Handbook of Size Exclusion Chromatography and Related ...

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2.3 Particle Morphology As mentioned, reducing particle size was crucial in making liquid chromatography a high-performance technique. Early in the development of HPLC, small silica particles were obtained by grinding and sieving larger silica gels used in the purification of natural products by open-column liquid chromatography. Once the potential of “high-pressure” LC had been demonstrated (41,42), columns packed with 10-mm irregularly shaped silica became readily available. Although such particles are still widely used in routine analyses, most analyses and column development work in academia and industry is performed with spherical 5-mm particles. In recent years, 5-mm particles have become widely available for gel filtration of proteins. The use of even smaller particle sizes in SEC has been advocated by Guiochon and Martin (36) and Engelhardt and Ahr (43), who investigated the optimum particle size for analysing proteins. One of the main advantages of a column packed with spherical particles is that the pressure drop is lower by as much as a factor of 2 compared with a column packed with irregular particles of the same average size. Also, although the hardness of silica depends mainly on the size of the pores together with the pore volume per particle, there is some evidence for the widely held belief that irregular particles are more prone to breakage during the column-packing process (44). It is also considered more difficult to prepare a well-packed column with irregular particles (45). Particle shape does not influence the kinetic and thermodynamic properties that describe the chromatographic process. The relationship between particle size and column efficiency is now well understood, although the exact form of the equations, including the Knox equation [see Eq. (3)], is still debated (46). The 3–5 mm particle size of modern HPLC columns allows fast analysis of small molecular weight compounds at near optimal column efficiency. As discussed, larger molecular weight compounds, because of their smaller diffusion coefficients, require much lower flow rates to elute with maximum column efficiency. Because of the usual variation in polymer molecular weight, it is not possible to operate the column at the optimal speed for all components in the sample. 2.4 Column Dimensions A common internal diameter for an SEC column is 7.5 or 7.8 mm vs. 4.6 mm for non-SEC columns. The length of an SEC column has traditionally been 30 cm, but 60-cm columns have also been available for 10-mmm packings. Initial packing studies showing higher efficiencies for larger bore columns contributed to the choice of 7–8 mm as the internal diameter for most high-performance SEC columns (47,48). Advantages of such larger ID columns are (1) a reduction of the importance of extra column contributions to the volume of the sample band, (2) increased sample capacity for preparative purposes, and (3) the ability to © 2004 by Marcel Dekker, Inc.
operate at aflow rate that can easily be maintained with the available HPLC instrumentation. Recent studies have demonstrated that capillary SEC columns can be packed with equivalent or higher efficiencythan SEC columns of standard dimensions. An example is shown in Fig. 2, in which the efficiency of 28- and 50-mm ID columns were evaluated using bovine serum albumin (BSA), chicken ovalbumin, and bovine a-chymotrypsinogen as test solutes at linear velocities (based on atotally excluded solute) varying from 0.01 to 0.9 mm/s (49). The microcolumnswerepackedwith4.5-mm,150 A ˚ ,ZorbaxGF-250XLparticlesthat were treated with azirconium salt and derivatized with adiol functionality.The diffusion coefficients ( 10 7 cm 2 /s) for these proteins, ranging in molecular weight from 69,000 to 43,000 and 26,000, were experimentally determined to be 5.65,6.68,and8.23,respectively.Notethattheoptimumreducedplateheightwas as low as 2for BSA and as high as 4for a-chymotrypsinogen. In all cases, the reduced velocity at hmin was approximately 5. As measured by the half-height method, the efficiency of a30 cm 50 mm ID column compared favorably with that of astandard 25 cm 9.4 mm ID column filled with the same packing material, and the performance of the capillary column was much better when calculated by statistical moments or based on the Dorsey–Foley equation (50). BecauseofthelargerIDwhenoperatingastandarddiameterSECcolumnat aflowrateof1mL/min,thelinearvelocityis2.5timeslowerthanwhenthesame flowrateisusedona4.6-mmIDcolumn.Thus,anSECcolumnisoperatedcloser to the velocity at which the column performs at optimal efficiency.As discussed, however, at least a10-fold drop in flow rate is required for the column to perform near its optimum for most proteins. This effect is illustrated in Fig. 3, in which a protein test mixture is separated at various flow rates on a25 cm 4.1 mm ID column packed with 10-mm, 250 A ˚ ,amide-bonded silica (51). Clearly,resolution improves with decreasing flow rate: the optimum efficiency had not yet been reached at aflow rate of 65 mL/min or alinear velocity at 0.13 mm/s. AccordingtoEq.(2),reducedvelocityisinverselyproportionaltothesolute diffusion coefficient. Under the same conditions, solutes of varying molecular weightshowoptimalcolumnperformanceatdifferentflowrates.Thisisillustrated in Fig. 4. The relationship between the logarithm of molecular weight (MW) and the otimal flow rate is plotted for 50 peptides and glycine (MW 50–10,000) analyzed under denaturing mobile-phase conditions (52). As shown, the optimal flow rate is inversely and linearly related to log MW. Over the narrow molecular weight range, the optimum flow rate decreases roughly 2-fold for a 10-fold increase in molecular weight. 2.5 Porosity Except for nonporous particles, all packing materials contain a variation of pore sizes around a mean value. This pore size distribution determines the range of © 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
