Increasing the Sensitivity of the Anthrone Method for Carbohydrate
The anthrone method for determining carbohydrate (1,2) has a number of unsatisfactory features. Maximum colour development is achieved at a point in time where the rates of formation of the chromogen and its destruction in hot acid just balance. This point is different for different sugars, and for ketoses both processes are much faster than for aldoses (see Fig. 3). Even at room temperature the rate of colour production from ketoses is sufficient to allow them to be determined without external heating (3). Although this formation of colour at room temperature from ketoses is slow, the ratio of the rates of chromogen formation and destruction is much greater at low temperatures than at high temperatures. Hence by working in the cold and using reaction periods of -12 hr it is possible to obtain a maximum optical density at 630 nm from fructose that is three to four times that obtained at 100ยฐC and about eight times that obtainable from an equimolar amount of glucose at 100ยฐC.
Since assay solutions containing glucose and other aldoses have to be heated for any significant colour development to occur, it is apparent that the sensitivity of their determination is inherently low compared to what can be achieved for ketoses and, further, that the determination of mixtures of sugars with very different times and amounts of maximum colour development is quite unreliable. The decomposition of the chromogen leads to the appearance of material with a complex optical absorption spectrum covering the range 400-800 nm and enhances the background against which the absorption peak at 630 nm is ever less clearly differentiated as heating proceeds (see Fig. 2). This leads not only to further loss of sensitivity but to increasingly erratic results. In addition, the anthrone reagent itself darkens and discolours on heating, at a rate somewhat dependent on the batch of reagent and its age.
This communication describes a procedure that considerably enhances the sensitivity and reproducibility of the anthrone determination by both increasing the rate of formation of chromogen and reducing the background. It was discovered accidentally during an attempt to estimate total carbohydrate in hydrolysates of glycoproteins in hydrochloric-formic acid mixtures. The conditions giving maximum colour yield from known sugars were then determined by varying the concentrations of hydrochloric and formic acids in the assay mixture.