Handbook of Size Exclusion Chromatography and Related ...

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Figure 6 Substantialperformancelosscausedbyreducingcolumnlengthfrom30 cmto 5cm compared to aconventional SEC column (sample and conditions similar to Fig. 3). They used a4mm ID column (half the diameter of their conventional column) to investigate the effects of reduced column size on polymer separation efficiency and run time. A4mm ID column was chosen because it is compatible with existing instruments. It does not require m-bore ready equipment, which is not available for RI detection (e.g., low cell volumes). Figure 7 shows the raw chromatogram obtained under otherwise identical experimental conditions. As expected,theruntimeinthisexperimentissignificantlyreduced(toabout3min). Figure 7 Efficiency loss and poor peak shapes caused by a column with 4 mm internal diameter (sample and conditions similar to Fig. 3). © 2004 by Marcel Dekker, Inc.
Sincethereductionoftheinternaldiameterdrasticallyreducestheporevolumeof the column (squared relationship), asubstantial influence on performance has to be expected. This is clearly visible in the raw chromatogram. Resolution is much poorer as compared to the reference chromatogram of the conventional column in Fig. 3. Moreover, the peak shapes are badly affected as can be seen in the relative change of the peak heights of the standards. The higher the molar mass of a standard the broader the peak gets (and the lower the corresponding peak height will be). This is caused by the high linear flow rate inside the column, which is necessary to drive the eluent through it; please note that the pump flow rate was kept constant at 1.0 mL/min. Samples with high molar mass will be affected by shear degradation under such conditions. The limited efficiency in this setup is alsoseeninthepoorseparation ofsamplepeaks fromthesolventpeaks attheend of the chromatogram. 3.1.5 Changing Column Aspect Ratio The simultaneous adaption of column length and diameter allows the internal volume of the separation system to be kept constant. This is important for SEC, because the column volume and the pore volume of the packed bed are directly related. As pointed out above, the pore volume is one of the major factors influencing peak resolution. Cutting down the column length and increasing the internal dimension of the column at the same time can, in theory, reduce the chromatographic run time while maintaining the efficiency of the separation. Obviouslysuchcolumnshavetobeoperatedathigherflowratestoreducetherun times. Solvent cannot be saved significantly without interfering with resolution. Moreover, other effects can influence the separation. In such ascenario, the accessibility of the pores in the packing will be most important. If the architecture of the pores in the column will restrict diffusional migration of the solutes, the column will not and cannot perform as expected. Wall effects might also influence the separation and have to be watched closely as column length is reduced. PSS studied such short wide-bore columns extensively for their use in fast separations. Figure8showstheseparationonatestcolumnof50 mmlengthand20 mm ID run at a flow rate of 6 mL/min. The same packing material as in all other columns was used in this experiment. All other experimental parameters were kept constant. The first impression of the separation is favorable: . All peaks are present in their correct height (concentration) ratios, . A good peak symmetry is kept throughout the whole chromatogram, . No indication of sample degradation at high molar masses, . Separation is carried out in less than 2 min, and . Sufficient separation of solvent peaks from sample. © 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
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 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: chromatogram at 4mL/min, which is f
Page 585 and 586: Table 3 Summary of Column Performan
Page 587 and 588: Figure 11 Dependence of column perf
Page 589 and 590: . Two-dimensional chromatography ru
Page 591 and 592: The standard deviations for the mol
Page 593 and 594: Figure 15 Comparison of time requir
Page 595 and 596: Figure 17 Accuracy and precision of
Page 597 and 598: polymer analysis. Samples are “ru
Page 599 and 600: just ignoring the fractionation of
Page 601 and 602: 20 Automatic Continuous Mixing Tech
Page 603 and 604: out at low enough concentration tha
Page 605 and 606: educed viscosity, h r, to be comput
Page 607 and 608: © 2004 by Marcel Dekker, Inc. Figu
Page 609 and 610: Figure 2 Raw ACOMP signals from mul
Page 611 and 612: Figure 4 Mw vs. monomer conversion,
Page 613 and 614: decreases smoothly and monotonicall
Page 615 and 616: Figure 7 Effects of fluctuating rea
Page 617 and 618: Finally, the use of a light-scatter
Page 619 and 620: Figure 8 Linear and cubic increases
Page 621 and 622: where r(t) is now given by 2 6 r(t)
Page 623 and 624: Figure 11 Light scattering from sol
Page 625 and 626: 3.1 ASimple System: Equilibrium Cha
Page 627 and 628: Figure 13 (a) ACM applied to the ch
Page 629 and 630: Also of note in Fig. 13b is how hig
Page 631 and 632: 15. JM Starita, CL Rohn. Rheologica
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52. M Hulden. Hydrophobically modif
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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
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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
Page 661 and 662:
fractionated sample. They represent
Page 663 and 664:
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
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
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and concomitant clogging of columns
Page 677 and 678:
Figure 5 Concentration dependence o
Page 679 and 680:
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
Page 689 and 690:
Figure 2 Calibration curve for the
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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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