Carbohydrates analytical methods

Longland, A.C. (2001) Plant carbohydrates analytical methods and nutritional implications for equines. Internal communication, Institute of Grassland and Environmental Research, Aberystwyth, UK. 

Periodic acid oxidation (Section 15 12) finds extensive use as an analytical method m carbohydrate chemistry Structural information is obtained by measuring the number of equivalents of periodic acid that react with a given compound and by identifying the reaction products A vicinal diol consumes one equivalent of penodate and is cleaved to two carbonyl compounds... 

The earliest examples of analytical methods based on chemical kinetics, which date from the late nineteenth century, took advantage of the catalytic activity of enzymes. Typically, the enzyme was added to a solution containing a suitable substrate, and the reaction between the two was monitored for a fixed time. The enzyme s activity was determined by measuring the amount of substrate that had reacted. Enzymes also were used in procedures for the quantitative analysis of hydrogen peroxide and carbohydrates. The application of catalytic reactions continued in the first half of the twentieth century, and developments included the use of nonenzymatic catalysts, noncatalytic reactions, and differences in reaction rates when analyzing samples with several analytes. 

In order to define this variety of food matrices, chemical composition differences that primarily influence chemical analytical measurements have to be considered. Major food components determining basic chemical make-up are the proximate composition of fat, protein, carbohydrate, ash, and moisture. Variations in ash content in general have a minor influence on analytical methods for other constituents and impact of moisture content can be controlled. Thus the major components influencing analytical performance are the relative levels of fat, protein, and carbohydrate. 

As in the other -omics, analyses may be directed at a specific metabolite, at all metabolites in a given system in a shot-gun approach, or at accessible groups of molecules in profiling experiments. In that also the technology varies. In addition, the chemistry of different metabolites is very heterogeneous since it involves hydrophobic lipids, hydrophilic carbohydrates, ionic inorganic species, and other secondary natural products and already the choice of solvent in metabolite extraction dictates which types of molecules will be present (Fig. 10.8). Therefore, total metabolome profiling is not possible, because no analytical method will be able to accommodate all the different molecule classes at once. 

The chemical or biological basis of an analytical method may not permit a simple, direct relationship between the reading and the concentration of the analyte and failure to appreciate the limitations and constraints of a method can lead to significant systematic error. Carbohydrates may be quantified, for instance, using a method based on their reducing properties but results will tend to be higher than they should be if non-carbohydrate reducing substances are also present in the samples. 

Difficulties are encountered in the qualitative and quantitative analysis of carbohydrate mixtures because of the structural and chemical similarity of many of these compounds, particularly with respect to the stereoisomers of a particular carbohydrate. As a consequence, many chemical methods of analysis are unable to differentiate between different carbohydrates. Analytical specificity may be improved by the preliminary separation of the components of the mixture using a chromatographic technique prior to quantitation and techniques such as gas-liquid and liquid chromatography are particularly useful. However, the availability of purified preparations of many enzymes primarily involved in carbohydrate metabolism has resulted in the development of many relatively simple methods of analysis which have the required specificity and high sensitivity and use less toxic reagents. 

The preparation of the sample prior to its analysis will depend upon the nature of both the sample and the analytical method chosen and may involve the disruption of cells, homogenization and extraction procedures as well as the removal of protein or other interfering substances. It may be necessary to prevent the decomposition and degradation of the carbohydrate content during such treatments or during storage by the addition of antibacterial agents such as thymol or merthiolate, or substances such as fluoride ions, which will inhibit the enzymic transformation of the carbohydrates. 

The dehydration reactions initiated by eliminating a hydroxyl group from an enediol are discussed in the present article. The products (usually dicarbonyl compounds) of these eliminations are normally transient intermediates, and undergo further reaction. The final products formed are determined by the carbohydrate reacting, the conditions of reaction, and the character of the medium. Except for a Section on analytical methods (see p. 218), the subject matter is restricted to aqueous acids and bases. The presence of compounds other than the carbohydrate under study has only been considered where it has helped to elucidate the mechanism involved. The approach here is critical and interpretative, with emphasis on mechanism. An attempt has been made to demonstrate how similar reactions can logically lead to the various products from different carbohydrates a number of speculative mechanisms are proposed. It is hoped that this treatment will emphasize the broad functions of these reactions, an importance that is not fully recognized. No claim is made for a complete coverage of the literature instead, discussion of results in the articles that best illustrate the principles involved has been included. 

