Characterization Methods and Techniques 115

protein content by a generic multiplier of 6.25 although, a more accurate multiplier

between 5.1 and 6 has been determined for a number of species [61, 62]. ASL is analyzed

in the filtrate using UV–vis spectroscopy be relating the absorbance at휆=240–320 nm

to the total concentration, through the absorptivity coefficient (maintaining a given

path length). Note that this analysis is based on the Beer–Lambert Law and is a highly

accurate and simple method to determine the concentration. Careful selection of the

absorbance range (usually below 1) and absorptivity coefficient is required based on

the biomass type and the pretreatment method.

4.8.1 Analyzing Filtrate by HPLC for Monosaccharide Contents

Many of the monosaccharides are epimers of each other; hence they have the exact same

molecular formula, similar ring structures but differ in conformations of the hydroxyl

and hydrogen groups attached to the different carbons on the ring. This similarity

makes it difficult to determine the exact amount of components without separating

them in some manner. A common method of separation is liquid chromatography that

partitions components between two phases, typically a mobile fluid phase and a solid

stationary phase. Chromatography was first developed by M. Twsett to separate chloro-

phyll pigments, as their separation on a CaCO 3 column left bands of color (chroma

is color in Greek). Nowadays high-performance liquid chromatography equipment

couples a column system to an online detection system to determine the concentration.

These detection systems can be simple for light absorbing compounds, such as UV–vis

detectors, to detectors that measure the change in refractive index (RI) of the solution

as a function of concentration, or a pulsed amperometric detector that measures the

change of electrical current. The latter has gained in popularity for systems for dedicated

sugar analysis as it is very sensitive for detection of sugars at very low concentrations.

4.8.2 Choosing the HPLC Column and Its Operating Conditions

Figure 4.2 shows the chromatogram of a mixture of cellobiose, glucose, xylose, and

arabinose using Bio-Rad®Aminex 87Hcolumn and detected with an RI detector.

This column can separate cellobiose, glucose, xylose, and arabinose. The column is not

suitable for the analysis of lignocellulose with high mannose content, such as softwoods

because mannose is co-eluted with glucose. The advantage of this column is that it is

robust and will not be greatly impacted by fluctuations of the pH of the eluent. It is

commonly used to analyze glucan digestibility of biomass, as glucose is released based

on the specificity of the cellulase hydrolyzing cellulose. Figure 4.2 shows an example of

the monosaccharide elution profile using RI detector.

To analyze samples with high mannose content, the Bio-Rad

®

Aminex 87Pcolumn is

effective to separate out all the hydrolyzed monosaccharides (glucose, xylose, galactose,

arabinose, and mannose); however, rigorous protocols must be used to limit pH

fluctuations that will impact the life of the column.

Other components of biomass can make up a significant mass portion. In hardwoods,

the hemicelluloses are highly acetylated and they can make up to 10% of the mass of the

isolated xylan corresponding to 2–5% of the wood. Additionally, uronic acid branches

of hemicelluloses can compose upwards of 2–6% of the wood [63]. Acetyl groups can

be readily determined by measuring the acetic acid concentration in the hydrolysis

liquor via HPLC (high-performance liquid chromatography) with an RI detector. The