Tools for the Characterization of Biomass at the Nanometer Scale - The Nanoscience and Technology of Renewable Biomaterials - page 70

Table3.1 Affinityandswellingpowerofdifferentsolventstowardbiomass(4,65).

Solvent Liquid-holding capacity Liquid-holding capacity Tangential swelling

of α -Cellulose (%) a of sulfite pulp (%) b of spruce wood (%) c

Water 85.0 403.0 8.4

Methanol 60.5 253.6 8.2

Ethanol 41.7 109.4 7.0

Acetone 13.4 47.6 5.7

a

α -Cellulose is pure cellulose.
b Sulfite pulp contains some hemicellulose and lignin residue; cellulose is less crystalline than α -Cellulose.
c Tangential swelling is relative to oven dry.

by liquid nitrogen (59, 61, 62). While this kind of super-fast cooling is essential for live
cells, this method is rarely used when preparing biomass samples, even though there is
evidence that slow or normal freezing changes pore structure (31).
Another approach to avoiding liquid:vapor surface tension during drying is to use
critical point drying (CPD). In this method, water is replaced by a transitional solvent
(ethanol or acetone), and then CO 2 . When CO 2 is pressurized to the critical point, there
is no surface tension between the liquid and gas phases, allowing evaporation of all the
liquid with no surface tension (64). If proteins and lipids are present, fixation before

CPD is suggested.
The primary problem with either CPD or low-temperature embedding is the need
to change solvent. Besides the obvious problem of dissolving cellular components,
changing solvents changes the stability of solvated structures, resulting in bulk swelling
and shrinking and unknown changes to nanostructures. The extent of biomass swelling
in a few typical solvents is given in Table 3.1.
Exposing wood to a solvent may change some aspects of the nanostructure, but it
can also be useful in revealing nanoscale features. Removing extractives (resins, waxes,
gums, fats) by acetone or ethanol, or ethanol/toluene is common as these extractives can
interfere with chemical analysis. Tokareva showed how various specimen preparation
procedures, including extraction by acetone or critical point drying, improved their ability
to see fine structure such as cellulose macrofibrils (66).

3.4.1.2 Microtoming

Preparing 20- to 30-nm cross sections of homogeneous materials with a microtome
is technically challenging, but the heterogeneity and fibrous nature of biomass makes
producing these specimens from biomass even more difficult. The microtome knife is
really initiating a crack that will grow along the path of least resistance. Biomass is
difficult to microtome without distortion because fibrous structures and very nonuni-
form mechanical properties of different cell wall components redirect the progress of
the crack.
Distortions introduced by microtoming have been systematically examined (67), and
means to minimize distortion with diamond and glass knives have been reported (68,
69). H. Sitte has extensively studied microtomy and directed the design of commercial
instruments and has thoroughly reviewed microtomy practice (70).