Carmela Ariane D. Aliazas

Classical MethodMany of the earliest methods available for the

measurement of carbohydrates were based on their chemical reactivity and involved the addition of a particular reagent with the subsequent formation of a colored product.

These methods are inherently non-specific and often involve the use of substances that are now recognized as hazardous.

It is essential that the hazard assessment is undertaken before using such techniques.

The lack of specificity is largely attributed to the fact that many monosaccharides have similar chemical composition and properties.

The numerous isomeric forms in which the hexoses exist, all having identical chemical composition, with some even showing interconversion to an entirely common structure in alkaline solution, demonstrate the difficulties encountered in attempting to measure an individual member of the group in a sample containing others.

Chemical methods are not even capable of differentiating effectively between classes of monosaccharides because the reactions of the carbonyl group are used.

This function is common to all monosaccharides and thus they may give similar or sometimes identical reaction products.

The use of chemical methods is justified in a variety of situations.

Reaction of the Carbonyl GroupThe reactions of the carbonyl group form the

basis of many qualitative methods for the detection of carbohydrates and several have been used quantitatively.

The reagents or reaction conditions have been modified to improve specificity.

Reduction methodsReduction 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.

The distinction between reducing and non-reducing disaccharides can be used to advantage in qualitative tests.

One of the most common methods is the reduction of cupric ions (Cu2+)to cuprous ions (Cu+).

In this method, alkaline solution form yellow cuprous hydroxide, which is in turn converted by the heat of the reaction to insoluble red cuprous oxide (Cu2O)

The production of a yellow or orange-red precipitate indicates the presence of a reducing carbohydrate.

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 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 colored complex produced is related to the concentration of the reducing substances in the original sample. The color 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-1-10-phenanthroline hydrochloride (neocuproine), which produces a stable color and is specific for cuprous ions.

Ferricyanide ions (yellow solution) are reduced to ferrocyanide ions (colorless solution) by reducing carbohydrates when heated in an alkaline solution. The concentration of the carbohydrate can be related to the decrease in absorbance at 420nm.

The precision of this type of method in which quantitation involves inverse calorimetry is questionable, especially at low concentrations of the analyte because of the difficulty of measuring slight absorbance differences from the high blank reading.

Reactions with aromatic aminesVarious aromatic amines will condense whit aldoses

and ketoses in glacial acetic acid to form colored 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 individual sugars.

Aromatic amines that have been used include o-toluidine, p-aminosalicylic acid, p-aminobenzoic acid, diphenylamine, and p-aminophenol.

These reagents have proved particularly useful for the visualization and identification of carbohydrates after separation of mixtures by paper or thin-layer chromatography, when color variations and the reaction aid the interpretation of the chromatogram.

Reactions with strong acids and a phenolWhen heated with a strong acid, pentoses and hexoses

are dehydrated to form furfural and hydroxymethylfurfural derivatives, the aldehyde groups of which will then condense with a phenolic compound to form a colored product.

This reaction forms the basis of some of the oldest qualitative tests for the detection of carbohydrates.

Seliwanoff’s test uses hydrochloric acid and either resorcinol or 3-indolyacetic

acid to measure fructose with minimal interference from glucose.

The color produced by pentoses with orcino (Bial’s reagent) or p-bromoaniline is sufficiently different from that produced by hexoses to permit their quantitation in the presence of hexoses.

Some 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.

STRUCTURAL STUDIES OF POLYSACCHARIDESThe quantitation of polysaccharides is usually achieved by

hydrolysis of the glycosidic linkages with the release of the individual components.

This can be accomplished by heating at 60ᵒC with concentrated hydrochloric acid for 30 minutes followed by the quantitation of the monosaccharide using a suitable method. This hydrolysis procedure may result in the destruction of some carbohydrates and milder conditions using acetic acid or lower concentrations of HCl are often preferable.

Methanolysis under mild conditions will yield the methyl glycosides , which are suitable for analysis by gas-liquid chromatography.

Enzymatic hydrolysis of specific linkages is of great value in the investigation of the structure and composition of hetero- and homopolysaccharides, it may also be important to measure any non-carbohydrate moieties or monosaccharide derivatives that have been released by chemical or enzymatic hydrolysis.

Structural investigations into the degree of branching and into the position and nature of glycosidic bonds and of non-carbohydrate residues in polysaccharides may include periodate oxidation and other procedures such as exhaustive mehtylation.

X-ray diffraction and spectroscopic techniques such as nuclear magnetic resonance and optical rotatory dispersion also give valuable information especially relating to the 3D structures of these polymers.

