Handbook of Size Exclusion Chromatography and Related ...

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The LALLS device developed by Kaye provides three significant changes that make it amenable as an SEC molecular weight detector: an intense, monochromaticlightsource(aHeNelaser,l¼632:8nm)isused,thecellvolume is reduced to 10 mL and the scattering volume to 0.1 mL (26), and the single scatteringangleemployedisintherangeof2–78.Thenetresultisthatthedeviceis extremely sensitive; it can readily distinguish scattering due to an individual dust particle flowing through the cell from that due to the sample, and the angular dependence is removed from the Debye equation. The latter follows from the fact that the value of sin 2 (u=2) for a small angle is essentially zero. Under this condition the Debye equation becomes or Kc ¼ DRu 1 þ2A2C (24) M W 1 M W¼ Kc=DRu 2A2C (25) andMWcanbeobtainedatasinglefiniteconcentrationprovidedthatA2 isknown from the literature or is determined from the slope of Eq. (24) using aseries of concentrations. However, the removal of the angular variability from the LALLS detector means that it cannot be used to determine molecular size, that is, ks 2 l. TheSEC/LALLSexperimentisthenconductedasfollows.TheLALLSand concentration detectors are connected in series after the SEC column set and interfaced with the computing system. Time slice data from both detectors is acquired, as shown in Fig. 6, so as to have corresponding time slices in each distribution. In order to accomplish this the time delay between the detectors must be accurately known. The instantaneous concentration in either detector, ci, may be computed using ci ¼ mAi V P i Ai (26) where m is the sample mass injected, V is the effluent volume passing through the cell in the time of a single time slice, and Ai is the area of a concentration detector time slice. If one assumes that each time slice is sufficiently narrow so as to be monodisperse, then the instantaneous molecular weight is determined using Eq. (25). This data collectively constitute the absolute molecular weight distribution calibration. It is generally acknowledged that LALLS used either as a stand-alone lightscattering photometer or as an SEC detector provides accurate values for M W. Yet in 1987 a number of independent workers reported that the ability of SEC/LALLS to accurately determine M N was dependent on the polydispersity of the sample: the © 2004 by Marcel Dekker, Inc.
Figure 6 Overlay of time-sliced peak output from a dual (DRI/LALLS) detector system. greater the polydispersity, the poorer the estimate of M N (28–30). In performing SEC/LALLS on high molecular weight poly(vinyl pyrrolidone), Senak et al. (28) demonstrated that this phenomenon is caused by the lack of sensitivity of the LALLS detector toward the low molecular weight portion of a broad distribution (D ¼ 6:0). As shown in Fig. 7, the DRI detector is still responding (the shaded area) in a region where the LALLS detector is not. As discussed by Hamielec et al., Figure 7 Relative sensitivity of a LALLS vs. a DRI detector for a broadly dispersed sample of poly(vinyl pyrrolidone). © 2004 by Marcel Dekker, Inc.
Page 1 and 2: MARCEL DEKKER Handbook of Size Excl
Page 3 and 4: CHROMATOGRAPHIC SCIENCE SERIES A Se
Page 5 and 6: 60. Modern Chromatographic Analysis
Page 7 and 8: Preface to the Second Edition Gel p
Page 9 and 10: Preface to the First Edition Molecu
Page 11 and 12: Contents Preface to the Second Edit
Page 13 and 14: 17. Size Exclusion Chromatography o
Page 15 and 16: James F. Curry Analytical Departmen
Page 17 and 18: Iwao Teraoka, Ph.D. Othmer Departme
Page 19 and 20: The graphical data display typicall
Page 21 and 22: polymer molecule can elute. The tot
Page 23 and 24: individual states) allowed to them.
