Qualitative Tests

Different qualitative tests were done to be able to determine and have an idea of
what different groups are present in each solution.
Positive results for each test are as follows: violet for Biuret test, purple/violet for
Ninhydrin test, yellow for Xanthoproteic test and peach red for Sakaguchi test. While the
negative for each are characterized by its colorless solution.
Table X. Results of the Qualitative Tests on Different Solutions
Qualitative Test Solution
P1 HP1 P2 HP2
Biuret Test + -* + -*
Ninhydrin Test + + + +
Xanthoproteic Test + + + +
Sakaguchi Test + + + +
*Obtained data does not conform to the theoretical result.

Figure X. Biuret test positive control and negative control respectively.

Figure X. Biuret test for P1, P2, HP1 and HP2 all arranged accordingly from left
to right.
 Biuret test is a qualitative test for the presence of peptide bonds. In the
experiment, all resulted positive in this test when theoretically, the hydrolyzed solutions,
which are HP1 and HP2, should have yielded a colorless solution since they only
contain free amino acids. Complete hydrolysis breaks peptide bonds. Incomplete
hydrolysis my have resulted to the errors of this test.
This test forms a violet-purplish color when copper (II) ions from CuSO4 react
with substances that contains at least 2 peptide bonds which forms a stable complex
under basic conditions. The solution will remain clear for a long period of time if the
solution is not under alkaline condition since it was observed that the when the reaction
has higher alkali concentrations, the faster the development of color. But the
concentration of sodium hydroxide should not be high to prevent the precipitation of the
complex that was formed. And it is also said that the intensity of the color is proportional
to the concentration of proteins in the solution (Bardawill, David & Gornall, 1948;
Harding & Warneford, 1916; Milio & Lofredo, 1995; Nielsen, 2017; Watters, 1978).

Figure X. Protein-copper (II) complex (Milio & Lofredo, 1995)

Figure X. Ninhydrin test negative control, arginine postive control and tryptophan
positive control respectively.
 Figure X. Ninhydrin test for P1, P2, HP1 and HP2 all arranged accordingly from
left to right.
Ninhydrin test is a qualitative test that determines the presence of amino acids. In
the experiment, all resulted positive in this test which is theoretically correct.
Ninhydrin test is the reaction of α-amino acids or proteins with free amino groups
that reacts with triketohydrindene hydrate when warmed that gives off a blue-purple
color. Ninhydrin reagent is a strong oxidizing agent that decarboxylates and deaminates
the amino acid forming the colored complex (Galewska et. al., 2013; Harding &
Warneford, 1916; Milio & Lofredo, 1995).

Figure X. Reaction of amino acid with ninhydrin (Milio & Lofredo, 1995).

Figure X. Xanthoproteic test positive control and negative control respectively.

 Figure X. Xanthoproteic test for P1, P2, HP1 and HP2 all arranged accordingly
from left to right.
Xanthoproteic test is simply the nitration of the aromatic ring of the amino acids
forming yellow- colored nitro derivatives. The amino acids that readily undergo nitration
has activated rings since some amino acids have aromatic groups which are derivatives
of benzene, so we expect it to have the same characteristics with it (Galewska et. al.,
2013; Milio & Lofredo, 1995).

Figure X. Nitration of tyrosine (Milio & Lofredo, 1995).

In the experiment, all exhibited a yellow color upon nitration and are therefore
positive for the presence of aromatic amino acids.

SAKAGUCHI

References:

Bardawill, C., David, M. & Gornall, A. (1948). Determination of serum proteins by

means of the biuret reaction. Retrieved from
https://pdfs.semanticscholar.org/07a4/2e4dd0fe329c979199d885b74a896ad13975.pdf

Galewska, Z., Gogiel, T., Malkowski, A., Romanowicz, L., Sobolweski, K., Wolanska,
M., & Bankowski, E. (2013). Biochemistry workbook. Retrieved from
https://www.umb.edu.pl/photo/pliki/WL_jednostki/zaklad_biochemii_lekarskiej/pdf/bioch
emistry_workbook.pdf

Harding, V.J., & Warneford, F.H.S. (1916). The ninhydrin reaction with amino acids
and ammonium salts. Retrieved from http://www.jbc.org/content/25/2/319.full.pdf

Milio, F. R., & Loffredo, W.M. (1995). Qualitative tests for amino acids and proteins.
Palmyra, PA: Qualitative Tests for Amino Acids and Proteins

Nielsen, S. (2017). Food analysis. New York, NY: Springer International

Publishing.

Waters, C. (1978). A one-step biuret assay for protein in the presence of

detergent. Vermont USA: Elsevier Inc.

