Handbook of Size Exclusion Chromatography and Related ...

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Concentration-sensitive detectors (104,105) as well as low-angle laser light scattering (LALLS) detectors (106,107) have been used during the last decade. Cellulose acetate is commonly produced by reaction with acid anhydride usingacatalystsuchaszincchlorideorsulfuricacid(35).Inthissolutionprocess, the derivative formed is dissolved in glacial acetic acid or dichloromethane. For cellulose triacetate (CTA) (DS 2.8–3.0) the fibrous process is commonly applied. This process uses perchloric acid to catalyse the reaction and anonsolvent of the derivative to maintain the fiber structure. Cellulose diacetate (CDA) can also be made by deacetylation of CTA. The chemical and enzymatic reactivity is highly dependent on the DS of the cellulose acetate. The depolymerization of cellulose is catalyzed by sulfuric acid; that is, the latter does not only catalyze the acetylation reaction. By using GPC-LALLS, Shimamoto et al. (107) found that the depolymerization reaction is faster during the early stages of acetylation than for the fully substituted derivative, and also that the depolymerization of CTA proceeds randomly whereas the hydrolysis of cellulose does not. The degree of biodegradability is also closely connected to the DS, the lower DS the more biodegradable the cellulose acetate becomes (105). Depending on application, the target DS is in the range 1.2–3. The solubility depends on the DS but also on the distribution of substituents between the three possible positions (108). CDA (DS 2.2–2.7) is soluble in acetone and THF whereas CTA requires chloroform or dichloromethane for dissolution. From light-scattering studies, various degrees of aggregation of dilute solutions of CTA in m-creosol, tetraethane, and mixtures of dichloromethane–methanol have been shown (109). SEC characterizations have been performed primarily on cellulose diacetates and triacetates using THF (110,111), chloromethane (112), or acetone (106) as solvent, but also polar solvents such as DMAc (104,105) or N-methylpyrrolidone (NMP) (104) with or without addition of salt have been used in the past (Table 5). Owing to the high viscosity of cellulose acetate solutions of DMAc or NMP, the chromatography is performed at elevated temperature. A general problem encountered with SEC of cellulose acetate is the presence of extra humps and/or shoulders on the high molecular mass range of the main distribution and, in addition, a gel fraction (104,106,110–112). Whereas the gel fraction may be found in solutions of cellulose acetate samples from both cotton linter and wood pulps, the other anomalies are commonly only observed in cellulose acetates from wood pulps. The observed prehumps correlate with the hemicellulose content of the sample and can be reduced by optimizing the reaction conditions during acetylation or removed by fractional precipitation (110,111). The extra peaks have also been attributed to ionic effects caused by sulfate groups in the CDA solutions of acetone (106,113). The prehumps could only be © 2004 by Marcel Dekker, Inc.
observed using column materials having aslightly anionic charge (GMPWXI and CPG-10) and not when neutral column material (Toyopearl-75HW) was used (106),indicatinganexclusioneffectoftheformercolumnmaterial.Byadditionof CaI2 or NaI, the prehumps in the chromatograms were eliminated. Fleury et al. (104) thoroughly studied the humps of CDA from cotton linter and wood pulp samples seen in SEC by using polystyrene divinylbenzene (PS/DVB) columns and DMAc or NMP as mobile phase. The first eluting hump, the gel fraction, of the cellulose acetate samples was isolated by ultracentrifugation of acetone solutions of the samples prior to SEC. The amount of microgels in thewood pulp acetate was more than twice that of the corresponding cotton linter sample. After hydrolysis of the microgel fraction, it was found that the cotton linter sample consists almost exclusively of glucose while that of wood pulp also contains xylose, mannose, and galactose. By x-ray and electron diffraction analysis it was foundthatthemicrogelfractionfromthecottonlintercorrespondstoCTAandthe microgel fraction from the wood pulp sample is amixture of CTA and xylan diacetate. The reason for the remaining prehumps was attributed to ionic associations of remaining sulfate groups on the CDAwith residual calcium. The latter component proved to be directly correlated with size. By addition of 0.01 M LiBrorLiCltoDMAcorNMP,theioniceffectswereeliminatedandprehumpsin the chromatogram were circumvented. Thus, the problems encountered with SEC characterizationsofacetatescanberegardedassolvedutilizingLi-salt addition to the mobile phase, it being DMAc or NMP. 4.4 Cellulose Tricarbanilate The first report concerning SEC application for characterization of cellulose triphenylcarbamate or tricarbanilate (CTC) was in 1968 (114). CTC is still the mostutilizedderivativeforSECstudiesondifferentkindsofcellulosesamples,for example, microcrystalline cellulose, cotton linters, dissolving pulps, paper grade pulps, paper, ramie, and linen. The advantages in using CTC for cellulose characterizations are complete substitution, no depolymerization during the derivatization procedure, the stability of the formed derivative, and solubility and stability in THF.Fully substituted cellulose has anitrogen content of 8.09%, corresponding to a repeating unit molecular mass of 519. Thus, the large molecular mass is important to consider in order to select columns with appropriate exclusion limits. The columns used are exclusively packed with porous crosslinked polystyrene particles. Owing to the aromatic group in the carbanilate, UV detection has frequently been used for SEC of CTC. Differential refractiveindexdetectors(DRI)andon-linelight-scatteringdetectors,suchaslowangle laser light scattering (LALLS) and multi-angle laser light scattering (MALLS)detectors,havebeenusedoverthepastfewyears.CommonlyusedSEC conditions for characterization of CTCs are exemplified in Table 6. © 2004 by Marcel Dekker, Inc.