However, during the past three decades, an analytical method has been developed that currently rivals and may soon surpass the traditional liquid chromatographic techniques in importance for analytical separations. This technique, high-performance liquid chromatography (HPLC), is ideally suited for the separation and identification of amino acids, carbohydrates, lipids, nucleic acids, proteins, pigments, steroids, pharmaceuticals, and many other biologically active molecules. 

Ionization constants have been determined for numerous simple carbohydrates (10,13, i5, 25, 45), as well as for cellulose (32, 43), wheat starch (43), and alginate (43). Selected carbohydrates with their corresponding pK values are presented in Table I. The analytical methods involved in these determinations include conductimetry, potentiometric titration, thermometric titration, and polarimetry. Polarimetry was used by Smolenski and co-workers (45) to calculate a first and a second ionization constant for sucrose at 18°C (Ki = 3X 10"13 K2 = 3 X 10"14). 

Some basic food analytical methods such as determination of °brix, pH, titratable acidity, total proteins and total lipids are basic to food analysis and grounded in procedures which have had wide-spread acceptance for a long time. Others such as analysis of cell-wall polysaccharides, analysis of aroma volatiles, and compressive measurement of solids and semi-solids, require use of advanced chemical and physical methods and sophisticated instrumentation. In organizing the Handbook of Food Analytical Chemistry we chose to categorize on a disciplinary rather than a commodity basis. Included are chapters on water, proteins, enzymes, lipids, carbohydrates, colors, flavors texture/ rheology and bioactive food components. We have made an effort to select methods that are applicable to all commodities. However, it is impossible to address the unique and special criteria required for analysis of all commodities and all processed forms. There are several professional and trade organizations which focus on their specific commodities, e.g., cereals, wines, lipids, fisheries, and meats. Their methods manuals and professional journals should be consulted, particularly for specialized, commodity-specific analyses. 

What factors can be used to predetermine the quality and utility of a method An analyst must consider the following questions Do I need a proximate analytical method that will determine all the protein, or carbohydrate, or lipid, or nucleic acid in a biological material Or do I need to determine one specific chemical compound among the thousands of compounds found in a food Do I need to determine one or more physical properties of a food How do I obtain a representative sample What size sample should I collect How do I store my samples until analysis What is the precision (reproducibility) and accuracy of the method or what other compounds and conditions could interfere with the analysis How do I determine whether the results are correct, as well as the precision and accuracy of a method How do I know that my standard curves are correct What blanks, controls and internal standards must be used How do I convert instrumental values (such as absorbance) to molar concentrations How many times should I repeat the analysis And how do I report my results with appropriate standard deviation and to the correct number of significant digits Is a rate of change method (i.e., velocity as in enzymatic assays) or a static method (independent of time) needed ... 

In this sense, HPLC is an analytical method that cannot be put aside in the determination of carbohydrates, since the wide variety of these species and their inherent polydispersity and heterogeneity require separation techniques of high resolving power and high selectivity (7). 

Fermentation proceeds via the development of microorganisms that the food industry controls and corrects to obtain the desired results. The main substratum that is transformed is made up of carbohydrates, which may undergo various types of fermentation, with the production of more simple substances that are very important in the determination of the quality of the final product. In some cases, the formation of some substances may indicate undesired processes. There is, therefore, the need to intervene rapidly so that the necessary corrections may be made to the process. Fast analytical methods are required that can in most cases ascertain the content of various sugars, organic acids, glycerol, and alcohols (for example, methanol, ethanol, higher alcohols). 

Many investigations were made by early workers using techniques such as starvation and pancreatectomy, but these for the most part gave equivocal results (for summary see Williams35), probably because the close interrelation of fat, protein and carbohydrate metabolism was not then fully appreciated and because the analytical methods used were often... 

Analytical methods used to identify monomers have improved significantly from those that quantify whole classes of compounds, such as amino acids, peptides, proteins, and primary amines (Undefriend et al., 1972) or carbohydrate-like compounds (Johnson and Sieburth, 1977) to ones that are molecule-specific (Table II). Most of these methods are based on combining chromatography techniques that can separate complex mixtures of molecules with highly sensitive detectors that can approach the nanomolar or picomolar range. Monomers are usually present at low concentrations, so... 

Soluble and insoluble lignin were determined with the NREL standard biomass analytical methods for the analysis of acid-insoluble and acid-soluble lignin inbiomass, LAP-003 and LAP-004, respectively (13-14). NREL s laboratory analytical procedure for the determination of carbohydrates in biomass, LAP-002, without correction for hydrolysis losses was performed to determine the sugar content (15). 

---