Handbook of Size Exclusion Chromatography and Related ...

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Figure 8 Calibration curves of solid-bead HdC columns. Columns, ¼3mm, 4 ¼ 5mm, W ¼ 10mm; mobile phase, THF with 250ppm BHT, at 1.0mL/min; column temperature, 508C. range of the three columns are approximately the same. The curve of the 3mm column turns upward above 3 10 6 MW,while both 5mm and 10mm columns do not reach their upper MW limits with the available PS standards, up to 7:5 10 6 .It seems that the 10mm column would separate the highest MW range among three columns. For an ideal SEC/HdC column, it is possible to adjust the SECseparationsothattheportionofthecalibrationcurvefor5 10 5 andbelowis colinear with the HdC high MW portion of the curve. This adjustment can be accomplished by controlling the total pore volume of the packing gel. As discussed previously,the slope of an SEC calibration curve depends on the pore volume. Figure 2shows the calibration curve of an SEC separation in a © 2004 by Marcel Dekker, Inc.
egular SEC column in which the pore volume is roughly 30 to 40% of the entire column volume. In the curve, the linear portion above 3 10 5 is due to the HdC mechanism separating large molecules while the portion below 5 10 4 is due to the SEC mechanism separating lower MW species. It can be seen that the portion of the curve due to the SEC mechanism has a flatter slope than that region due to the HdC mechanism. In order to obtain a linear calibration curve over the entire MW range, the total pore volume must be reduced so that the slope of the lower MW portion of the curve matches that of the upper region. From experience, the ratio of the total pore volume to total column for such an ideal SEC/HdC column should be roughly one-third that of a regular SEC column. Figure 9 Calibration curve of SE/HdC. Column, 10mm solid-bead column (250 10mm) þ 5mm PLgel MIXED-D column (300 7:5mm); mobile phase, THF with 250ppm BHT, at 0.5mL/min; column temperature, 508C. © 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
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Figure 11 Molecular weight distribu
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2 CHEMICAL, MACROMOLECULAR AND MORP
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Table 2 The Relative Composition of
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considered to be 3. After the DS ha
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een extensively studied by methods
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The silylation was performed in LiC
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observed using column materials hav
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Table 6 (Continued) Packing materia
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4.5 Other Cellulose Derivatives Bes
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Table 8 SEC Conditions for Characte
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dextrans swell too much in cadoxen
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stirring at room temperature for an
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Figure 2 MMDofunbleached(HP)andblea
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Figure 4 MMDofbirchkraftpulpdegrade
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adequate when evaluating the influe
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differential refractive index (DRI)
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30. HA Krässig. Effects of structu
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67. E Sjöholm, K Gustafsson, A Col
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104. B Fleury, J Dubois, C Léonard
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136. D Miller, D Senior, R Sutcliff
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166. SS Cutié, CG Smith. Determina
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200. R Berggren, F Berthold, E Sjö
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13 Molar Mass and Size Distribution
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measurements, and vapor pressure os
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The calibration of SEC columns by c
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The division according to eluent ty
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Apolymer produced by UV irradiation
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Because DMAC/LiCl is also agood sol
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compoundsandwasexplainedbyadsorptio
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isolated lignins because they are e
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The Separon HEMA and Separon HEMA B
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Figure 4 GPC elution curves of the
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liquor is highest throughout the co
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topological anisotropy in the polym
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29. M Ristolainen, R Alen, J Knuuti
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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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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
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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
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Page 549 and 550:
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However, strong adsorption of macro
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elution step may be sufficient for
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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
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1 Segmental interaction energy para
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58. BG Belenkii. Pure Appl Chem 51:
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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
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Sincethereductionoftheinternaldiame
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Table 3 Summary of Column Performan
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Figure 11 Dependence of column perf
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. Two-dimensional chromatography ru
Page 591 and 592:
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
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polymer analysis. Samples are “ru
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just ignoring the fractionation of
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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
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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
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Figure 7 Effects of fluctuating rea
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Finally, the use of a light-scatter
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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
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Figure 13 (a) ACM applied to the ch
Page 629 and 630:
Also of note in Fig. 13b is how hig
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15. JM Starita, CL Rohn. Rheologica
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52. M Hulden. Hydrophobically modif
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21 Light Scattering and the Solutio
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therefore, is to remove any lingeri
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is often found in the literature wh
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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: Table 1 Comparison Between SEC and
Page 697: Such an ideal separation can be obt
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