2.4.24
Remove
AOAC Official Method 976.01
Biuret in Fertilizers
Atomic Absorption Spectrophotometric Method
First Action 1976
Final Action 1980
A. Apparatus and Reagents
(a) Atomic absorption spectrophotometer.—Instrument with Cu
hollow cathode lamp.
(b) Copper sulfate solution.—Dissolve 15 g CuSO4⋅5H2O in
H2O and dilute to 1 L.
(c) Buffer solution.—pH 13.4. Dissolve 24.6 g KOH and 30 g
KCl in H2O and dilute to 1 L.
(d) Starch solution.—Treat 1 g soluble starch with 10 mL cold
H2O, triturate to thin paste, and pour gradually into 150 mL boiling
H2O containing 1 g oxalic acid. Boil until solution clears, cool, and
dilute to 200 mL. Prepare fresh weekly.
(e) Bromocresol purple indicator.—Dissolve 0.1 g bromocresol
purple in 19 mL 0.1M NaOH and dilute to 250 mL with H2O.
(f) Biuret.—See 960.04A(c) (see 2.4.23).
(g) Biuret standard solution.—0.4 mgmL. Dissolve 0.4000 g
recrystallized biuret in warm H2O, cool, transfer to 1 L flask, and di-
lute to volume.
(h) Copper standard solutions.—Dilute aliquots of Cu stock so-
lution, 965.09B(b) (see 2.6.01), with H2O to obtain ≥4 standard so-
lutions within range of determination, 1–4 µg Cu/mL final
solution.
B. Preparation of Standard Curve
Transfer aliquots of biuret standard solution containing 0, 2,
4, 6, 8, 10, and 12 mg biuret to separate 100 mL volumetric
flasks, dilute to ca 30 mL with H2O, and add 25 mL alcohol to
each. While stirring with magnetic stirrer, add 2 mL starch solu-
tion, 10 mL CuSO4 solution, and 20 mL buffer solution.
stirring bar, rinse, dilute to volume, mix thoroughly, and let
stand 10 min. With vacuum, filter ca 50 mL through dry 150 mL
medium porosity fritted glass funnel into dry flask. Transfer 25 mL
aliquots of each filtrate to 250 mL volumetric flasks, acidify with
5 mL 1M HCl, and dilute to volume with H2O. Proceed as in
965.09 (see 2.6.01), using standard solutions, A(h), to determine
complexed Cu in solution by AA spectrophotometry after adding
equivalent amounts of alcohol, KOH solution, buffer solution,
and 1M HCl. Take ≥3 readings of each solution. From mean value
of Cu concentration, prepare standard curve relating mg Cu found
to mg biuret added. Redetermine daily.
C. Determination
(a) In urea.—Accurately weigh test portion containing <10 mg
biuret, dissolve in H2O, transfer to 100 mL volumetric flask, add
25 mL alcohol, and proceed as in B, beginning “While stirring with
magnetic stirrer, . . .”. From Cu found, calculate biuret concentra-
tion, using standard curve.
(b) In mixed fertilizers.—Transfer accurately weighed test por-
tion containing <40 mg biuret to 250 mL beaker and add 1 mL H2O
for each g of test sample (5 g maximum). Warm, add 65 mL alcohol
and 7 drops bromocresol purple, and adjust pH to first blue color
(pH 6–7) with 20% KOH w/v. Place on hot plate, heat to bp, cool,
and, if pH has changed, make final adjustment to first blue. Vac-
uum-filter through alcohol-washed paper pulp pad into 100 mL vol-
umetric flask. (If filtrate is not clear, improper pH adjustment has
been made. Add HCl and readjust to pH 6–7.) Wash pad and precipi-
tate with alcohol and dilute to volume with alcohol. Transfer 25 mL
aliquot to 100 mL volumetric flask, and proceed as in B, beginning
“While stirring with magnetic stirrer, . . .”. From Cu found, calculate
biuret concentration, using standard curve and appropriate dilution
factors. (Final aliquot can be varied to give Cu concentration be-
tween 1 and 4 µg/mL.)
References: JAOAC 59, 22(1976); 62, 153(1979); 63, 222(1980).
CAS-108-19-0 (biuret)
© 2000 AOAC INTERNATIONAL