Handbook of Size Exclusion Chromatography and Related ...

measurements, and vapor pressure osmometry (VPO). Recently, MALDI-TOF-MS
(10) and electron spray/mass spectrometric (11) methods have been taken into use.
These different absolute methods are used when calibrating different HP/SEC
systems either by on-line detection using light-scattering techniques (MALLS,
LALLS) and viscosity measurements for universal calibration or by isolating
preparatively fractions with narrow MMDs for absolute molar mass determinations.
Absolute molar mass values are obtained by using LALLS detectors (7). By
this method the size of the lignin molecules is obtained, which includes the
possible occurrence of lignin aggregates, and the Mw and Mz can be calculated
from the results together with the dimensions of the molecules. The possible
occurrence of lignin aggregates emphasizes the importance of knowing the
physicochemical properties of the lignin molecules in different eluents. During LS
measurements no fluorescence should occur in the samples (12). It is possible to
measure the absolute weight-average molar mass Mw, second virial coefficient, and
the z-average root-mean-square radius of gyration. The theory and applications of
light scattering for calculating sizes of lignin molecules have been described by
Pla (7). The use of pulsed field gradient NMR has also been used in the
determination of the size of the lignin molecules (13).
The theory of the sedimentation equilibrium has been described in detail
earlier (6). If the lignin/THF system is considered as an ideal solution, then the
following expression describing sedimentation–diffusion equilibrium in the
centrifuge cell can be used.
Mapp ¼
2RT
[(1 v2r)w2]
d ln c
dr2
When solutes are polydisperse, as is the case for SEC of lignin, it is useful to
recognize that M app becomes M wr, which is the weight-average molar mass at any
given radial distance r from the center of rotation.
Absolute number-average molar mass (Mn) is obtained by vapor pressure
osmometry, which, however, is restricted to lignins of low molar mass,
approximately 500–10,000 g/mol. The best results are obtained if the lignins are
soluble in organic solvents such as toluene or THF. This is often only partly the
case, when acetylating and/or methylating the solublilities of lignin samples
increase. The theory and application of this method are described by Pla (7).
Recently MALDI-TOF-MS (10) and electron-spray method mass
spectrometry (11) have been applied. These methods are used for the
determination of absolute molar masses for narrow fractions collected during
MMD measurements. The heterogeneity of the kraft lignin makes it difficult to
detect separate MALDI-MS peaks for the different components in the lignin. No
structural information is therefore obtained for the lignin sample from the
MALDI-TOF-MS spectrum. The electron-spray/mass spectrometry (ESI/MS)
© 2004 by Marcel Dekker, Inc.
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