Shodex™ HPLC Approach for Biomass Related Analysis
Kanna Ito, Shodex™/Showa Denko America, Inc.
Data provided by Showa Denko K.K.
Introduction
Biomass is a renewable energy source obtained from biological materials. One type of useful product is biofuel. Biofuel is a general term used for the bioethanol and biodiesel. Bioethanol is the majority (~90%) of the biofuel used worldwide. It is considered as an ECO-friendly automobile fuel produced from sugar and starch in plants. Ideally the main source of biofuel is none-food source such as wood, algae, and inedible part of crops.
There are different biofuel production methods already used in industries, and the others that are under development. Most starting product is polysaccharide which needs to be broken down to monosaccharide in order to produce ethanol through fermentation process. Throughout the production, it is important to analyze various components generated. They are for quality assurance purpose of raw material, final product, byproduct, and impurities, as well as for understanding of degradation mechanism and for recovery monitoring of final product. HPLC analysis is the most frequently used qualification and quantification method. Several methods using Shodex™ columns are introduced in this article.
Contents
- Saccharides in wood
- Separation of hexose and pentose
- Hydrolyzed dextran I
- Hydrolyzed dextran II
- Fermentation inhibitors (Organic acids)
- Fermentation inhibitors (Furfurals)
- Biodiesel composition analysis
- Biomass (Lignin)
- Biomass (Xylan)
- Impurities (Glycerides)
- Impurities (Organic acids)
Results
1. Saccharides in Wood
Polysaccharide hydrolyzation is a prerequisite for biomass production: As oligomers need to be further processed into monosaccharides in order to produce bioethanol. Separation of oligosaccharides and monosaccharides can be achieved under simple HPLC conditions using Shodex SUGAR SP0810 column. It works under size exclusion and ligand exchange modes. This method is used as a laboratory analytical procedure at National Renewable Energy Laboratory (http://www.nrel.gov/biomass/analytical_procedures.html: Technical Report NREL/TP-510-42618 Determination of Structural Carbohydrates and Lignin in Biomass 2008).
Fig. 1. Separation of saccharides in wood showing peaks for Cellobiose, Glucose, Xylose, Galactose, Arabinose, and Mannose.
| Column | Shodex SUGAR SP0810 |
| Eluent | H2O |
| Flow Rate | 0.6 mL/min |
| Column Temp. | 85 °C |
| Detector | RI |
2. Separation of Hexose and Pentose
Both hexose and pentose are the source of bioethanol. However, there is less number of enzymes that can decompose pentose compared to the enzyme that can decompose hexose. Shodex Asahipak NH2P-50 4E, an amino HILIC column can be used to separate several hexose and pentose under isocratic elution.
Fig. 2. Isocratic separation of hexose and pentose including Fructose, Xylose, Fructose, and Glucose.
| Column | Shodex Asahipak NH2P-50 4E |
| Eluent | CH3CN/H2O=80/20 |
| Flow Rate | 1.0 mL/min |
| Column Temp. | 30 °C |
| Detector | RI |
3. Hydrolyzed Dextran I
Hydrolyzation process can be monitored by analyzing oligosaccharides with different molecular sizes. Shodex SUGAR KS series columns are suitable for the separation of polysaccharides under size exclusion mode. The same column can be also used to separate oligosaccharides from ethanol.
Fig. 3a. Hydrolyzed starch showing oligosaccharide distribution from Dp-10 to Dp-1.
| Column | Shodex SUGAR KS-802 × 2 |
| Eluent | H2O |
| Flow Rate | 1.0 mL/min |
| Column Temp. | 80 °C |
| Detector | RI |
Fig. 3b. Separation of Cellopentaose, Cellotetraose, Cellotriose, Cellobiose, Glucose, and Ethanol.
| Column | Shodex SUGAR KS-802 |
| Eluent | H2O |
| Flow Rate | 0.6 mL/min |
| Column Temp. | 80 °C |
| Detector | RI |
4. Hydrolyzed Dextran II
Amino HILIC column is also suitable for hydrolyzation process monitoring. Gradient method provides better separation compared to an isocratic method. Evaporative light scattering detector (ELSD) is feasible for the detection of saccharides under gradient separation mode.
Fig. 4. Comparison of (A) isocratic vs (B) gradient elution for hydrolyzed starch analysis using Asahipak NH2P-50 4E column with ELSD detection.
| Column | Shodex Asahipak NH2P-50 4E |
| Eluent | (A) CH3CN/H2O=50/50 (B) 0 to 7min CH3CN/H2O=57/43 to 50/50 (Linear gradient), 7 to 16min CH3CN/H2O=50/50 |
| Flow Rate | 1.0 mL/min |
| Column Temp. | 30 °C |
| Detector | ELSD |
5. Fermentation Inhibitors (Organic Acids)
Organic acids are one of biomass hydrolyzation byproducts. They prevent microorganism (i.e., enzyme) growth, and thus inhibit fermentation process. Shodex SUGAR SH1821 column works under size exclusion and ion exclusion modes. It is suitable for the simultaneous separation of saccharides, organic acid, and alcohol. Elution time of organic acids can be controlled by optimizing the sulfuric acid concentration. For a better separation of DP4 and DP3, slower flow rate (0.6 mL/min instead of 1.5 mL/min) is recommended.
