Quantitative tests for carbohydrates
Several methods have been established to determine amount of sugars in a
sample. For accurate determination of a particular sugar, it may be necessary
to separate out the various carbohydrates in a mixture or a tissue extract, i.e.,
by chromatographic techniques and then estimate them individually.
Generally, to quantify amount of carbohydrates in a given sample is also
based on the reaction of carbohydrate’s functional group (usually hydroxyl
group). The principle is that the products obtained from the reaction are
directly proportional to the amount of present carbohydrates in the sample.
With this understanding, many qualitative assays are useful and can be used
for this purpose, for example, Benedict’s solution, Fehling’s solution, Nelson’s
reagent, and dinitrosalicylate reagent (DNS). Alternatively, analysis of
amount of products can be characterised by titration, colourimetry,
polarimetry, and HPLC. Another interesting way to determine amount of the
sugars is to utilise enzymatic technique. The major advantage of this
approach is that prior separation of individual sugars is no longer required as
one of the distinctive properties of the enzymes is substrate specificity.
Therefore, the chosen enzyme will only catalyse that particular sugar as the
substrate. Due to different property of obtained products, it is necessary to
select the suitable method for analysis.
For this experiment, amount of carbohydrates will be examined using DNS.
Under alkaline condition, the DNS will be reduced by sugar (reducing sugars)
and thus yielding colourful complex substances. These products can then be
observed from the absorption at 540nm. This method is appropriate in
determining amount of reducing sugars and, in particular, is useful if the
sugar samples are derived from hydrolysis of carbohydrase since DNS can stop
enzyme’s activity.