Handbook of Size Exclusion Chromatography and Related ...

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Table 2 Molecular Weights of PVP K value Mw Mn K-15 7,000–12,000 22,500 K-30 40,000–65,000 210,000 K-60 350,000–450,000 2100,000 K-90 900,000–1,500,000 2360,000 K-120 2,000,000–3,000,000 — If a K value vs. absolute weight-average molecular weight equation or table is available for PVP, then the K value can be easily determined from relative viscosity. Such a relationship, developed by Senak et al., is shown in the equation log Mw ¼ 2:82 log K þ 0:594 and in Table 3 for commercial grades of unfractionated PVP (L Senak, CS Wu, EG Malawer, unpublished results). It should be pointed out here that the K value is a function not only of molecular weight but also of molecular weight distribution and branching. 2.2 Molecular Weights of VP-Based Copolymers Most VP-based copolymers are also characterized by K value. However, the literature on molecular weights of VP copolymers is very sparse. Wu and Senak reported in 1990 (5) the absolute molecular weights of cationic copolymers of quaternized vinyl pyrrolidone and dimethylaminoethyl methacrylate by size Table 3 K Value vs. Weight-Average Molecular Weight for PVP a K value Mw (AMU) K value Mw (AMU) 10 2,594 70 626,869 15 8,139 75 761,505 20 18,319 80 913,511 25 34,371 85 1,083,831 30 57,475 90 1,273,397 35 88,771 95 1,483,135 40 129,363 100 1,713,957 45 180,326 105 1,966,770 50 242,714 110 2,242,474 55 317,558 115 2,541,955 60 405,870 120 2,866,099 65 508,646 a The calculations are based on the regression formula logMw ¼ 2:82 log K þ 0:594. © 2004 by Marcel Dekker, Inc.
exclusion chromatography with low-angle laser light scattering (SEC/LALLS) and SEC with universal calibration (Table 8). The molecular weights (relative to polyethylene oxide standards) of nonionic copolymers of vinyl pyrrolidone and vinyl acetate, anonionic terpolymer of vinyl pyrrolidone, dimethylaminoethyl methacrylate,andvinylcaprolactam,andanioniccopolymersofvinylpyrrolidone and acrylic acid were also reported in 1991 (6) by Wu et al. (Tables 6and 7). 3 MOLECULAR WEIGHT DISTRIBUTION OF VP-BASED POLYMERS BY SIZE EXCLUSION CHROMATOGRAPHY Many important properties of polymers depend not only on molecular weight but also on molecular weight distribution. For example, both viscosity and its dependence on the shear rate of polymer melt and concentrated polymer solution are dependent on molecular weight distribution. SEC is the most practical and the best method for determining the molecular weight distribution of a polymer without going through the tedious classic fractionation procedure using nonsolvent precipitation. 3.1 SEC of PVP: Historical Review The SEC of PVP is not straightforward because of the polar nature of the polymer. Various interactions between PVP and columns, such as adsorption, partition, and electrostatic interactions, must be eliminated by prudent choice of column and mobile phase to obtain true separation by size with 100% recovery and compliance with universal calibration. The SEC behavior of PVP has been of interest to many researchers. In the 10-year period from 1975 to 1984, seven papers, using seven different kinds of columns with various surface modifications and in both aqueous and nonaqueous mobile phases with and without modifiers and salts, were reported for the SEC of PVP with different degrees of success. Some of the columns used are commercially available; others are specially made. Belenkii et al. reported in 1975 (7) the SEC of PVP with unspecified molecular weight using Pharmacia Sephadex G-75 and G-100 columns and a 0.3% sodium chloride solution as the mobile phase. Deviations from universal calibration behavior were noticed from PVP, dextran, polyethylene oxide (PEO), and polyvinyl alcohol. With the development of the important semirigid polymer gel, Toyo Soda TSK-PW columns for water-soluble polymers, Hashimoto et al. reported in 1978 (8) the SEC of PVP K-30 and K-90 using TSK-PW 3000 and two 5000 columns an 0.08 M Tris–HCl buffer (pH ¼ 7.94) as mobile phase and PEO and dextran as calibration standards. By using an E. Merck LiChrospher SI300 column, modified with an amide group chemically bonded to the surface, Englehardt and Mathes reported in 1979 (9) the SEC of PVP with molecular weights from 10,000 to 360,000 AMU. A 0.1 M © 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
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 283 and 284: 10 Size Exclusion Chromatography of
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 and 302: The limits of detection of the PVA
Page 303 and 304: Figure 12 PVAc broad molecular weig
Page 305 and 306: 4 SUMMARY Advances in SEC character
Page 309: 2.1 Molecular Weight Grades of PVP
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
Page 353 and 354: Figure 4 MMDofbirchkraftpulpdegrade
Page 355 and 356: adequate when evaluating the influe
Page 357 and 358: differential refractive index (DRI)
Page 359 and 360: 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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Page 455 and 456:
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
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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
Page 547 and 548:
Page 549 and 550:
Page 551 and 552:
However, strong adsorption of macro
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elution step may be sufficient for
Page 555 and 556:
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:
Page 573 and 574:
19 Methods and Columns for High-Spe
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Table 1 Summary of Methods for Incr
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not savealot of time and solvent, d
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Figure 3 Conventional SEC chromatog
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chromatogram at 4mL/min, which is f
Page 583 and 584:
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
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The standard deviations for the mol
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Figure 15 Comparison of time requir
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Figure 17 Accuracy and precision of
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
Page 605 and 606:
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
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
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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
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
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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
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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