**5.2.1 Methodology**

Reagents: Resorcinol reagent, 1g resorcinol and 0.25g thiourea in 100mL. This solution must be kept in the dark to keep its stability. Hydrochloride acid is diluted as 5mL HCl and 1mL water.

Spectrophotometry as a Tool for Dosage Sugars in Nectar of Crops Pollinated by Honeybees 279

according to stoichiometry of the second reaction (Figure 4), the spectrophotometric

Analysis of glucose is according to the following principle: hexokinase, a first enzyme, catalyzes the phosphorylation of glucose to glucose-6-phosphate, with the participation of the enzyme glucose-6-phosphate dehydrogenase and nicotinamide adenine dinucleotide (NAD) is further specifically oxidized to gluconate-6-phosphate. According to the stoichiometry of the last reaction (Figure 4), the photospectrometrically quantified amount of reduced nicotinamide adenine dinucleotide (NADH) is representative for the amount of glucose. Fructose is always determined subsequently to the glucose determination. Fructose undergoes phosphorylation to fructose-6-phosphate, with the same enzyme hexokinase, which is further converted to glucose-6-phosphate with phosphoglucose isomerase. Further oxidation to gluconate-6-phosphate as described above generates a supplementary amount

All methodology of this analysis is performed following instructions of each enzymatic kit. For these enzymatic analysis must carried out the assays criteriously, because the order of addiction of reagents, the time of analysis and reading on spectrophotometer are

quantity of NADH is corresponding to glucose quantity.

of NADH that is stoichiometric with the amount of fructose.

Fig. 4. Enzymatic reactions in enzymatic methods using hexokinase.

determinants for an adequate analysis.

Getting an aliquot 80µg as maximum of the sample, add 0.5mL resorcinol reagent, shake in vortex, add 3.5mL hydrochloride acid, shake again, let in water bath at 80°C for 10 minutes, after that, read the absorbance in spectrophotometer at 520nm.