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MARCEL DEKKER Handbook of Size Excl
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CHROMATOGRAPHIC SCIENCE SERIES A Se
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60. Modern Chromatographic Analysis
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Preface to the Second Edition Gel p
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Preface to the First Edition Molecu
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Contents Preface to the Second Edit
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17. Size Exclusion Chromatography o
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James F. Curry Analytical Departmen
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Iwao Teraoka, Ph.D. Othmer Departme
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The graphical data display typicall
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polymer molecule can elute. The tot
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individual states) allowed to them.
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unsaturation while the IR detector
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Sampleconcentrationshouldbeminimize
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averagesdefinedintermsofthemolecula
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information regarding appropriate M
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Analytical. Two models, the KMX-6 a
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Figure 6 Overlay of time-sliced pea
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accurately for very large and very
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4 GENERAL REFERENCES The interested
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46. TA Chamberlin, HE Tuinstra. US
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materials are commercially availabl
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Table 1 Commercial Column Packing M
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necessarily comparable. For this re
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adjusted such that the pore size di
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narrowmolecular weight range, indiv
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Figure 8 Effect of particle size on
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Figure 9 SEC calibration using poly
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Table 4 Typical Eluent Systems for
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Figure 12 Analysis of poly-2-vinyl
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24. E Meehan, S O’Donohue. The ro
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into accepted laboratory techniques
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Table 1 Trace Metal Impurities in C
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mass and biological activity, for m
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Experimental efficiency vs. velocit
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Table 3 Properties of SEC Silica Ge
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operate at aflow rate that can easi
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Figure 3 Efficiency of amide-bonded
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mechanically stable packing materia
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Figure 5 Pore size distributions of
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Figure 6 Separation of polystyrenes
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60-120 A ˚ are large enough to be
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Table 6 Selected Silica-Based Colum
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Table 7 Separation Ranges for Polym
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the decline in permeability is perm
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sample components can be varied by
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Figure 10 Diol bonding reactions. I
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ecause of the higher relative molec
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valuesfork1,k2,anda,theunknownmolec
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water-soluble and detergent-soluble
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the importance of secondary retenti
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discussed in Table 8(33). Based on
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individual dispersion processes ins
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The detector time constant can dist
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5.3 Mobile Phase A mobile phase is
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less steep and still linear at high
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35. P Bristow, J Knox. Chromatograp
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112. K Gooding, K Lu, G Vanecek, F
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for branched polymers or copolymers
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Figure 1 “Bridge design” flow-r
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Figure 3 A light-scattering photome
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Figure 5 SEC universal calibration
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where hn is the hydrodynamic volume
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It should be noted that dn=dc also
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sensitivity for many samples compar
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that is, log[h] vs. logM,generated
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Figure 8 Effect of band broadening
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Table 1 Measurement of Molecular We
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Figure 10 Differential refractomete
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Figure 11 (A) Mark-Houwink plot of
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Table 3 Measurement of Molecular We
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size information is derived from un
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Table 5 Molecular Weight Distributi
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By measuring the scattered intensit
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8 APPENDIX: INSTRUMENT COMPANIES 8.
