Handbook of Size Exclusion Chromatography and Related ...

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useful information in the analysis of linear polyolefins at high temperature. They also demonstrated, for the polymer studied, that no single detector was able to give a complete picture of the MWD because of different sensitivity ranges. Jackson and co-workers (218) showed that the Mark–Houwink exponent of polystyrene in toluene could be measured with a relative standard deviation of less than 1% with a single injection of a broad MWD standard. As discussed earlier, a combination of a right-angle laser light-scattering detector and a viscometer has proved to be a useful system for determining not only molecular weight and intrinsic viscosity data but also radius of gyration of linear polymers (11). Light-scattering measurements made at 908 simplify the design of light-scattering instrumentation and, in principle, give a less noisy signal by reducing spikes from particle contamination and stray light. In fact, a commercially available HPLC fluorescence detector can be employed for these measurements (212). 6 SUMMARY The use of molecular weight sensitive detectors has increased dramatically the information content that can be obtained from an SEC analysis. With these detection systems, accurate measurements of fundamental molecular parameters, both average and distributed values, can be determined readily. Furthermore, the use of light-scattering detectors, and viscosity detectors for IVD, eliminates the need for column calibration, which greatly increases the precision and reliability of these measurements. However, as, in any other analytical instrumental procedure, good chromatographic practise must be exercised: signal-to-noise ratio of detector outputs must be maximized, defined polymer solutions injected, and instrument calibration parameters and proper interdetector volumes established. In addition to applications in the area of synthetic polymers, we foresee exciting uses of molecular weight sensitive detectors for biopolymer characterization and with interactive modes of separation, such as reversed-phase gradient elution or ion-exchange chromatography. Finally, the combination of online spectroscopic detectors, including UV-diode array, Fourier transform infrared, mass spectrometry and possibly nuclear magnetic resonance with molecular weight sensitive detectors represent a significant breakthrough for the characterization of complex polymeric materials. 7 ACKNOWLEDGEMENTS The authors gratefully acknowledge the valuable input and discussions with our colleague Wallace W. Yau. We also thank the Corporate Center for Analytical Sciences of the DuPont Company for giving us opportunity to prepare this chapter. © 2004 by Marcel Dekker, Inc.
8 APPENDIX: INSTRUMENT COMPANIES 8.1 Light-Scattering Detectors for SEC Brookhaven Instruments Corp., 750 Blue Point Rd, Holtsville, NY 11742: CCD-based seven-angle light-scattering detector. Precision Detectors, Inc., 34 Williams Way, Bellingham, MA 02019: dynamic light-scattering and dual-angle light-scattering detectors. Viscotek Corp., 15600 West Hardy Rd, Houston, TX 77060: right-angle and dual-angle light-scattering detectors. Wyatt Technology Corp., 30 South La Patera Lane, Santa Barbara, CA 93117: dynamic light scattering, multiangle and triple-angle lightscattering detectors. 8.2 Viscometers for SEC Viscotek Corp., 15600 West Hardy Rd, Houston, TX 77060: four capillary bridge design. Waters Corp., 34 Maple St., Milford, MA 01757: flow-referenced capillary design. 9 REFERENCES 1. WW Yau, JJ Kirkland, DD Bly. Modern Size Exclusion Liquid Chromatography. New York: John Wiley and Sons, 1979. 2. P Kratochvil. Classical Light Scattering from Polymer Solutions. New York: Elsevier, 1987. 3. M Bohdanecky, J Kovar. Viscosity of Polymer Solutions. New York: Elsevier, 1982. 4. H Yamakawa. Modern Theory of Polymer Solutions. New York: Harper and Row, 1971. 5. H Fujita. Polymer Solutions. New York: Elsevier, 1990. 6. MA Haney. J Appl Polym Sci 30:3023, 1985; 30:3037, 1985. 7. JL Ekmanis, RA Skinner. J Appl Polym Sci, Appl Polym Symp 48:57, 1991. 8. WW Yau, SD Abbott, GA Smith, MY Keating. ACS Symp Ser 352:80, 1987. 9. BH Zimm. J Chem Phys 16:1093, 1948. 10. ML McConnell. Am Lab 10(5):63, 1978. 11. MA Haney, C Jackson, WW Yau. Proc Int Gel Permation Chromatography Symp 1991, San Francisco, 1993, p 49. 12. Precision Detectors, Application Notes, Amherst, MA. 13. PJ Wyatt, DL Hicks, C Jackson, GK Wyatt. Lab 20(6):108, 1988. 14. PJ Wyatt, C Jackson, GK Wyatt. Am Lab 20(5):86, 1988. 15. Z Grubisic, P Rempp, H Benoit. J Polym Sci, Part B, Polym Lett 5:753, 1967. 16. AE Hamielec, AC Ouano. J Liq Chromatogr 1:111, 1978. 17. CJ Stacy. J Appl Polym Sci 32:3959, 1986. © 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
