Handbook of Size Exclusion Chromatography and Related ...

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Figure 1 MALDI-MS positive ion spectra of (A) a whole Indulin AT sample, (B) a narrow MMD fraction of an Indulin AT sample isolated by SEC. (From Ref. 10.) detectors are continuously developed. New column materials are also developed, falling into three different classes of column packing materials. The rigid crosslinked polystyrene-based materials may be derivatized in order to obtain hydrophilic properties. Semirigid synthetic hydrophilic packing materials can generally be used both with aqueous and weakly alkaline eluents and also with aprotic organic eluents. Soft packing materials are mainly based on different crosslinked polysaccharides and can be used in aqueous and alkaline eluents, but it should be pointed out that when using alkaline eluents the stability of the packing materials should be continuously monitored. In several systems there are possibilties for connecting up to four different detectors that all detect the same sample as a function of Vh of the polymer molecule but measure different properties of the lignin molecule. The RI detector determines the concentration of the lignin molecules at a certain elution volume, the UV/VIS also measures the concentration of the lignin and is dependent on the extinction coefficient of the lignin, the light-scattering detectors (MALLS, LALLS) determine the size of the lignin molecules and also the interactions between lignin and the eluent. The viscosity detectors relate the hydrodynamic volume to the molar mass of lignin. The MALDI-TOF-MS and ESI/MS detectors give absolute molar masses by mass spectrometry of fractions obtained during SEC of lignins. Some recent applications of known SEC systems using different types for mesurements of MMs and MMDs for lignins of different origin will be presented. © 2004 by Marcel Dekker, Inc.
The division according to eluent type is applied because the solubilty of lignin samples of different origin varies as do the interactions between lignin, eluents, and column packing material. Some new approaches to the physicochemical behavior of lignins in aqueous solutions will also shortly be mentioned. 3.1 SEC Systems Using Organic Eluents (Mostly THF) as Eluent THF is the most used eluent for HPSEC (high performance/size exclusion chromatography) systems using crosslinked polystyrene gels as column packing material. The solubilty of native lignins (milled wood lignin) and methylated or acetylated lignins and, in general, lignin fractions with low molar mass is good (9). Underivatized technical lignins and also enzymatically isolated native lignins are, however, only partly soluble in THF, as are hydrophilic technical lignins. The RI, UV, viscosity and light-scattering detectors and the new MALDI-TOF and electronspray detectors have all been applied in HP/SEC systems using THF as eluent. THF has been used as eluent (9) in the investigation of several commercial and semicommercial technical lignins, using the universal calibration with a differential viscosimeter including an RI detector. The solubility of partially soluble lignin samples was improved by acetylating and/or methylating of lignin samples. The measurements with the viscometric detector indicated that the lignin acetates are compact spherical molecules in THF. This seems to be a general property for lignin acetates in THF because according to light-scattering measurements (14) organosolv spruce lignin acetates are more compact in THF than in acetone. HPSEC measurements of lignin using THF as eluent have been described in an overview by Gellerstedt (15). In the interpretations of the results the lack of a full understanding of concentration, association, adsorption, and exclusion effects and their relationship to the hydrodynamic volume of lignin derivatives should be kept in mind. Himmel et al. (16) have demonstrated that commercially available molar mass standards, as well as low molar mass lignins, all follow the universal calibration curve. The universal calibration gives a more complete picture of molar masses and molecular size of lignins than conventional calibration using the elution volumes of monodisperse polystyrenes for calculation of the calibration line. However, conventional calibration has been used succesfully in several investigations for both native and technical lignins (7,9,17) when relative values for the molar masses are enough, which is often the case when following changes during specific processes. The difficulties in calibrating an HPSEC/THF system with linear polystyrenes on the one hand, and lignin fractions, lignin-like molecules, and lignin models on the other have been demonstrated. It was seen that for THF only a low amount of association between lignin molecules occurred but interactions between lignin, eluent, and packing materials did occur (18). © 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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A relatively new technique utilizin
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where ais the exponent of the Mark-
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Figure 2 TDS chromatograms for full
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Figure 5 Comparison of molecular we
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Table 3 Summary of TDS Molecular We
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thepartiallyhydrolyzedPVAwiththecol
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Table 5 Summary of Conformation and
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a¼0.627andlog(K) ¼23.249.Theseare
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The limits of detection of the PVA
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Figure 12 PVAc broad molecular weig
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4 SUMMARY Advances in SEC character
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2.1 Molecular Weight Grades of PVP
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exclusion chromatography with low-a
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weightandmolecularweightdistributio
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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
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 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
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: The calibration of SEC columns by c
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
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
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an Ubbelohde-viscometer for concent
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Rheological investigations of stabi
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shifts to applied laser light, whic
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Table 8 Characteristic Parameters f
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Table 8 (Continued) and H2O. A part
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10. SG Ball, MHBJ van de Wal, RGF V
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52. W Praznik, R Beck. J Chromatogr
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3 PROTEIN PARTITIONING IN SEC 3.1 G
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Figure 1 Plot of Kav vs. log M for
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yconsideringtheproteinsolutestobesp
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globular proteins and selected flex
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silanol groups (52-54); even capped
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most proteins to be maximally stabl
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Figure 4 Chromatograms of BSA and P
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concentrated) steps of protein puri
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Table 3 Characteristics of Dextran-
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preparative SEC. The capabilities o
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37. GR Noll, N Nagle, JO Baker, DJ
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Figure 2 Separation of total E. col
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Figure 3 Separation of HaeIII-cleav
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The recovery of DNA fragments has b
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Endotoxin should also be removed fr
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Table 3 Best Columns for the Separa
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Figure 10 Dependence of HETP on flo
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Appendix (Continued ) Polymer Colum
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REFERENCES 1. CT Wehr, SR Abbott. J
Page 495 and 496:
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
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
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Table 1 Summary of Methods for Incr
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not savealot of time and solvent, d
Page 579 and 580:
Figure 3 Conventional SEC chromatog
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chromatogram at 4mL/min, which is f
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
Page 593 and 594:
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
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out at low enough concentration tha
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educed viscosity, h r, to be comput
Page 607 and 608:
Page 609 and 610:
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
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where r(t) is now given by 2 6 r(t)
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Figure 11 Light scattering from sol
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3.1 ASimple System: Equilibrium Cha
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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