Xanthoproteic Test

 The aromatic rings of phenylalanine, tyrosine and tryptophan under the effect of nitric acid form
yellow-coloured nitro derivatives. This process is called the xanthoproteic reaction. (Galewska et. al.,
2013)
 Some amino acids contain aromatic groups that are derivative of benzene. These
aromatic groups can undergo reactions that are characteristic of benzene and
benzene derivatives. One such reaction is the nitration of benzene ring with nitric
acid. The amino acid phenylalanine also contains a benzene ring , but he ring is
not activated and therefore does not readily undergo nitration.
The nitration reaction, when used to identify the presence of an activated
benzene ring, is commonly known as xanthoproteic test, because the produced
product is yellow. Xanthoproteic comes from the Greek word Xanthos, which
means “yellow.” The intensity of the yellow color deepens when the reaction
occurs in the basic solution.
This reaction is one of the reaction that occurs if you spill a concentrated nitric
acid onto your skin. The proteins in skin contain tyrosine and tryptophan, which
become nitrated and turn yellow (Milio & Lofredo, 1995).

Biuret Test
 An alkaline-copper reagent is described which forms a soluble, stable complex
with protein (Watters, 1978)
 the concentration of NaOH has been reduced by 0.75x to prevent the
precipitation of the detergent or the copper-protein-detergent complex (Watters,
1978)
 Peptide bond is complexed with cupric ions under alkaline conditions to give
color that is quantitated by spectroscopy Less expensive, faster, and simpler
 than Kjeldahl method. Does not detect nopeptide or nonprotein sources. Few
interferences (Nielsen, 2017)
 A violet-purplish color is produced when cupric ions are complexed with peptide
bonds (substances containing at least two peptide bonds, i.e., oligopeptides,
large peptides, and all proteins) under alkaline conditions. The absorbance of the
color produced is read at 540 nm. The color intensity (absorbance) is
proportional to the protein content of the sample (Nielsen, 2017)
 The importance of sodium hydroxide in the biuret reaction has long been
recognized and was studied in some detail by Rising and Johnson (13). Before
the development of stabilized reagents, the biuret reaction was generally carried
out in a medium containing about 3 per cent alkali. It was necessary to separate
a precipitate of cupric hydroxide before color comparisons could be made. With
low concentrations of alkali, a reaction mixture will remain clear for several days.
When levels above about 2 per cent are used, the mixture may show, but only
after 12 to 24 hours, a slight flocculent coagulum. This precipitation, which occurs
when alkali alone is added to serum, has no significance for readings made at 30
minutes. (Bardawill, David & Gornall, 1948)
 Reacts with copper (II) ions in a basic solution to form deep violet complex. The
peptide linkages in proteins resemble those in biuret and also form deep violet
complexes with basic copper (II) ions in solution. The general or biuret complex
formed between the protein linkages and the copper (II) ion of the biuret test is
shown. (Milio & Lofredo, 1995)
 The biuret reaction is a commonly used colour reaction, used for the detection and
quantification of peptides and proteins. It is characteristic of structures that have at least two
peptide bonds. In the presence of peptide or protein, the biuret reagent, which is a solution of
CuSO4, NaOH and sodiumpotassium tartrate changes colour from blue to purple. In an alkaline
environment, forms a complex of Cu2+ with a peptide or protein and with tartrate. The last
increases the solubility of the complex. The colour intensity is proportional to the concentration
of proteins in the solution. (Harding & Warneford, 1916)

Ninhydrin test

 The detection of the hydrolysis products of the circulating foreign protein is usually carried out
by the dialysis method, and depends ultimately on the detection of these products after dialysis,
by the now so called ninhydrin reaction. This latter reaction was discovered by Ruhemann3 who
found that a-amino-acids when warmed with triketohydrindene hydrate gave a fine blue color.
p- and r-amino-acids hardly reacted at all, and Lu-aminoacids when substituted on the amino or
carboxyl group, gave a negative reaction. (Harding & Warneford, 1916)
 Amino acids contain a free amino group and a free carboxylic acid group that
react together with ninhydrin to produce a colored product. When an amino group
is attached to the first, or alpha, carbon on the amino acid’s carbon chain, the
amino group’s nitrogen atom is part of a blue-purple product as shown in eq.
 Proteins also contain free amino groups on the alpha-carbon and can react with
ninhydrin to produce blue-purple product.
Amino acids that have secondary amino group attachments also react with
ninhydrin. However, when the amino group is secondary, the condensation
product is yellow. Blue-purple and yellow reaction products positively identify free
amino groups in amino acids and proteins. (Milio & Lofredo, 1995)
 The chemical properties common to all amino acids are due to the presence of the α-carboxyl
group and the α-amino group in their molecules. All amino acids, containing a free α-amino
group, in a reaction with ninhydrine form products of a violet-blue colour, while proline and
hydroxyproline, containing the imino group, create yellow-coloured products. During a
ninhydrine reaction, the amino acid decarboxylates and deaminates, and the released ammonia
is fixed with ninhydrine to form a violet-bluecoloured product. (Galewska et. al., 2013)