**4.1.1 Methodology**

Reagents:

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

Spectrophotometry as a Tool for Dosage Sugars in Nectar of Crops Pollinated by Honeybees 277

unstable. Immediately, this first chromophore formed after react with tryptophan hydrochloride forming now a chromophore of pink colour that has greater stability

The reaction is also sensitive because can detect a low quantities like 1µg of fructose. The reaction also is specific because aldohexoses, aldopentoses, and ketopentoses do not interfere, however these compounds do not react even if its concentrations are higher, as 5mg.mL-1. The sensitivity of the method is from 1 to 50µg of fructose. A standard solution of fructose is used to build the calibration curve and to get the equation of the linear regression

Reagents: sulphuric acid 75%, cysteine hydrochloride 2.5%, tryptophan solution in hydrochloride acid to formation of tryptophan hydrochloride (100µg.mL-1 in HCl 0.1M).

Getting an aliquot 50µg.mL-1 of the sample, add 2.8mL of sulphuric acid 75%, shake in vortex, add 0.1mL cysteine hydrochloride solution 2.5%, shake in vortex again, let in waterbath 45-50°C for 10 minutes, cool at room temperature and add 1mL tryptophan

This sequence must be followed rigorously during assay because the formation of the final chromophore depends on the initial formation of the first formed chromophore by complexation with cysteine hydrochloride. After that, read the absorbance in

This reaction follows the same theoretical principles in which there is formation of furfural from hexoses and hydroxy-methyl-furfural (HMF) and from aldopentoses by acid dehydration (Fig. 1). These two products, singly are colorless, however, it is necessary a phenolic compound addition in the medium to develop a colored compound, in this case redness. This technique is firstly mentioned by Roe (1934), with some posterior modifications by Roe et al. (1949), becoming a quick reaction and with stable color. The reaction uses the hydrochloride acid (HCl) for carbohydrates dehydration and the resorcinol

This test allows distinguish aldoses from ketoses because the reaction with ketoses is faster and more intense than aldoses. Therefore, the formation of the furfural is easier than HMF

The sensitivity of the method ranges from 10 to 80µg.mL-1 fructose. A standard fructose solution is used to build the calibration curve and to get the equation of the linear regression

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

chemical than the first and stability of 48 hours.

hydrochloride solution, shake again in vortex.

**5.2 Resorcinol method (Roe et al., 1949)** 

is the phenolic compound that reacts with furfural and HMF.

spectrophotometer at 518nm.

formation.

water.

to quantity samples.

**5.2.1 Methodology** 

to quantity samples.

**5.1.1 Methodology** 

Reagent B – Weight 75g Potassium sodium tartrate (Rochelle salt), add 125mL of destiled water. Shake under heating until total dissolution.

Subsequently, add reagent A over the B, homogenize under heating until dissolve completely, and after cool the mix, complete the volume of the solution to 250mL.

Get an aliquot 500µg.mL-1 of the sample. Add 1mL of DNS, shake in vortex and let in a boiling bath during 5 minutes, put the material in ice bath until cool, add 3.75mL of destiled water, shake again, and read the absorbance in spectrophotometer at 540nm.

An adaptation of the DNS method to determine the total sugar can be getting from a previous hydrolysis before dosage. The hydrolysis is made with 0.5mL HCl concentrate and incubation in water bath at 60°C for 10 minutes. After then, the solution must be neutralized with NaOH 2M and cooled with ice bath until room temperature.

The procedure for total sugar quantification follow the same described for reducing sugar used above.
