galactosamine). Once liberated from the larger polymeric structures,
accurately quantifying these sugars can be incredibly challenging. The
spectrophotometric determination of carbohydrates is a technique
that is easier to adopt and does not require specialized equipment.
One of the most commonly used spectrophotometric methods is the
phenol-sulfuric acid (PSA) method [8, 9], but this method is susceptible to varying sensitivity to microalgae-specific carbohydrates and
does not produce consistent peak shapes under visible spectroscopy
[10]. The PSA method is also prone to interference from other
microalgal biochemical constituents, such as protein and lipids.
Because of the above limitations, we developed a novel use for
the spectrophotometric 3-methyl-2-benzothiazolinone hydrazone
(MBTH), which has been shown to give an almost equal response of
different aldehyde sugars in solution [10]. At the same time, we
demonstrated that the MBTH quantification matched HPLC quantification of complex mixtures of microalgae-specific monosaccharides. Spectrophotometric methods are quick and can provide a
researcher with an overall quantitation of a component such as carbohydrates. Though to derive fully integrated options on downstream
conversion and fermentation of carbohydrates, a detailed understanding of the monomeric makeup is necessary [4]. Identification
and quantification of the respective monomers by chromatography
provide the required details; however, this is not straightforward
[1]. The utilization of high-performance anion-exchange chromatography (HPAEC) has proven instrumental in providing the resolution of the unusual monosaccharides typically found in algae [1, 6,
10]. We discuss here a process for whole biomass analytical hydrolysis
to monomeric constituents and subsequent quantification of total
carbohydrates by either MBTH derivatization and spectrophotometric detection or HPAEC with pulsed amperometric detection (PAD)
for monomeric sugar quantification.
2 Materials
2.1 Hydrolysis
Apparatus
1. Analytical balance, accurate to 1 mg or 0.1 mg.
2. Vortex mixer.
3. Water bath, set to 30 Æ 3
C.
4. Autoclave, suitable for autoclaving liquids, set to 121 Æ 3
C.
5. 10 mL glass tubes and caps, heavy walled and capable of withstanding high pressure in an autoclave (see Note 1).
6. Adjustable pipet(s), covering 250 μL and 7 mL.
7. Disposable plastic ~3 mL syringes and compatible 0.2 μm
nylon filters, if planning to filter as opposed to centrifuging to
remove solids.
192
S. Van Wychen and L.M.L. Laurens
accurately quantifying these sugars can be incredibly challenging. The
spectrophotometric determination of carbohydrates is a technique
that is easier to adopt and does not require specialized equipment.
One of the most commonly used spectrophotometric methods is the
phenol-sulfuric acid (PSA) method [8, 9], but this method is susceptible to varying sensitivity to microalgae-specific carbohydrates and
does not produce consistent peak shapes under visible spectroscopy
[10]. The PSA method is also prone to interference from other
microalgal biochemical constituents, such as protein and lipids.
Because of the above limitations, we developed a novel use for
the spectrophotometric 3-methyl-2-benzothiazolinone hydrazone
(MBTH), which has been shown to give an almost equal response of
different aldehyde sugars in solution [10]. At the same time, we
demonstrated that the MBTH quantification matched HPLC quantification of complex mixtures of microalgae-specific monosaccharides. Spectrophotometric methods are quick and can provide a
researcher with an overall quantitation of a component such as carbohydrates. Though to derive fully integrated options on downstream
conversion and fermentation of carbohydrates, a detailed understanding of the monomeric makeup is necessary [4]. Identification
and quantification of the respective monomers by chromatography
provide the required details; however, this is not straightforward
[1]. The utilization of high-performance anion-exchange chromatography (HPAEC) has proven instrumental in providing the resolution of the unusual monosaccharides typically found in algae [1, 6,
10]. We discuss here a process for whole biomass analytical hydrolysis
to monomeric constituents and subsequent quantification of total
carbohydrates by either MBTH derivatization and spectrophotometric detection or HPAEC with pulsed amperometric detection (PAD)
for monomeric sugar quantification.
2 Materials
2.1 Hydrolysis
Apparatus
1. Analytical balance, accurate to 1 mg or 0.1 mg.
2. Vortex mixer.
3. Water bath, set to 30 Æ 3
C.
4. Autoclave, suitable for autoclaving liquids, set to 121 Æ 3
C.
5. 10 mL glass tubes and caps, heavy walled and capable of withstanding high pressure in an autoclave (see Note 1).
6. Adjustable pipet(s), covering 250 μL and 7 mL.
7. Disposable plastic ~3 mL syringes and compatible 0.2 μm
nylon filters, if planning to filter as opposed to centrifuging to
remove solids.
192
S. Van Wychen and L.M.L. Laurens