Three elements, many forms and functions
Together with proteins, lipids and nucleic acids, carbohydrates are designated as macromolecular biomolecules — substances essential to one or more fundamental biological processes. As the name implies, carbohydrates are all assembled from carbon, hydrogen and oxygen. They vary greatly in structure and complexity, however, as well as their specific requirements for in vitro purification and subsequent analysis.
Carbohydrates of higher complexity are typically assembled from simpler ones, and can be categorized in terms of degree of polymerization, i.e., the number of these smaller monomeric units that they contain.
Due to their vast structural variety, carbohydrates are among the most difficult to purify and analyze using methods that can be easily generalized or scaled up for high-throughput operations. Multiple approaches to isolation and analysis have been developed, and these can be loosely categorized based on the relative size and complexity of the target biomolecule:
Monosaccharides and Oligosaccharides
Chromatographic and electrophoretic methods
- Thin-layer (TLC), gas (GC), high-performance liquid chromatography (HPLC) are effective at determining type and concentration
- Gel electrophoresis separates carbohydrates based on size
Chemical methods (used with reducing-agent carbohydrates)
- Titration – e.g., Lane-Eynon method using copper sulfate and methylene blue indicator
- Gravimetric – e.g., Munson-Walker method using copper sulfate and alkaline tartrate
- Colorimetric – e.g., Anthrone and phenol/sulfuric acid methods, where light absorbance (concentration) is measured following color-generating chemical reactions
Enzymatic methods (typically kit-based, highly sensitive and specific)
- • D-glucose/D-fructose – using hexokinase and ATP, followed by spectrophotometric analysis
- • Maltose/sucrose – α-glucosidase treatment yields glucose, fructose for analysis
Additional analytical methods for smaller carbohydrates include physiochemical approaches (e.g., polarimetry, refractive index, infrared absorbance, density) and immunoassay techniques that are fast, sensitive, specific and easy to use.
Polysaccharides and Fiber
Larger carbohydrates such as starch can be isolated by dispersing sample material in hot 80% ethanol; while the mono- and oligosaccharides readily dissolve, starch does not, allowing its purification through subsequent filtration and centrifugation steps.
Actual analysis of starch can then be accomplished through several methods, including:
- Enzymatic treatment, which breaks starch down into glucose and allows the initial starch concentration to be found by quantitating remaining glucose
- Iodine treatment, which results in starch-iodine complexes that can be analyzed gravimetrically or through titration
- Physical methods, such as density, polarimetry or refractive index in cases where higher solution purity allows these
Analysis of other large carbohydrates such as dietary fiber can be accomplished after a few essential purification steps are complete:
- Removal of lipids by solvent extraction
- Removal of proteins by treatment with enzymes or strong acid/alkali solutions
- Removal of starch by enzyme or acid/alkali treatment followed by filtration and/or selective precipitation of insoluble fiber
The purified fiber can then be analyzed by chemical or gravimetric means described above.