The reactions of the carbonyl group form the basis of many qualitative methods for the detection of carbohydrates and several have been used quantitatively. These are general methods that often only measure the total amount present in the sample. However, in some cases, reagents or reaction conditions have been modified to improve specificity.
Carbohydrates that have a potentially free aldehyde or ketone group exist in solution at equilibrium with the enediol form. At a slightly alkaline pH this conversion is favoured and the resulting enediol is an active reducing agent (Figure 9.19). Reduction methods can be used for disaccharides provided that the aldehyde or ketone group of at least one of the monosaccharides has not been eliminated in the glycosidic bond. Sucrose is an example of a disaccha-ride in which the anomeric carbon atoms of both monosaccharides are involved in the glycosidic bond and the reducing power is lost. However, this distinction between reducing and non-reducing disaccharides can sometimes be used to advantage in qualitative tests.
One of the commonest methods involves the reduction of cupric ions (Cu2+) to cuprous ions (Cu+), which in alkaline solution form yellow cuprous hydroxide, which is in turn converted by the heat of the reaction to insoluble red cuprous oxide (Cu20). In the qualitative tests based on this reaction, the production of a yellow or orange-red precipitate indicates the presence of a reducing carbohydrate. It is necessary to keep the cupric salts in solution and to this end Benedict's reagent incorporates sodium citrate while Fehling's reagent uses sodium potassium tartrate. Under carefully controlled reaction conditions, the amount of cuprous oxide formed may be used as a quantitative indication of the amount of reducing carbohydrate present, although different carbohydrates will result in the formation of different amounts of cuprous oxide.
The methods of measuring the amount of cuprous oxide formed are numerous but the one most frequently used involves the reduction of either phosphomolybdic acid or arsenomolybdic acid by the cuprous oxide to lower oxides of molybdenum. The intensity of the coloured complex produced is related to the concentration of the reducing substances in the original sample. The colour produced with arsenomolybdic acid is more stable and the method is more sensitive than with phosphomolybdic acid.
The neocuproine method for the measurement of the cuprous oxide is more sensitive than the phosphomolybdic acid reagent and uses 2,9-dimethyl-l, 10-phenanthroline hydrochloride (neocuproine), which produces a stable colour and is specific for cuprous ions.
Although a variety of oxidants other than copper salts have been used ferricyanide is the only other one of note. Ferricyanide ions (yellow solution) are reduced to ferrocyanide ions (colourless solution) by reducing carbohydrates when heated in an alkaline solution. The concentration of the carbohydrate can be related to the decrease in absorbance at 420 nm.
The precision of this type of method in which quantitation involves inverse colorimetry (i.e. the absorbance decreases with increasing concentration of the analyte) is questionable, especially at low concentrations of the ana-lyte, because of the difficulty of measuring slight absorbance differences from the high blank reading.
In addition to the lack of specificity of such reduction methods already mentioned, non-carbohydrate reducing substances present in the sample will also react similarly resulting in positive error. Over the years, workers have modified the reagent composition, sample preparation and even the shape of the test-tubes in attempts to reduce interference. Such names as Fehling, Benedict, Nelson, Somogy, Folin and Wu are still associated with these reduction methods. Such considerations are of little consequence nowadays, when reduction methods are less frequently used and the more specific and precise enzymic or chromatographic methods are preferred.
Various aromatic amines will condense with aldoses and ketoses in glacial acetic acid to form coloured products whose absorbance maxima are often characteristic of an individual carbohydrate or group. The use of different aromatic amines or absorbance measurements at alternative wavelengths gives a degree of specificity for individuai sugars. The carcinogenic nature of some aromatic amines has ruled out their use as laboratory reagents.
Aromatic amines that have been used include o-toluidine, /»-aminosalicylic acid, p-aminobenzoic acid, diphenylamine and p-aminophenol. Their ability to react preferentially with a particular carbohydrate or class of carbohydrate is often useful, e.g. p-aminophenol, which shows some specificity for ketoses compared with aldoses and is useful for measuring fructose. These reagents have proved particularly useful for the visualization and identification of carbohydrates after separation of mixtures by paper or thin-layer chromatography, when colour variations and the presence or absence of a reaction aid the interpretation of the chromatogram.
When heated with a strong acid, pentoses and hexoses are dehydrated to form furfural and hydroxymethylfurfural derivatives respectively (Figure 9.20), the aldehyde groups of which wiil then condense with a phenolic compound to form a coloured product. This reaction forms the basis of some of the oidest qualitative tests for the detection of carbohydrates, e.g. the Molisch test using concentrated sulphuric acid and a-naphthol.
By careful choice of both the reaction conditions and the phenolic compound used, it may be possible to produce a colour that is characteristic of a particular carbohydrate or related group, so giving some degree of specificity to the method. Thus, Seliwanoff's test uses hydrochloric acid and either resor-cinol or 3-indolylacetic acid to measure fructose with minimal interference from glucose. The colour produced by pentoses with orcinol (Bial's reagent) or p-bromoaniline is sufficiently different from that produced by hexoses to permit their quantitation in the presence of hexoses. However, none of the methods based on the formation of furfural or its derivatives can be considered to be entirely specific.
There may be non-carbohydrate substances present in a biological sample that will decompose on heating under the acidic conditions and will react in a similar manner to a carbohydrate. Glucuronic acid is an example and is often present in abundance in urine and may give a false positive reaction for carbohydrates.
h2coh hc -
Furfural hc= o c=0
Formation of furfural and 5-hydroxymethylfurfural. 5-Hydroxymethylfurfural
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