Fig. 5. Simultaneous separation of saccharides (Maltotetrose, Maltotriose, Maltose, Glucose), organic acids (Lactic acid, Acetic acid), and alcohols (Glycerol, Methanol, Ethanol).
| Column | Shodex SUGAR SH1821 |
| Eluent | 0.5 mM H2SO4 (aq) |
| Flow Rate | 0.6 mL/min |
| Column Temp. | 75 °C |
| Detector | RI |
6. Fermentation Inhibitors (Furfurals)
Depending on the type of biomass and processing mechanisms, furfural and 5-hydroxymethylfurfural (5-HMF) may be generated. They are considered as fermentation inhibitors. Shodex SUGAR SH1821 is suitable for the separation of celloligosaccharides and furfurals, using a combination of size exclusion and ion exclusion separation modes.
Fig. 6. Separation of celloligosaccharides, organic acids (Glyceric acid, Acetic acid), and furfurals (Furfural, 5-HMF).
| Column | Shodex SUGAR SH1821 |
| Eluent | 2 mM H2SO4 (aq) |
| Flow Rate | 0.6 mL/min |
| Column Temp. | 75 °C |
| Detector | RI |
7. Biodiesel Composition Analysis
Fatty acid methyl esters are the components of biodiesel. They were analyzed using a polymer-based reversed-phase column; Shodex RSpak DS-413.
Fig. 7. Separation of fatty acid methyl esters: Methyl linoleate, Methyl palmitate, Methyl oleate, and Methyl stearate.
| Column | Shodex RSpak DS-413 |
| Eluent | CH3CN/THF/H2O=45/30/25 |
| Flow Rate | 1.0 mL/min |
| Column Temp. | 40 °C |
| Detector | RI |
8. Biomass (Lignin)
Lignin is present in the cell wall of wood and other plants. Although it is an undesired component for the production of biofuel, the degradation products of lignin have potential use in biopolymers. Shodex GPC LF-804 is a linear-calibration type GPC column, suitable for a wide range of MW determination analysis.
Fig. 8. GPC chromatogram of lignin showing molecular weight distribution.
| Column | Shodex GPC LF-804 |
| Eluent | 20 mM H3PO4 + 20mM LiBr in DMSO/DMF=80/20 |
| Flow Rate | 0.6 mL/min |
| Column Temp. | 50 °C |
| Detector | RI |
9. Biomass (Xylan)
Xylan is a type of hemicellulose and present in most plants' cell wall. Hemicellulose, as well as cellulose, is used as a biofuel source. Hemicellulose is easily decomposed by weak acid or weak base and enzymes. Shodex GPC LF-804 is suitable for the MW determination and/or for xylan decomposition monitoring.
Fig. 9. GPC chromatogram of xylan showing molecular weight distribution.
| Column | Shodex GPC LF-804 |
| Eluent | 20 mM H3PO4 + 20mM LiBr in DMSO/DMF=80/20 |
| Flow Rate | 0.6 mL/min |
| Column Temp. | 50 °C |
| Detector | RI |
10. Impurities (Glycerides)
Glycerides are one of impurities found in biodiesel. Shodex Asahipak GF-310 HQ works under size exclusion mode to separate mono-, di- and tri-palmitins.
Fig. 10. Separation of Tripalmitin, Dipalmitin, and Monopalmitin.
| Column | Shodex Asahipak GF-310 HQ |
| Eluent | THF |
| Flow Rate | 0.6 mL/min |
| Column Temp. | 30 °C |
| Detector | RI |
11. Impurities (Organic Acids)
Acetic, propionic, and formic acids are impurities of biofuel. They may cause fuel tank corrosion. Shodex IC SI-52 4E is a strong-anion chromatography column. It provides simultaneous separation of organic acids, oxyhalides, and other anions.
Fig. 11. Simultaneous separation of organic acids (Acetic, Propionic, Formic, Malonic) and various anions (F-, Cl-, NO2-, Br-, NO3-, PO43-, SO42-, ClO2-, BrO3-, ClO3-).
| Column | Shodex IC SI-52 4E |
| Eluent | 3.6mM Na2CO3 (aq) |
| Flow Rate | 0.8 mL/min |
| Column Temp. | 45 °C |
| Detector | Suppressed conductivity |
Conclusions
Shodex™ HPLC columns offer a number of separation modes to analyze various components generated during biomass production. Presented methods include the analysis of raw material, final products, byproducts, impurities, as well as methods for saccharification/hydrolization monitoring.
Concentration and composition of mobile phase, temperature, and/or flow rate influence the separation performance of particular HPLC column. Therefore, for the specific set of target analysis, optimization of HPLC condition is highly recommended.
Key Applications Covered
- Raw Material Analysis: Saccharides in wood, biomass composition (lignin, xylan)
- Process Monitoring: Hydrolyzation monitoring, oligosaccharide distribution
- Final Product Analysis: Bioethanol recovery, biodiesel composition
- Byproduct Analysis: Fermentation inhibitors (organic acids, furfurals)
- Impurity Detection: Glycerides, organic acids, anions in biofuels
- Multiple Separation Modes: Size exclusion, ion exclusion, HILIC, reversed-phase, ion chromatography