Handbook of Size Exclusion Chromatography and Related ...

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Narrow columns require lower flow rates for optimal operation. Figure 10 shows the same columns as above, but this time under identical linear flow velocity. This means that the standards travel with identical speed through each of the different columns and all columns perform close to the optimum in the van Deemter plot. This view allows the comparison of the efficiency of the separation and its time requirement. Because narrow-bore columns need lower volume flow rates for best performance, almost no time can be gained. The resolution difference between the conventional analytical column and the narrow-bore column in Fig. 10 can, in part, be attributed to the fact that the same instrument was used that was not optimized for narrow-bore use. The shorter run time for the narrow-bore column must only be attributed to its shorter column length (25 cm instead of 30 cm for the analytical column). The fastest column is the short wide-bore column, which is about six times faster than the other columns in the comparison and nevertheless shows satisfactory resolution. Figure 10 Overlay of SEC chromatograms of columns with different dimensions tested with identical polystyrene standards in THF at identical linear flow rates reveals time requirements and related column efficiency. [1] Traditional analytical SEC column (8 300 mm), [2] short SEC column (8 50 mm), [3] narrow-bore SEC column (4 250 mm). © 2004 by Marcel Dekker, Inc.
Figure 11 Dependence of column performance parameters on SEC column dimensions and pore volume. [1] Traditional analytical SEC column (8 300 mm), [2] short SEC column (8 50 mm), [3] narrow-bore SEC column (4 250 mm), and [4] short wide-bore SEC column (20 50 mm). The column with the best overall performance is certainly the conventional column. This is no surprise since this product has been optimized for highest performance/price and has been in use in many laboratories for years. The conventional column performs extremely well in all areas studied. The next best design for polymeric applications is the short wide-bore column. It is quite obvious that the resolution must be enhanced by using a specially designed packing with optimized pore architecture. This can also be seen in Fig. 11. As a general rule the following conclusions can be drawn: . For least time requirement a short wide-bore column should be used, and . For lowest eluent consumption a short and thin column is best. 4 HOW CAN HIGH-SPEED SEC COLUMNS BE MADE? The previous chapter has shown the potential and shortcomings of various methods to overcome the time restraints in conventional SEC experiments. In order to utilize the short wide-bore columns best, new packing materials have to be designed that overcome the pore access limitations of conventional packings. Conventional analytical columns have been optimized for their typical flow rates © 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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© 2004 by Marcel Dekker, Inc. Figu
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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
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 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: Table 3 Summary of Column Performan
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
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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
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5 THE IMPORTANCE OF SOFTWARE Like a
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standard deviations of the measured
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These include the differential weig
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Figure 8 Data of Fig. 7corrected fo
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Figure 11 Data of Fig. 10 with spik
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Figure 13 Fitstodatacollectedatasli
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Table 1 Errors (in %) Characteristi
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homogeneous copolymer, that is, tak
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process may be used to identify the
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fractionated sample. They represent
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Figure 16 An example of poor SEC ch
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on) methods, whereby the average di
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23. WH Stockmayer, LD Moore Jr, M F
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condition, as is explained in this
Page 671 and 672:
Figure 2 Partition coefficients KL
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4 COLUMNS FOR HOPC Detailsaregiveni
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and concomitant clogging of columns
Page 677 and 678:
Figure 5 Concentration dependence o
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Table 1 Solution Volume, Polymer Ma
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in Fig. 4a. The middle fractions (8
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Figure 10 SEC chromatograms for som
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appropriate for the early fractions
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23 Size Exclusion/ Hydrodynamic Chr
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Figure 2 Calibration curve for the
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Figure 5 Elutionbehaviorofpolystyre
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Table 1 Comparison Between SEC and
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egular SEC column in which the pore
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Such an ideal separation can be obt
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