Page 97 and 98: valuesfork1,k2,anda,theunknownmolec
Page 99 and 100: water-soluble and detergent-soluble
Page 101 and 102: the importance of secondary retenti
Page 103 and 104: discussed in Table 8(33). Based on
Page 105 and 106: individual dispersion processes ins
Page 107 and 108: The detector time constant can dist
Page 109 and 110: 5.3 Mobile Phase A mobile phase is
Page 111 and 112: less steep and still linear at high
Page 113 and 114: 35. P Bristow, J Knox. Chromatograp
Page 115 and 116: 112. K Gooding, K Lu, G Vanecek, F
Page 117 and 118: for branched polymers or copolymers
Page 119 and 120: Figure 1 “Bridge design” flow-r
Page 121 and 122: Figure 3 A light-scattering photome
Page 123 and 124: Figure 5 SEC universal calibration
Page 125 and 126: where hn is the hydrodynamic volume
Page 127 and 128: 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: By measuring the scattered intensit
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 and 180: Figure 4 Result of the PBT analysis
Page 181 and 182: Figure 8 Result of natural spider s
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
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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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8 Size Exclusion Chromatography of
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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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Page 255 and 256:
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
Page 277 and 278:
Page 279 and 280:
Page 281 and 282:
46. CJ Kim, A Hamielec, A Bendek. J
Page 283 and 284:
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
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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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Page 309 and 310:
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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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
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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
Page 341 and 342:
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
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)
Page 359 and 360:
30. HA Krässig. Effects of structu
Page 361 and 362:
67. E Sjöholm, K Gustafsson, A Col
Page 363 and 364:
104. B Fleury, J Dubois, C Léonard
Page 365 and 366:
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
Page 371 and 372:
13 Molar Mass and Size Distribution
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measurements, and vapor pressure os
Page 375 and 376:
The calibration of SEC columns by c
Page 377 and 378:
The division according to eluent ty
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Apolymer produced by UV irradiation
Page 381 and 382:
Because DMAC/LiCl is also agood sol
Page 383 and 384:
compoundsandwasexplainedbyadsorptio
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isolated lignins because they are e
Page 387 and 388:
The Separon HEMA and Separon HEMA B
Page 389 and 390:
Figure 4 GPC elution curves of the
Page 391 and 392:
liquor is highest throughout the co
Page 393 and 394:
topological anisotropy in the polym
Page 395 and 396:
29. M Ristolainen, R Alen, J Knuuti
Page 397 and 398:
eds. Proc Int Symp 1998. Canton, Pe
Page 399 and 400:
92. L Jurasek. Towards a three dime
Page 401 and 402:
Table 1 Selected Industries Connect
Page 403 and 404:
into C3-metabolites, which then may
Page 405 and 406:
Table 4 Key Enzymes in the Biosynth
Page 407 and 408:
Table 5 Cloned Mutants of Starch En
Page 409 and 410:
Figure 3 Modeled x-ray diffraction
Page 411 and 412:
In the native environment (plant ce
Page 413 and 414:
Figure 6 (a) Large starch granules
Page 415 and 416:
number of branching points); crossl
Page 417 and 418:
Both absolute approaches are extrao
Page 419 and 420:
Table 7 (Continued) Approach Experi
Page 421 and 422:
structures, excluded volumes, and t
Page 423 and 424:
Figure 10 (a) SEC elution profile o
Page 425 and 426:
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
Page 439 and 440:
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
Page 461 and 462:
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
Page 467 and 468:
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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