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Handbook of Size Exclusion Chromatography and Related ...

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The calibration <strong>of</strong> SEC columns by conventional calibration, universal<br />

calibration, <strong>and</strong> sedimentation equilibrium studies have been compared for native<br />

lignins <strong>and</strong> acetylated organosolv lignins (5). Conventional SEC analysis<br />

calibratedwithapolystyrenest<strong>and</strong>ardgavethelowestmolarmassvalues.Theuse<br />

<strong>of</strong> universal calibration gave molar mass estimates higher by factors <strong>of</strong> 1.5–2.5<br />

than conventional SEC. The sedimentation equilibrium studies gave values <strong>of</strong><br />

Mw,app that were roughly similar to those obtained by universal calibration. These<br />

resultswerenotconsideredtobesurprisingbecauseconventionalHPSECpredicts<br />

the effective Vh <strong>of</strong> the lignin derivative, not its molar mass, <strong>and</strong> also because<br />

conventional calibration was performed relative to polystyrene st<strong>and</strong>ards.<br />

Universal calibration uses the relationship between V h([h]M) <strong>and</strong> elution volume<br />

for aspecificcolumnset throughout awiderange <strong>of</strong>polymer structures <strong>and</strong>sizes.<br />

ThehigherMMobtainedbyuniversalcalibrationthanbyconventionalcalibration<br />

isinlinewiththepossibilitythatligninsarebranchedpolymers.Itisapparentthat<br />

branched polymers <strong>of</strong> higher molar mass may occupy the same Vh as alinear<br />

polymer <strong>of</strong> lower molar mass.<br />

Using THFas eluent <strong>and</strong> Styragel columns packed with crosslinked divinyl<br />

benzene–polystyrene, Jacobs <strong>and</strong> Dahlman (10) investigated matrix-assistedlaser-desorption-ionization<br />

time-<strong>of</strong>-flight mass spectrometry (MALDI-TOF-MS)<br />

for determination <strong>of</strong> absolute molar masses <strong>of</strong> lignins <strong>and</strong> hemicelluloses. During<br />

the SEC runs <strong>of</strong> lignins, fractions <strong>of</strong> different molecular size are collected <strong>and</strong><br />

introduced in the the MALDI-TOF-mass spectrometer, <strong>and</strong> by this system it is<br />

possible to obtain absolute molar masses <strong>of</strong> the different fractions, whichare then<br />

used for the calibration <strong>of</strong> the columns. The results were compared with apparent<br />

molarmassesobtainedusingmonodispersepolystyrenesforcalibration.Themain<br />

features <strong>of</strong> the MALDI technique are high sensitivity,wide mass range, relatively<br />

simple sample preparation, rapid generation <strong>of</strong> results, <strong>and</strong> almost no<br />

fragmentation <strong>of</strong> the molecules during the analyses. The heterogeneity <strong>of</strong> the<br />

kraftlignin makesitimpossibletodetectseparateMALDI-TOF-MSpeaks for the<br />

different components in the lignin polymer distribution, therefore no structural<br />

information besides the molar mass distribution can be obtained for the lignin<br />

sample from the MALDI-MS spectrum (Fig. 1).<br />

It is possible to determine absolute molar masses <strong>of</strong> narrow lignin fractions<br />

directly by MALDI-TOF-MS.<br />

3 DIFFERENT SEC METHODS FOR DIFFERENT<br />

LIGNIN SAMPLES<br />

Currently, very few new experimental methods for SEC measurements <strong>of</strong> lignins<br />

have been developed, although several new applications have been reported.<br />

Programs for data collection <strong>and</strong> treatment <strong>of</strong> raw data obtained from different<br />

© 2004 by Marcel Dekker, Inc.

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