Basic Concepts in Clinical Biochemistry-A Practical Guide.7z

16.3 Principle........................................

Urea reacts directly with diacetyl monoxime under strong acidic conditions to give a
yellow-colored condensation product. Firstly, diacetyl is released from diacetyl
monoxime, and then urea reacts with diacetyl in hot acidic medium to give diazine
complex. Diazine is stabilized as pink-colored compound by thiosemicarbazide and
ferric ions. The acid reagent of H 3 PO 4 and H 2 SO 4 acid is used to provide acidic
medium for these reactions, andfinal pink product has absorption maximum at
520 nm in proportion to amount of urea.

CH 3

C=O

O

H+

Fe3+

O

Water

Thiosemicarbazide

Heat

Diazine

(yellow)

O

O

N Heat

Diacetyl monoxime

Diacetyl

Pink-red coloured

product

Diacetyl

OH

O

O

C

+ +

C

C N

C N

C C

C

C

C

CH 3

H 2 N

H 2 N

CH 3

CH 3

2H 2 O

CH 3

CH 3

CH 3

Urea CH 3

16.4 Reagents........................................

1.Acid reagent
Sol. A–Dissolve 0.5 g of ferric chloride (FeCl 3 ) in 2 ml of distilled water. Add
10 ml H 3 PO 4 acid to it. Then mix and makefinal volume to 25 ml with
distilled water. Store at room temperature in a brown bottle.
Sol. B–Prepare 20% aqueous solution of H 2 SO 4. Then mix 0.25 ml of solution A
with 500 ml of solution B to prepare acid reagent.
2.Color reagent
Sol. A–Prepare 2% diacetyl monoxime solution in distilled water.
Sol. B–Prepare 0.5% thiosemicarbazide solution in distilled water.

68 16 Estimation of Urea in Serum and Urine