Samples were analyzed chemically according to the
official methods of analysis described by the Association of Analytical Chemist
(AOAC. 18th edition, 2005). All analysis were:
CRUDE PROTEIN DETERMINATION (AOAC OFFICIAL METHOD 988.05)
The
crude protein in the samples were determined by the routine semi micro
Kjeldahl, procedure/technique. This consists of three techniques of analysis
namely digestion, distillation and titration.
APPARATUS: Analysis Balance, Digestion tubes, Digestion Block Heaters, 50ml
Burette, 5ml Pipette, 10ml measuring Cylinder, 100ml Beakers, Fume Cupboard.
REAGENTS: Conc.
H2SO4, 0.01NHCL, 40% (W/V) NaOH, 2% Boric Acid Solution,
Methyl Red Bromocresol green mixed indicator, Kjeldahl Catalyst tablet.
DIGESTION
0.5g of each finely ground sample was weighed
carefully in the Kjeldahl digestion tubes to ensure that all sample materials
got to the bottom of the tubes. To this were added 1 Kjeldahl catalyst tablet
and 10ml of Conc. H2SO4. These were set in the
appropriate hole of the Digestion blocks Heaters in a fume cupboard. The Digestion
was left for 4 hours, after which a clear colorless solution was left in the
tubes. The digest was cooled and carefully transferred into 100ml volumetric
flask, thoroughly rising the digestion tube will distilled water and the flask
was made up to mark the distilled water.
DISTILLATION
The distillation was done
with Markham Distillation Apparatus which allow volatile substances such as
ammonia to be steam distilled with complete collection of the distillate. The
apparatus was steamed out for about ten minutes. The steam generator is then
removed from the heat source to all the developing vacuum to removed condensed
water. The steam generator was placed on the heat source (i.e. Heating mantle)
and each component of the apparatus was fixed up appropriately.
Determination : 5ml portion of the digest above was pipette into the body of the
apparatus via the small funnel aperture. To this was added 5ml of 40% (W/V)
NaOH through the same opening with 5ml pipette.
The mixture was steam distilled
for 2 minutes into a 5ml conical flask containing 10ml of 2% Boric Acid plus
mixed indicator solution placed at the receiving up of the condenser. The Boric
Acid plus the indicator solution changes color from red to green showing that
all the ammonia liberated have been trapped.
TITRATION
The green colored solution obtained was
then titrated against 0.01N HCL contained in a 50ml Burette. At the end point
or the equivalent point the green color turns to wine color which indicator
that all the nitrogen trapped as Ammonia Borate [(NH4)2BO3)]
have been removed as
Ammonia chloride (NH4CL).
The percentage nitrogen in this analysis
was calculated using the formula:
%N = Titre value x Atomic mass of
nitrogen x Normality of HCL used x 4
Or %N = Titre value x Normality/Morality
of HCL used x Atomic mass of
N x Volume of flask containing the
digest x 100
Weight of sample digested in milligram x
Vol. of digest for steam distillation. The crude protein content was determined
by multiplying percentage Nitrogen by a constant factor of 6.25 i.e. % CP= %N x
6.25.