ENZYMIC METHODS OF CARBOHYDRATE ANALYSISLarge numbers of enzymes that are capable of modifying

carbohydrates or carbohydrate derivatives, and may be used in various analytical methods.

Enzymic methods for the quantitation of monosaccharides are employed when a higher degree of specificity is required than can be achieved by the majority of the chemical methods.

Absolute specificity of an enzyme for only one substrate is rare and there may be several monosaccharides present in a sample that can be acted on to varying degrees by the same enzyme.

The specificity of a particular enzyme may also vary according to the source from which it was prepared and the method of commercial preparation can affect the purity and thus may influence the results obtained when the enzyme is used.

Assay of Glucose using glucose oxidaseGlucose oxidase catalyses the oxidation of β-D-glucose

by atmospheric oxygen to produce D-glucolactone, which is converted to gluconic acid with the production of hydrogen peroxide.β-D-glucose + O2 + H2O D-gluconic acid +H2O2

The inclusion of aldose-1-epimerase (glucomutarotase) in the glucose oxidase reagent will permit rapid restoration of the α-β equilibrium effectively enabling the reaction to go to completion.

The rate of oxidation of other monosaccharides by glucose oxidasehas been shown to be negligible or zero but some derivatives of glucose react slightly.

Quantitation of glucose using glucose oxidase is achieved by measurement of either the hydrogen peroxide formed or the oxygen consumed during the reaction, both of which are proportional to the β-D-glucose content of the sample.

Assay of glucose using glucose dehydrogenaseGlucose can be measured bacterial glucose

dehydrogenase, which catalyses the dehydrogenation of β-D-glucose to D-glucolactone, the hydrogen being transferred to NAD+ or NADP+.

The method involves measuring the increase in absorbance at 340 nm caused by the production of NADH using either a kinetic or fixed time assay technique.

A mutarotase should be included in the assay to accelerate the conversion of the α to the β form.

A colorimetric version of the method uses a tetrazolium salt.

The oxidation of the NADH is coupled to the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide by a diaphorase and a deep blue formazan develops.

The absorbance is read at a wavelength between 540 and 600 nm.

This is more sensitive than the ultraviolet method but is more prone to interference , especially from any reducing agents present in the sample, which will result in a positive error.

Assay of glucose using hexokinaseHexokinase catalyses the phosphorylation of

glucose to produce glucose-6-phosphate with ATP as the phosphate donor and magnesium ions as an activator.

The rate of formation of glucose-6-phosphate can be linked to the reduction of NADP by the enzyme glucose-6-phosphate dehydrogenase.

Hexokinase is not specific for glucose and is capable of converting some other hexoses to their corresponding 6-phosphate derivatives.

The specificity of this enzyme vary slightly depending on its source. Yeast hexokinase will catalyse the phosphorylation of a number of other hexoses.

Miscellaneous methods for the measurement of hexose

An enzymic method for the measurement of fructose using hexokinase together with the method for mannose and glucose.

Galactose oxidase catalyses the oxidation of β-D-galactose in a similar manner to the oxidation of β-D-glucose by glucose oxidase and forms the basis of an identical quantitative method.

The specificity is less than that of glucose oxidase and although glucose is not a substrate, other monosaccharides and glycosides may also be oxidized depending upon the source of the enzyme.

An assay using galactose dehydrogenase, which catalyses the conversion of D-galactose to D-galactonolactoce in the presence of the coenzyme NAD+, is more specific and enzyme preparations are available for which the only other substrates are α-L-arabinose and β-D-fructose.

SEPARATION AND IDENTIFICATION OF CARBOHYDRATE MIXTURESTechniques such as the formation of osazones and the

demonstration of fermentation have contributed significantly to the separation and identification of carbohydrates.

Observation of the characteristic crystalline structure and melting point of the osazone derivative, prepared by reaction of the monosaccharide with phenylhydrazine, was used in identification.

This method is not completely specific, because the reaction involves both carbon atoms 1 and 2 with the result that the three hexoses, glucose, fructose, and mannose will yield identical osazones owing to their common enediol field.

Fermentation tests are based on the ability of yeast to oxidize the sugar to yield ethanol and carbon dioxide.

Modern chromatographic techniques are much more acceptable and paper and thin-layer techniques are useful for routine separation and semi-quantitation of carbohydrate mixtures.

Paper and Thin-layer Chrpmatography

Both ascending and descending paper chromatographic tecniques have been used and, when thin-layer supports are employed, the use of either silica gel or cellulose is applicable.