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 and 34: Analytical. Two models, the KMX-6 a
Page 35 and 36: Figure 6 Overlay of time-sliced pea
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: Table 3 Properties of SEC Silica Ge
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
Page 97 and 98: valuesfork1,k2,anda,theunknownmolec
Page 99 and 100: water-soluble and detergent-soluble
Page 101 and 102: the importance of secondary retenti
Page 103 and 104: discussed in Table 8(33). Based on
Page 105 and 106: individual dispersion processes ins
Page 107 and 108: 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
Page 113 and 114: 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
Page 119 and 120: Figure 1 “Bridge design” flow-r
Page 121 and 122: Figure 3 A light-scattering photome
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Figure 5 SEC universal calibration
Page 125 and 126:
where hn is the hydrodynamic volume
Page 127 and 128:
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
Page 133 and 134:
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
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
Page 169 and 170:
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
Page 191 and 192:
© 2004 by Marcel Dekker, Inc. Figu
Page 193 and 194:
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
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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
Page 247 and 248:
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
Page 253 and 254:
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,
Page 261 and 262:
Figure 1 Size exclusion chromatogra
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Table3 MWandMWDofaBroad-MWDPAMStand
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Another commercially available MW d
Page 267 and 268:
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
Page 275 and 276:
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
Page 287 and 288:
where ais the exponent of the Mark-
Page 289 and 290:
Figure 2 TDS chromatograms for full
Page 291 and 292:
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
Page 303 and 304:
Figure 12 PVAc broad molecular weig
Page 305 and 306:
4 SUMMARY Advances in SEC character
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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
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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
Page 327 and 328:
Page 329 and 330:
2 CHEMICAL, MACROMOLECULAR AND MORP
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Table 2 The Relative Composition of
Page 333 and 334:
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
Page 339 and 340:
observed using column materials hav
Page 341 and 342:
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
Page 357 and 358:
differential refractive index (DRI)
Page 359 and 360:
30. HA Krässig. Effects of structu
Page 361 and 362:
67. E Sjöholm, K Gustafsson, A Col
Page 363 and 364:
104. B Fleury, J Dubois, C Léonard
Page 365 and 366:
136. D Miller, D Senior, R Sutcliff
Page 367 and 368:
166. SS Cutié, CG Smith. Determina
Page 369 and 370:
200. R Berggren, F Berthold, E Sjö
Page 371 and 372:
13 Molar Mass and Size Distribution
Page 373 and 374:
measurements, and vapor pressure os
Page 375 and 376:
The calibration of SEC columns by c
Page 377 and 378:
The division according to eluent ty
Page 379 and 380:
Apolymer produced by UV irradiation
Page 381 and 382:
Because DMAC/LiCl is also agood sol
Page 383 and 384:
compoundsandwasexplainedbyadsorptio
Page 385 and 386:
isolated lignins because they are e
Page 387 and 388:
The Separon HEMA and Separon HEMA B
Page 389 and 390:
Figure 4 GPC elution curves of the
Page 391 and 392:
liquor is highest throughout the co
Page 393 and 394:
topological anisotropy in the polym
Page 395 and 396:
29. M Ristolainen, R Alen, J Knuuti
Page 397 and 398:
eds. Proc Int Symp 1998. Canton, Pe
Page 399 and 400:
92. L Jurasek. Towards a three dime
Page 401 and 402:
Table 1 Selected Industries Connect
Page 403 and 404:
into C3-metabolites, which then may
Page 405 and 406:
Table 4 Key Enzymes in the Biosynth
Page 407 and 408:
Table 5 Cloned Mutants of Starch En
Page 409 and 410:
Figure 3 Modeled x-ray diffraction
Page 411 and 412:
In the native environment (plant ce
Page 413 and 414:
Figure 6 (a) Large starch granules
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number of branching points); crossl
Page 417 and 418:
Both absolute approaches are extrao
Page 419 and 420:
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
Page 425 and 426:
Figure 12 Wheat glucans: DRI elutio
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Figure 13 Wheat glucans. Normalized
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Figure 16 Wheat glucan. Normalized
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Figure 18 Wheat glucan.Elution prof
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Figure 20 Wheat glucans. Absolute m
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Figure 22 Wheat glucan. (a) Intrins
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Figure 24 Wheat PA-glucan. Elution
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an Ubbelohde-viscometer for concent
Page 441 and 442:
Rheological investigations of stabi
Page 443 and 444:
shifts to applied laser light, whic
Page 445 and 446:
Table 8 Characteristic Parameters f
Page 447 and 448:
Table 8 (Continued) and H2O. A part
Page 449 and 450:
10. SG Ball, MHBJ van de Wal, RGF V
Page 451 and 452:
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
Page 461 and 462:
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
Page 515 and 516:
largerispressuredropandresultingexp
Page 517 and 518:
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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