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Determination of Phosphorus in feeds and fodder by UV-VIS

spectrophotometry.

Method · July 2022

DOI: 10.13140/RG.2.2.15461.27361

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Manika Debnath
Department of Livestock services, Bangladesh
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Manika Debnath
Principal author & Researcher
QC Lab, Savar, Dhaka, Bangladesh
Quality Control Laboratory
Animal Feed Quality Section
DLS, Savar, Dhaka-
www.qclabdls.gov.bd
Phone:02-0000000, Email:QClab.dls@gmail.com

SOP QCLab_DLS_FQCS_SOP -28 Version: 01

number Model: UV-1900, Shimadzu
corporation
SOP Determination of Phosphorus in feeds and fodder by UV-VIS
title spectrophotometry.
Source FAO, Animal production and health, 14, ISSN 1810-1119

Name Designation Date

Reviewer Md. Mosharaf Hossain SSO 03/03/2022
Authorizer Dr. Abu Sayeed Md PSO 03/03/2022
Abdul Hannan

Effective Date: 03/03/2022

Review Date: 03/03/2022

Prepared by

Name Designation Signature Date

Manika Debnath S.S.O 03/03/2022

pg. 1
Manika Debnath
Principal author & Researcher
QC Lab, Savar, Dhaka, Bangladesh

1.0 Title of the SOP

Determination of Phosphorus in feeds and fodder by UV-VIS spectrophotometry.
2.0 Purpose of the SOP
To analyze the Urea using visible light and ultraviolet rays. The sample is suspended in distilled water
with a clarifying agent.
The P content is determined using a spectrophotometer after addition of Ammonium monovanadate
reagent.
Scope
The method is suitable for feeds and forages.
Definitions
A spectrophotometer is a special type of spectrometer, which is used to measure the intensity of light,
and the intensity is proportional to the wavelength of chemical structure of substances.
3.0 Responsibilities
Scientific Officer (SO)/Senior Scientific Officer (SSO) of the Animal feed quality section will
responsible for the execution of this SOP. Principal Scientific Officer (PSO) will supervise the
execution.
4.0 Requirements
4.1 Equipment and glass wares:
a) UV-VIS spectrophotometer
b) 2mm sieve grinder
c) Digital balance, accurate to 0.1mg.
d) Spatula
e) Volumetric flask, 100 ml and 500 ml
f) Test tube with ground-glass stopper.
g) Hot Water bath
h) Filter paper
4.2 Chemicals Required:
a) 37% HCL
b) ammonium heptamolybdate tetrahydrate[(NH4)6Mo7O24•4H2O
c) Ammonium Monovanadate
d) Distilled water
e) Phosphorus standard(1000ppm)
4.3 Solutions / Reagents required:
a) Ammonium molybdate Solution (kept a maximum period of 2 weeks):
Dissolve 25 g of Ammonium molybdate in 300 ml water. Mixing and boiling to dissolve. Cool at
room temperature.
b) Ammonium Monovanadate:
Dissolve 1.25 g of b) Ammonium Monovanadate and 330 ml of HCL acid.
c) Mix a+b and add water upto 1 L.
d) P Standard (total volume 25ml each):
Blank, 0.5-15pmm (maximum 30ppm)
pg. 2
Manika Debnath
Principal author & Researcher
QC Lab, Savar, Dhaka, Bangladesh
Add 5ml solution C and required standard+ Distilled water =25 ml

5. Preparation of sample for analysis:

Sample preparation:
For Green forage:

 Dry the sample at 60± 2º C, 24 hours in the oven with a partially open lid.
 Move to desiccator for cooling until room temperature (15-20 min).
For Feeds and feed ingradients:

 Add approximately 0.5-1 gm sample in a porcelain crucible and ash at 550C for 3 hours.
 After cooling the ash add 10ml 1:1 nitric acid and boil 2 min.
 Cool at room temperature and filter with Whatman filter paper.
 Volume 50/100 ml.
 Take 1 ml sample aliquot into another 25 ml volumetric flask.
 Add 5 ml Solution (Reagent) C and add water to volume.
 Rest 30 minutes to settle down the color into yellow.

6. Calibration curve:

 Set Photometric method in Software.

 Type wave length 400 nm.
 Run the prepared sample with cuvet.
 Measure absorbance of solutions against the blank solution at 400 nm with spectrophotometer.
 Record the absorbance at R2 value near to 1.
Calculation:
Dilution factor 1= (1st volume/Sample wt)
Dilution Factor 2= (Final volume/ Volume taken from 1st volume)
P %= (Dilution factor 1* Dilution factor 2* concentration)/10000

7. Quality Control
A control standard sample (QC sample) should be analysed in order to confirm the method. Laboratory
QC samples should be prepared by mixing an animal feed/forage sample that is essentially similar to
the nature samples to be analysed. Take 3~4 kg of the QC sample, grind sample to the size that can pass
through a 1 mm sieve and store in a dry and cool place. The analysis acceptable range is determined by
standard ± 2 SD obtained by repeating the QC sample analysis 15~20 times. Samples should be analysed
twice.
The difference between the values of two parallel determinations carried out on the same sample should
be less than 5% of the average value.

8. Remarks
pg. 3
 Manika Debnath
Principal author & Researcher
QC Lab, Savar, Dhaka, Bangladesh
a. Other digestion methods may be used if similar results have been demonstrated (e.g. use of
different acids or microwave digestion).
b. Participating in the proficiency testing program is strongly recommended to maintain the accuracy
of laboratory analysts and validate the measurement process. For example, AAFCO for animal
feed and pet feed
9. References
AOAC 965.17. 1990. Phosphorus in Animal Feed and Pet Food, Photometric Method. Gaithersburg,
MD, USA.
Commission Regulation (EC) No 152/2009. 27 Jan 2009. Laying down the methods of sampling and
analysis for the official control of feed. Annex III, H, Official Journal of the European Union L54/1
from 26/02/2009.
FAO Animal Production and Health Manual No. 14. 2011. Quality Assurance for Animal Feed Analysis
Laboratories, Food and Agriculture Organization of the United Nations, Rome, Italy.
ISO 6491. 1998. Animal feeding stuffs – Determination of phosphorus content – Spectrometric method.
Geneva, Switzerland.

pg. 4

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