A careful choice of solvent will generally make it unnecessary to perform the two-dimensional separations that are often needed when large numbers of substances are present.

Reference solutions of each carbohydrate can be made up in concentrations of approximately 2g/l dissolved in an isopropanol solvent and samples of about 10 µl should give discernible spots after separation.

Solvent SystemsSeveral monophasic solvent systems are useful for the separation

of carbohydrate mixtures.The distance moved by different oligosaccharides is a reflection of

the number of monosaccharide units of which they are composed, with the smallest molecules again moving the furthest.

The distances moved by all classes of carbohydrates are small and although the modification of solvent composition may result in greater overall mobility, the relative differences between the components is still low and it may be necessary to run the solvent off the end of the support to achieve a satisfactory separation.

Solvent composition proportions comments

Ethyl acetatePyridinewater

603020

Commonly used. Gives good separation of pentoses and hexoses. Will resolve glucose and galactose.

n-butanolPyridinewater

604030

Many variations in composition may be used to increase or decrease overall mobilities

Formic acidMethylethyl ketoneTertiary butanolwater

15304015

Gives good separation of monosaccharides and disaccharides

Ethyl acetateEthanolPyridineAcetic acidwater

7010101010

Useful for separation of pentoses and hexoses

Some monophasic solvents for thin-layer chromatography of carbohydrates

Locating reagentsA variety or reagents can be used for visualization of

the separated components and it may be useful to run duplicate chromatograms and use a different stain on each one to assist in identification of unknown spots.

The most commonly used reagents make use of the chemical reactions of carbohydrates already described in the section on quantitative methods and appropriate safety precautions must be taken when using the various locating reagents

In certain situations the use of a reagent that incorporates a specific enzyme may be advocated and it will be necessary to be aware of any apparent lack of enzyme specificity and to have a knowledge of all the substrates on which the enzyme will act

Color reaction of common mono- and disaccharidescarbohydrate Locating

reagents

naphthoresorcinol

4-Aminobenzoic acid

Aniline diphenylamine phosphate

Ribose --- Red/brown Blue/green

Xylose --- Violet Green/blue

Arabinose --- Red/brown Blue/green

Xylulose Green/brown Red/brown Blue/green

Glucose --- Brown Grey/blue

Galactose --- Brown Grey/blue

Fructose Red Pink Orange/brown

Sucrose Red Brown Brown

lactose --- brown blue

Gas-liquid chromatographyGas-liquid chromatography may be the method of choice when it is

necessary to identify or to quantitate one or more carbohydrates especially when they are present in small amounts.

Gas chromatography must be carried out using volatile derivatives of the carbohydrates and although many have been studied using a variety of stationary phases, the O-trimethylsilyl derivatives are probably used most frequently, although circumstances may dictate the use of an alternative.

Carbohydrates require only weak silylating conditions, otherwise random isomerization will occur with the production of spurious peaks making interpretaion of the chromatographic trace very difficult.

The use of a mixture of hexamethyldisilazane, trimethylchlorosilane and pyridine is recommended although carbohydrates combiined with or coctaining amino,phosphate, carboxylic groups or nucleic acids will require a more powerful silylating agent in conjunction with trimethylchlorosilane and pyridine are widely used.

The choice of a stationary phase will depend upon the nature of the carbohydrates to be separated and whereas an OV-17 column (phenylmethylpolysiloxy gum) may give satisfactory isometric separations, a non-polar phase such as OV-1 (methylpolysiloxy gum) may be more useful for a wider range of carbohydrates.

High Performance Liquid ChromatographySeparation and quantitaion of carbohydrate mixtures may

be achieved using HPLC, a method that does not necessitate the formation of a volatile derivative as in GLC.

Both partition and ion-exchange techniques have been used with either ultraviolet or refractive index detectors.

Partition chromatography is usually performed in the reverse phase mode using a chemically bonded stationary phase and acetonitrile in 0.1mol/l acetile acid as the mobile phase

Carbohydrate form anionic complexes in alkaline borate buffers and quaternary ammonium anion-exchange resins in the hydroxyl form can be used for their separation.

Problems caused by rearrangement reactions in alkaline solution have been minimized by the use of boric acid/glycerol buffers at pH 6.7. it is possible to separate monosaccharides at this pH by elution with such a buffer to which NaCl has been added.

Sulpfonated cationic exchange resins with metal counter-ions are also useful for carbohydrate analysis.

Resins are available in a variety of forms. The calcium or lead form is generally recommended for monosaccharides and disaccharides whereas, for oligosaccharides, the silver or sodium form is preferred.