**3.2 Anthrone method (Dreywood, 1946)**

The anthrone is the reduction product of anthraquinone and was recognized first as specific reagent for several carbohydrates by Dreywood (1946) because sugar solution in concentrate sulphuric acid form the blue-greenish colour characteristic and since anthrone has been used widely as suitable and specific reagent for sugar colorimetric dosage.

The anthrone reaction is based on hydrolitic and dehydrating action of concentrate sulphuric acid on carbohydrates, in which glicosidic linkages are broken releasing free reducing sugar that are dehydrated and converted to furfural by pentoses and hydroxymethylfurfural by hexoses (Figure 1).

Spectrophotometry as a Tool for Dosage Sugars in Nectar of Crops Pollinated by Honeybees 275

Reagents: anthrone solution 0.2% (0.2g anthrone em q.s. to 100mL of sulphuric acid 95%). This solution must be kept at rest for 30 minutes with sporadic agitation until the solution to

Get an aliquot of 100µg.mL-1 from the sample, add 2mL of the anthrone, leave at ice bath and hereupon cool in cooled water and read the absorbance in spectrophotometer at 620nm.

This method was first mentioned by Summer & Sisler (1944) and modified by Miller (1959),

The sugar act as a chemical reductor due free aldehyde group or ketone group presence in its molecule. In an alkaline medium, the reducing sugars are able to reduce the 3-5 dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid, wherever, the aldehyde group is oxidized to aldonic acid (Figure 2). The 3-amino-5-nitrosalicylic acid is a orange color product, and the intensity of the color depends on the concentration of the reducing sugar. The sodium hydroxide provides the glucose reaction with 3-5-dinitrosalicylic acid by

Besides to 3-5-dinitrosalicylic acid, it is also used in this method the Rochelles salt (Potassium sodium tartrate), phenol, sodium bisulfite, and sodium hydroxide. The phenol optimize the quantity of the colour produced and sodium bisulfite stabilize the colour in the

The sensitivity of the method is from 100 to 500µg.mL-1 of reducing sugar. A standard glucose or fructose solution is used to build the calibration curve and get the straight line

HO <sup>+</sup>

OH

COOH

O2N NO2

**3-amino-5-dinitro**

OH

**salycilic acid aldonic acid**

+

HOH2C

H HO

C C C C COOH OH H H H OH

HOH2C

**(DNS) reductor sugar**

H HO HO

C C C C C

Fig. 2. Reaction of reducing sugar with 3,5-dinitro-salycilic acid reagent.

Reagent A – Weight 2.5g DNS (3,5 dinitrosalicylic acid) and add 50mL NaOH 2M.

O H

OH H H H OH

with this technique there is a possibility to dosage reducing sugar as total sugar.

**3.2.1 Methodology** 

medium alkalinization.

phenol presence (Miller, 1959).

equation to quantify samples.

OH

COOH

O2N NO2

**3,5-dinitro-salycilic acid**

**4.1.1 Methodology** 

Reagents:

be clear, the reagents must be used in 12 hours.

**4.1 3,5-dinitrosalicylic acid method (Miller, 1959)** 

**4. Reducing sugar determination** 

These substances are condensed with anthrone to hydroxyanthracene (9,10-dihydro-9-oxo anthracene) forming a blue-greenish product that have absorption maximum at 620nm.

The sensitivity of this method is from 0 to 100µg.mL-1. Glucose solution is used as standard to build calibration curve and get the straight line equation to quantify samples.

Fig. 1. Reaction of formation of the coloured complex in phenol-sulphuric and anthrone methods.
