters Alliance 2690 by Mendichi and Schieroni, was © 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
Page 39 and 40:
4 GENERAL REFERENCES The interested
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46. TA Chamberlin, HE Tuinstra. US
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materials are commercially availabl
Page 45 and 46:
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
Page 57 and 58:
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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Page 203 and 204:
Page 205 and 206:
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
Page 251 and 252: Asphalts 235 122. M-S Lin, JM Chaff
Page 253 and 254: 9 Size Exclusion Chromatography of
Page 255 and 256: Table 1 Applications of Acrylamide
Page 257 and 258: (AM/AA), and acrylamide/dimethyldia
Page 259 and 260: polymer, a large size filter of 5,
Page 261 and 262: Figure 1 Size exclusion chromatogra
Page 263 and 264: Table3 MWandMWDofaBroad-MWDPAMStand
Page 265 and 266: Another commercially available MW d
Page 267 and 268: Table 5 Weight-Average Molecular We
Page 269 and 270: Figure 6 (a) Size exclusion chromat
Page 271 and 272: compared to anormal product. By com
Page 273 and 274: StudyingtheKineticsofaChemicalReact
Page 275 and 276: this information, the higher MW pea
Page 277 and 278: Appendix (Continued) Polymer Column
Page 279 and 280: Appendix (Continued) Polymer Column
Page 281 and 282: 46. CJ Kim, A Hamielec, A Bendek. J
Page 285 and 286: A relatively new technique utilizin
Page 287 and 288: where ais the exponent of the Mark-
Page 289 and 290: Figure 2 TDS chromatograms for full
Page 291 and 292: Figure 5 Comparison of molecular we
Page 293 and 294: Table 3 Summary of TDS Molecular We
Page 295 and 296: thepartiallyhydrolyzedPVAwiththecol
Page 297 and 298: Table 5 Summary of Conformation and
Page 299 and 300: a¼0.627andlog(K) ¼23.249.Theseare
Page 301: The limits of detection of the PVA
Page 305 and 306: 4 SUMMARY Advances in SEC character
Page 309 and 310: 2.1 Molecular Weight Grades of PVP
Page 311 and 312: exclusion chromatography with low-a
Page 313 and 314: weightandmolecularweightdistributio
Page 315 and 316: © 2004 by Marcel Dekker, Inc. Figu
Page 317 and 318: Figure 4 PEOcalibrationofUltrahydro
Page 319 and 320: Figure 6 Overlay of GPC chromatogra
Page 321 and 322: plus Ultrahydrogel 120 A ˚ ,Shodex
Page 323 and 324: Figure 10 SEC traces of PVP/AA copo
Page 325 and 326: Figure 11 Molecular weight distribu
Page 329 and 330: 2 CHEMICAL, MACROMOLECULAR AND MORP
Page 331 and 332: Table 2 The Relative Composition of
Page 333 and 334: considered to be 3. After the DS ha
Page 335 and 336: een extensively studied by methods
Page 337 and 338: The silylation was performed in LiC
Page 339 and 340: observed using column materials hav
Page 341 and 342: Table 6 (Continued) Packing materia
Page 343 and 344: 4.5 Other Cellulose Derivatives Bes
Page 345 and 346: Table 8 SEC Conditions for Characte
Page 347 and 348: dextrans swell too much in cadoxen
Page 349 and 350: stirring at room temperature for an
Page 351 and 352: 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
Page 397 and 398:
eds. Proc Int Symp 1998. Canton, Pe
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92. L Jurasek. Towards a three dime
Page 401 and 402:
Table 1 Selected Industries Connect
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into C3-metabolites, which then may
Page 405 and 406:
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
Page 423 and 424:
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
Page 441 and 442:
Rheological investigations of stabi
Page 443 and 444:
shifts to applied laser light, whic
Page 445 and 446:
Table 8 Characteristic Parameters f
Page 447 and 448:
Table 8 (Continued) and H2O. A part
Page 449 and 450:
10. SG Ball, MHBJ van de Wal, RGF V
Page 451 and 452:
52. W Praznik, R Beck. J Chromatogr
Page 453 and 454:
Page 455 and 456:
3 PROTEIN PARTITIONING IN SEC 3.1 G
Page 457 and 458:
Figure 1 Plot of Kav vs. log M for
Page 459 and 460:
yconsideringtheproteinsolutestobesp
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globular proteins and selected flex
Page 463 and 464:
silanol groups (52-54); even capped
Page 465 and 466:
most proteins to be maximally stabl
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Figure 4 Chromatograms of BSA and P
Page 469 and 470:
concentrated) steps of protein puri
Page 471 and 472:
Table 3 Characteristics of Dextran-
Page 473 and 474:
preparative SEC. The capabilities o
Page 475 and 476:
37. GR Noll, N Nagle, JO Baker, DJ
Page 477 and 478:
Page 479 and 480:
Figure 2 Separation of total E. col
Page 481 and 482:
Figure 3 Separation of HaeIII-cleav
Page 483 and 484:
The recovery of DNA fragments has b
Page 485 and 486:
Endotoxin should also be removed fr
Page 487 and 488:
Table 3 Best Columns for the Separa
Page 489 and 490:
Figure 10 Dependence of HETP on flo
Page 491 and 492:
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 and 544:
where Cand Dare constants for apart
Page 545 and 546:
ThisisimportantwhenLCCCisappliedtoc
Page 547 and 548:
Page 549 and 550:
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
Page 599 and 600:
just ignoring the fractionation of
Page 601 and 602:
20 Automatic Continuous Mixing Tech
Page 603 and 604:
out at low enough concentration tha
Page 605 and 606:
educed viscosity, h r, to be comput
Page 607 and 608:
© 2004 by Marcel Dekker, Inc. Figu
Page 609 and 610:
Figure 2 Raw ACOMP signals from mul
Page 611 and 612:
Figure 4 Mw vs. monomer conversion,
Page 613 and 614:
decreases smoothly and monotonicall
Page 615 and 616:
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 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
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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