Page 25 and 26: unsaturation while the IR detector
Page 27 and 28: Sampleconcentrationshouldbeminimize
Page 29 and 30: averagesdefinedintermsofthemolecula
Page 31 and 32: information regarding appropriate M
Page 33: Analytical. Two models, the KMX-6 a
Page 37 and 38: accurately for very large and very
Page 39 and 40: 4 GENERAL REFERENCES The interested
Page 41 and 42: 46. TA Chamberlin, HE Tuinstra. US
Page 43 and 44: materials are commercially availabl
Page 45 and 46: Table 1 Commercial Column Packing M
Page 47 and 48: necessarily comparable. For this re
Page 49 and 50: adjusted such that the pore size di
Page 51 and 52: narrowmolecular weight range, indiv
Page 53 and 54: Figure 8 Effect of particle size on
Page 55 and 56: Figure 9 SEC calibration using poly
Page 57 and 58: Table 4 Typical Eluent Systems for
Page 59 and 60: Figure 12 Analysis of poly-2-vinyl
Page 61 and 62: 24. E Meehan, S O’Donohue. The ro
Page 63 and 64: into accepted laboratory techniques
Page 65 and 66: Table 1 Trace Metal Impurities in C
Page 67 and 68: mass and biological activity, for m
Page 69 and 70: Experimental efficiency vs. velocit
Page 71 and 72: Table 3 Properties of SEC Silica Ge
Page 73 and 74: operate at aflow rate that can easi
Page 75 and 76: Figure 3 Efficiency of amide-bonded
Page 77 and 78: mechanically stable packing materia
Page 79 and 80: Figure 5 Pore size distributions of
Page 81 and 82: Figure 6 Separation of polystyrenes
Page 83 and 84: 60-120 A ˚ are large enough to be
Page 85 and 86:
Table 6 Selected Silica-Based Colum
Page 87 and 88:
Table 7 Separation Ranges for Polym
Page 89 and 90:
the decline in permeability is perm
Page 91 and 92:
sample components can be varied by
Page 93 and 94:
Figure 10 Diol bonding reactions. I
Page 95 and 96:
ecause of the higher relative molec
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valuesfork1,k2,anda,theunknownmolec
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water-soluble and detergent-soluble
Page 101 and 102:
the importance of secondary retenti
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discussed in Table 8(33). Based on
Page 105 and 106:
individual dispersion processes ins
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The detector time constant can dist
Page 109 and 110:
5.3 Mobile Phase A mobile phase is
Page 111 and 112:
less steep and still linear at high
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35. P Bristow, J Knox. Chromatograp
Page 115 and 116:
112. K Gooding, K Lu, G Vanecek, F
Page 117 and 118:
for branched polymers or copolymers
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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.
Page 151 and 152:
53. G Marot, J Lesec. J Liq Chromat
Page 153 and 154:
125. D Slootmaekers, JAPP Van Dijk,
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200. JG Bindels, GJJ Bessems, BM De
Page 157 and 158:
possibly with hydrodynamic volume (
Page 159 and 160:
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
Page 187 and 188:
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
Page 197 and 198:
APPENDIX: SEC CONDITIONS FOR RUBBER
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Appendix (Continued) Polymer Column
Page 201 and 202:
Page 203 and 204:
Page 205 and 206:
5. J West, E Rodriguez. Rubber Chem
Page 207 and 208:
Page 209 and 210:
associate. Girdler (67) and Speight
Page 211 and 212:
Table 1 Fractions Obtained Using Co
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Figure 1 SEC analyses of an aged as
Page 215 and 216:
Apparently more polar compounds are
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40% was fractionated into four frac
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Figure 7 SEC analyses of an unaged
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Figure 9 Comparison of percentage L
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Figure 11 Comparison of SEC chromat
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Figure 12 Comparisons of SEC chroma
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percentage LMS at the other locatio
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the total area into up to 12 sectio
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attempted to develop an SEC method
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parameterisbasedontheinternalpressu
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5 MODIFIED ASPHALTS The addition of
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Oxidation of the rubber and asphalt
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Figure 20 SEC chromatogram for an a
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Figure 21 Comparisons of apparent m
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© 2004 by Marcel Dekker, Inc. S/
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PS/10 4 þ 10 3 þ 500 þ 100 THF/1
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Nevertheless, SEC of asphalts is es
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56. NW Garrick, RR Biskur. Trans Re
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Asphalts 235 122. M-S Lin, JM Chaff
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Page 255 and 256:
Table 1 Applications of Acrylamide
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(AM/AA), and acrylamide/dimethyldia
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polymer, a large size filter of 5,
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Figure 1 Size exclusion chromatogra
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Table3 MWandMWDofaBroad-MWDPAMStand
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Another commercially available MW d
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Table 5 Weight-Average Molecular We
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Figure 6 (a) Size exclusion chromat
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compared to anormal product. By com
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StudyingtheKineticsofaChemicalReact
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this information, the higher MW pea
Page 277 and 278:
Page 279 and 280:
Page 281 and 282:
46. CJ Kim, A Hamielec, A Bendek. J
Page 283 and 284:
Page 285 and 286:
A relatively new technique utilizin
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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
Page 307 and 308:
Page 309 and 310:
2.1 Molecular Weight Grades of PVP
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exclusion chromatography with low-a
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weightandmolecularweightdistributio
Page 315 and 316:
Page 317 and 318:
Figure 4 PEOcalibrationofUltrahydro
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Figure 6 Overlay of GPC chromatogra
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plus Ultrahydrogel 120 A ˚ ,Shodex
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Figure 10 SEC traces of PVP/AA copo
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Figure 11 Molecular weight distribu
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Page 329 and 330:
2 CHEMICAL, MACROMOLECULAR AND MORP
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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)
Page 359 and 360:
30. HA Krässig. Effects of structu
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67. E Sjöholm, K Gustafsson, A Col
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104. B Fleury, J Dubois, C Léonard
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136. D Miller, D Senior, R Sutcliff
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166. SS Cutié, CG Smith. Determina
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200. R Berggren, F Berthold, E Sjö
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13 Molar Mass and Size Distribution
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measurements, and vapor pressure os
Page 375 and 376:
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
Page 381 and 382:
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
Page 389 and 390:
Figure 4 GPC elution curves of the
Page 391 and 392:
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
Page 401 and 402:
Table 1 Selected Industries Connect
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into C3-metabolites, which then may
Page 405 and 406:
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
Page 411 and 412:
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
Page 423 and 424:
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
Page 439 and 440:
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
Page 447 and 448:
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
Page 457 and 458:
Figure 1 Plot of Kav vs. log M for
Page 459 and 460:
yconsideringtheproteinsolutestobesp
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globular proteins and selected flex
Page 463 and 464:
silanol groups (52-54); even capped
Page 465 and 466:
most proteins to be maximally stabl
Page 467 and 468:
Figure 4 Chromatograms of BSA and P
Page 469 and 470:
concentrated) steps of protein puri
Page 471 and 472:
Table 3 Characteristics of Dextran-
Page 473 and 474:
preparative SEC. The capabilities o
Page 475 and 476:
37. GR Noll, N Nagle, JO Baker, DJ
Page 477 and 478:
Page 479 and 480:
Figure 2 Separation of total E. col
Page 481 and 482:
Figure 3 Separation of HaeIII-cleav
Page 483 and 484:
The recovery of DNA fragments has b
Page 485 and 486:
Endotoxin should also be removed fr
Page 487 and 488:
Table 3 Best Columns for the Separa
Page 489 and 490:
Figure 10 Dependence of HETP on flo
Page 491 and 492:
Appendix (Continued ) Polymer Colum
Page 493 and 494:
REFERENCES 1. CT Wehr, SR Abbott. J
Page 495 and 496:
MALDI-MS has been applied to determ
Page 497 and 498:
Figure 1 Calibration curvesof SEC c
Page 499 and 500:
Figure 3 Chromatograms of epoxy res
Page 501 and 502:
wherenandn0 aretheRIsofthesample an
Page 503 and 504:
Figure 5 SEC chromatogram of n-alka
Page 505 and 506:
Figure 7 Refractive indexes of comm
Page 507 and 508:
Another detector recently applied i
Page 509 and 510:
Figure 11 Absolute MW calibration p
Page 511 and 512:
Figure 12 Chromatogram of Acrawax C
Page 513 and 514:
18 Two-Dimensional Liquid Chromatog
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largerispressuredropandresultingexp
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elations between molar masses and s
Page 519 and 520:
Figure 2 (Continued) chapter, one o
Page 521 and 522:
where Nis the column efficiency exp
Page 523 and 524:
composition/architecture/molar mass
Page 525 and 526:
solvent. Here again, solvent-solven
Page 527 and 528:
or repulsive interactions between m
Page 529 and 530:
macromolecules may strongly differ
Page 531 and 532:
temperature. The efficacy of an ads
Page 533 and 534:
however, aliphatic bonded groups ma
Page 535 and 536:
Figure 10 Schematic representation
Page 537 and 538:
packing materials for SEC of synthe
Page 539 and 540:
Quantitative relations between sila
Page 541 and 542:
Both strength and thermodynamic qua
Page 543 and 544:
where Cand Dare constants for apart
Page 545 and 546:
ThisisimportantwhenLCCCisappliedtoc
Page 547 and 548:
Page 549 and 550:
Page 551 and 552:
However, strong adsorption of macro
Page 553 and 554:
elution step may be sufficient for
Page 555 and 556:
Figure 16 Block scheme of a full ad
Page 557 and 558:
processing of 2D-HPLC data. Knowled
Page 559 and 560:
Figure 20 Set of full retention-elu
Page 561 and 562:
distributions of complex polymers a
Page 563 and 564:
constituents were not discriminated
Page 565 and 566:
(Sec. 7) and therefore they can be
Page 567 and 568:
2. Identify sample solvents. First
Page 569 and 570:
1 Segmental interaction energy para
Page 571 and 572:
58. BG Belenkii. Pure Appl Chem 51:
Page 573 and 574:
19 Methods and Columns for High-Spe
Page 575 and 576:
Table 1 Summary of Methods for Incr
Page 577 and 578:
not savealot of time and solvent, d
Page 579 and 580:
Figure 3 Conventional SEC chromatog
Page 581 and 582:
chromatogram at 4mL/min, which is f
Page 583 and 584:
Sincethereductionoftheinternaldiame
Page 585 and 586:
Table 3 Summary of Column Performan
Page 587 and 588:
Figure 11 Dependence of column perf
Page 589 and 590:
. Two-dimensional chromatography ru
Page 591 and 592:
The standard deviations for the mol
Page 593 and 594:
Figure 15 Comparison of time requir
Page 595 and 596:
Figure 17 Accuracy and precision of
Page 597 and 598:
polymer analysis. Samples are “ru
Page 599 and 600:
just ignoring the fractionation of
Page 601 and 602:
20 Automatic Continuous Mixing Tech
Page 603 and 604:
out at low enough concentration tha
Page 605 and 606:
educed viscosity, h r, to be comput
Page 607 and 608:
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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