STANDARD OPERATING PROCEDURE

FOR

FOOD
HEAVY METALS ANALYSIS
 1.0 IDENTIFICATION SHEET

DOCUMENT :

VERSION NO. : 01

ISSUED TO : Chemical Department

DATE OF ISSUE : 03.02.2021

COPY NO : 1

STATUS OF MANUAL : ACTIVE

Sl. Page Clause Date of Amendment Made Reasons Signature

Scope:

This method is applicable for determination of Lead, Copper, Cadmium, Iron, and Zinc in food by
Atomic Absorption Spectrophotometer using Flame and Furnace Technique.

Principle:

Test portions are dried and then ashed at 450°C under a gradual increase (about 50°C/hr) in
temperature, 6 N HCl (1+1) is added and the solution is evaporated to dryness. The residue is
dissolved in 0.1N HNO3 and the analytes are determined by flame and graphite procedures.

Apparatus:

(a) Atomic absorption Spectrophotometer – with an air – acetylene burner or nitrous oxide-
acetylene burner for flame and a graphite furnace for electro-thermal determinations with
appropriate background (non atomic correction). Instrument parameters are usually given by the
manufacturer in the manual provided with the instrument

(b) Hollow cathode or electrode less discharge lamps for all elements.

(c) Furnace – Programmable or muffle furnace with thermostat maintaining 450 ±25°C

(d) Hot plate – with heating control to heat upto 300°C

(e) Quartz or platinum dishes

(f) Polystyrene bottles – with leak proof closures – 100mL Carefully clean and rinse all glassware
and plastic ware with HNO3 or HCL to avoid metal contamination – First wash with water and
detergent, rinse with tap water, followed by distilled water, then with dilute acid ( 1 + 9 ) and
finally 3-4 times with distilled water.

NOTE: Microwave Digestion can be done since there is a chance of loss of target metals i.e. Lead, during
ashing.

Reagents:

(a) Water – redistilled or deionised

(b) Hydrochloric acid A.R (6N) – Dilute 500mL HCl to 1 litre with water

(c) Nitric Acid A.R 0.1M – dilute 7mL conc. acid to 1 litre

(d) Nitric acid concentrated (Sp. Gravity 1.40)

(It is suggested to use NIST traceable all solution of 1000mg/L. Prepare stock & working solution from
this. For the better result, working solution should be prepared in the digestion solution. Commercially
available standard solutions for AAS may be used for all metal standards).

(1) Lead Standard solution– 1mg/mL. Dissolve 1.000 gm Pb in 7 mL conc. HNO3 in 1litre volumetric
flask. Dilute to volume with water.

(2) Cadmium Standard solution – 1 mg/mL. Dissolve 1.000gm in 14mL water and 7mL conc. HNO3 in 1
litre flask. Dilute to volume with water.

(3) Zinc Standard solution – 1mg/mL. Dissolve 1.000gm Zinc in14mL water + 7mL conc. HNO3 in 1 litre
volumetric flask and dilute to volume with water.

(4) Copper Standard solution – 1mg/mL. Dissolve 1.000gm Copper in 7mL HNO3 in 1 litre flask. Dilute to
volume with water.

(5) Iron Standard solution – 1mg/mL. Dissolve1.000gm Iron in 14mL water and 7mL conc. HNO3 in 1
litre volumetric flask. Dilute to volume with water.

(e) Working Standard solution – For graphite furnace analysis dilute standard solutions with 0.1 M HNO3
to a range of standards that cover the linear range of the elements to be determined. For Flame analysis
dilute standard solutions with 0. 1 M HNO3 to a range of standards that covers the concentration of the
elements to be determined.

NOTE: Apart from the NIST traceable metal standard, internal standard may be used for accuracy of test
result

Preparation of Sample:

Sample may be prepared by wet digestion using microwave digestion system or by dry ashing as given
below. Weigh accurately about 25 gm of well homogenized sample into a clean silica dish. Add 25mL of
20% sulphuric acid. Mix thoroughly with a glass stirringrod ensuring all sample material is wetted by the
acid. Rinse stirring rod with water into silica dish. Dry the contents of the dish thoroughly on a steam bath
or in an oven around 110°C. When the sample is thoroughly dry, heat the contents of the dish with a soft
flame (such as that of a Bunsen burner) until all volatile or readily combustible matter has been removed.
Transfer the dish to a furnace set at 250°C. Slowly raise temperature to 500°C. Ash at this temperature for
about 6 to 8 hours. Remove the dish and cool. Ash should now be white or brownish red and essentially be
carbon free. If ash contains carbon particles, wash down sides of dish with water and add 2mL of HNO3
and mix well. Dry thoroughly on hot plate. Return dish to furnace at 500°C and ash for 30 minutes. Repeat
nitric acid treatment using 1mL increments of HNO3 until white/brownish red, carbon free ash is obtained.
When clean ash is obtained, remove the dish from furnace, cool and add 1mL HNO3 and 10mL of water.
Heat on hot plate till sample ash is dissolved. Quantitatively transfer the contents of the dish to a 50mL
volumetric flask, heat the dish with 10mL of HCl (1+1) and transfer the solution again to the same
volumetric flask to volume with water.

Prepare sample blank solution by following the same procedure as described for sample. Use same
quantities of reagents including water for both sample and blank. Subject both sample and sample blank to
identical treatment (even the length of time kept in furnace etc.)

NOTE: 1. Do not ash HNO3 in furnace. Always dry HNO3 (in the dish) on steam bath or hot plate and
then ash in furnace. 2. Do not allow sample to ignite during any stage of ashing. 3. If the calcium content
of the sample is high, then avoid the use of sulphuric acid (Ash aid) and ash at temperatures not exceeding
470°C.

Determination: Atomic Absorption Spectrophotometer: -

Lead and Cadmium in foods generally require graphite furnace AAS (GFAAS) for determination Zinc,
Copper and Iron can be determined by flame AAS

(1) Set the instrument as per the previously established optimum conditions /as per the guide lines given in
the Instruction Manual (provided along with the instrument). The standard conditions for Atomic
absorption spectrophotometer are given below.

(2)Determine absorbance of sample solution(s) and blank.

(3)Calculate the heavy metal content from standard curve. NOTE: calibrate AAS with copper solution
(NIST traceable) before use, for absorption value (pre defined).

Preparation of Standard Curve: Read the absorbance of a series of standard metal solutions in the Atomic
Absorption Spectrophotometer after setting the instrument as per optimum conditions. Plot absorbance
against μg of metal/mL solution.

Reference: -

 AOAC 19th edn, 2012 Official Method 999.11 Determination of Lead, Cadmium, Copper, Iron
and Zinc in Foods Atomic Absorption Spectrophotometry after dry ashing.