**3.2.1 Methodology**

274 Macro to Nano Spectroscopy

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

**Hexose Pentose**

**hydroxy-methyl-furfural furfural**

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

**+**

H2SO4

O

O

O

H

O

HOCH2 OH

H HO

H CH2OH

OH H

**Anthrone**

**Anthrone Method Color blue-green (max = 620 nm)**

to build calibration curve and get the straight line equation to quantify samples.

O

OH

H

OH

O

H

CH2OH

OH H

O

OH

**Phenol**

**Phenol-sulphuric Method Color orange-yellow (max = 390 nm)**

H

HO

HOCH2

methods.

H

H

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

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.
