Biomedical Engineering Reference
In-Depth Information
D.A.Harris, C.L.Bashford (ed.) (1987) Spectrophotometry and spectroflu-
orimetry: a practical approach, IRL Press, Oxford
P.K. Smith, R.I. Krohn, G.T. Hermanson et al. (1985) Anal Biochem 150:76-
85
1.1.6 K
JELDAHL
Protein Determination
Particularly suitable for insoluble proteins, protein in foods and
protein covalently immobilized on chromatographic supports.
The Kjeldahl totalnitrogendeterminationmethodisnotvery
sensitive, but it suits well for analyzing insoluble samples without
preceding disintegration. Automated Kjeldahl protein estima-
tions are used especially in food analysis.
A
selenium reaction mixture for nitrogen determination accord-
Solutions/Reagents
ing to Wi eninger
B
conc. sulfuric acid (98% w/w)
C
60% NaOH, 10% Na
2
S
2
O
3
(w/v) in ddH
2
O
D
2% boric acid (w/v) in ddH
2
O
E
Tashiro indicator (2 vol. 0.2% methyl red in 90% ethanol +
1 vol. 0.2% methylene blue in 90% ethanol)
F
0.010 N HCl (standard solution)
Mix 100 - 250 mg sample exactly weighed with 1.5 g catalyst A, and
then add 3 ml of concentrated sulfuric acid B. Heat the mixture
at the temperature of boiling sulfuric acid (about 180
◦
C)for2h.
Take care that the acid condenses in the middle of the neck of the
Kjeldahl flask.
Caution!
Strongly corrosive! Use a hood.
Put the flask into the distillation apparatus after cooling; there-
after add slowly 12 ml ddH
2
Ofollowedby12ml of Soln. C. Heat
the mixture to nearly 100
◦
C and the liberated ammonia is distilled
by steam for about 10 min through a condenser, the tip of which is
submerged in a flask containing 5 ml of Soln. D. When the distil-
lation is finished (total volume about 25 ml), add three drops of E
and titrate the ammonia with Soln. F. The results are calculated as
follows:
=
µ
=
1.0 ml 0.010 N HCl
10
Mol N
0.14 mg N
By means of the Kjeldahl factors
F
(Table 1.7) the amount of
protein is
=
mg protein
mg N
·
F
and the protein content c of the sample
100
weight of sample [mg]
(mg protein)
·
]
=
c
[
%