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53. G Marot, J Lesec. J Liq Chromat
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125. D Slootmaekers, JAPP Van Dijk,
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200. JG Bindels, GJJ Bessems, BM De
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possibly with hydrodynamic volume (
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An insurmountable limitation of SEC
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to sDR(V) when either nPS ¼nPB ¼c
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1,4-trans;and8%1,2-vynil.TheglobalC
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The goal is to find the MWD and CCD
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As expected, pS is closer to the no
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Figure 3 Example 3: MWD and BD of a
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9. BH Zimm, WH Stockmayer. The dime
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45. RO Bielsa, GR Meira. Linear cop
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solubility properties. They are use
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determination of the absolute molec
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Figure 4 Result of the PBT analysis
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Figure 8 Result of natural spider s
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7 Size Exclusion Chromatography of
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As pointed out earlier, rubber must
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Table 2 Various Solvents and Operat
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Figure 2 Typical GPC calibrations w
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© 2004 by Marcel Dekker, Inc. Figu
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Natural and Synthetic Rubbe177 vari
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Another example, shown in Fig. 5 (3
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APPENDIX: SEC CONDITIONS FOR RUBBER
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Appendix (Continued) Polymer Column
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5. J West, E Rodriguez. Rubber Chem
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associate. Girdler (67) and Speight
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Table 1 Fractions Obtained Using Co
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Figure 1 SEC analyses of an aged as
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Apparently more polar compounds are
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40% was fractionated into four frac
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Figure 7 SEC analyses of an unaged
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Figure 9 Comparison of percentage L
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Figure 11 Comparison of SEC chromat
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Figure 12 Comparisons of SEC chroma
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percentage LMS at the other locatio
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the total area into up to 12 sectio
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attempted to develop an SEC method
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parameterisbasedontheinternalpressu
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5 MODIFIED ASPHALTS The addition of
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Oxidation of the rubber and asphalt
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Figure 20 SEC chromatogram for an a
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Figure 21 Comparisons of apparent m
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© 2004 by Marcel Dekker, Inc. S/
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PS/10 4 þ 10 3 þ 500 þ 100 THF/1
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Nevertheless, SEC of asphalts is es
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56. NW Garrick, RR Biskur. Trans Re
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Asphalts 235 122. M-S Lin, JM Chaff
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Table 1 Applications of Acrylamide
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(AM/AA), and acrylamide/dimethyldia
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polymer, a large size filter of 5,
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Figure 1 Size exclusion chromatogra
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Table3 MWandMWDofaBroad-MWDPAMStand
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Another commercially available MW d
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Table 5 Weight-Average Molecular We
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Figure 6 (a) Size exclusion chromat
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compared to anormal product. By com
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StudyingtheKineticsofaChemicalReact
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this information, the higher MW pea
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46. CJ Kim, A Hamielec, A Bendek. J
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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 307 and 308: 11 Size Exclusion Chromatography of
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 327 and 328: 12 Size Exclusion Chromatography of
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: The silylation was performed in LiC
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
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
Page 367 and 368: 166. SS Cutié, CG Smith. Determina
Page 369 and 370: 200. R Berggren, F Berthold, E Sjö
Page 371 and 372: 13 Molar Mass and Size Distribution
Page 373 and 374: 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
Page 379 and 380: Apolymer produced by UV irradiation
Page 381 and 382: Because DMAC/LiCl is also agood sol
Page 383 and 384: compoundsandwasexplainedbyadsorptio
Page 385 and 386: isolated lignins because they are e
Page 387 and 388: 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
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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
Page 453 and 454:
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
Page 459 and 460:
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
Page 477 and 478:
Page 479 and 480:
Figure 2 Separation of total E. col
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Figure 3 Separation of HaeIII-cleav
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The recovery of DNA fragments has b
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Endotoxin should also be removed fr
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Table 3 Best Columns for the Separa
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Figure 10 Dependence of HETP on flo
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Appendix (Continued ) Polymer Colum
Page 493 and 494:
REFERENCES 1. CT Wehr, SR Abbott. J
Page 495 and 496:
MALDI-MS has been applied to determ
Page 497 and 498:
Figure 1 Calibration curvesof SEC c
Page 499 and 500:
Figure 3 Chromatograms of epoxy res
Page 501 and 502:
wherenandn0 aretheRIsofthesample an
Page 503 and 504:
Figure 5 SEC chromatogram of n-alka
Page 505 and 506:
Figure 7 Refractive indexes of comm
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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
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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