**5.1.1 Methodology**

276 Macro to Nano Spectroscopy

Reagent B – Weight 75g Potassium sodium tartrate (Rochelle salt), add 125mL of destiled

Subsequently, add reagent A over the B, homogenize under heating until dissolve

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

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

The procedure for total sugar quantification follow the same described for reducing sugar

This method is based on determination of reducing sugar before and after the digestion by invertase using acid for p-hydroxy benzoic acid hydrazide – PAHBAH. In this method, with some modifications, there is possible to perform the colorimetric determination of glucose, fructose and sucrose, during this analysis procedure with invertase. The major subject of the methodology described in this chapter is for determination of reducing sugar. The phydroxy benzoic acid hydrazide also has the advantage that glucose and fructose when reacting produce the same intensity of colour, and then all free monossacharides present in

Reagent A: 10g p-hydroxy benzoic acid dissolved in 60mL water, add 10mL hydrochloride

Reagent B: 24.9g trisodium citrate in 500mL water, 2.2g calcium chloride, 40g sodium

Get an aliquot 50µg.mL-1 of the reducing sugar sample, add 5mL p-hydroxy benzoic acid hydrazide reagent (mixed in the same day) shake in vortex, incubate in water bath at 100°C by six minutes, cool until room temperature, and read the absorbance in spectrophotometer

**5.1 Sulphuric acid-Cysteine-Tryptophan method (Messineo & Edward Musarra,1972)**  Fructose is dehydrated in acid medium for formation of furfural derivative, which complexs with cysteine hydrochloride to produce a chromophore at starting of green color that is

In the day of analysis, mix reagents A and B in proportion of 1:10, after keep at 4°C.

completely, and after cool the mix, complete the volume of the solution to 250mL.

water, shake again, and read the absorbance in spectrophotometer at 540nm.

**4.2 Hydrazide method of p-hydroxy-benzoic acid (Blakeney & Mutton, 1980)** 

with NaOH 2M and cooled with ice bath until room temperature.

water. Shake under heating until total dissolution.

used above.

the sample can be determined.

acid and complete volume to 200mL.

**5. Fructose determination** 

hydroxide, and to complete the volume to 2.000mL.

**4.2.1 Methodology** 

Reagents:

at 410nm.

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 hydrochloride solution, shake again in vortex.

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 spectrophotometer at 518nm.
