3.

Xanthoproteic Test
This test used to determine aromatic amino acids containing in a protein solution that
which gives a yellow color solution. By heating the solution with concentrated Nitric acid, the
aromatic benzene ring will undergo nitration process in which produces yellow colored product.
On adding an alkali in these nitro derivative salts, the color change from yellow to orange.

Table 3. Xanthoproteic test results:

Sample Figure Observation
Tryptophan (+) Yellow solution, became
orange upon addition of NaOH

Fig. 3.1 reaction with NaOH

Tyrosine (+) Light yellow solution,
became yellow upon addition
of NaOH

Fig. 3.2 reaction with NaOH

Glycine (-) Clear solution remains
colorless upon addition of
NaOH

Fig. 3.3 reaction with NaOH

Alanine (-) Clear solution remains

colorless upon addition of
NaOH

Fig. 3.4 reaction with NaOH

Phenylalanine (-) Clear solution remains
colorless upon addition of
NaOH

Fig. 3.5 reaction with NaOH

Results and Discussion

As shown in table 3, both figure 3.1 (tryptophan) and 3.2 (tyrosine) yields a positive result
that turned into yellow solution, which means the sample protein solution contains amino acids.
The intensity of the yellow color deepens when the reaction occurs by adding the basic solution
and the nitric acid gives a color yellow when heated with proteins, thus the color is due to nitration.

Figure 3.6 Chemical reaction of tyrosine and tryptophan

Nitration takes place on the benzene rings and the reaction is therefore specific for
aromatic nuclei (Gortner, 1929). Thus, this test is for proteins depending on the presence of amino
acids. Phenylalanine gives negative or weakly positive reaction though this amino acid contains
aromatic nucleus because it is difficult to nitrate under normal condition and benzene ring is not
activated which is colorless solution. (figure 3.5 shown) while figure 3.3 (glycine) and figure 3.4
(alanine) did not yield a positive result because there is an absence of phenol in their structure
that is needed in the chemical reaction of the sample.