SOP: Determination of Lead by the Dithizone Method
GIFSQ — Global Institute for Food Safety & Quality
Principle
When ammoniacal cyanide solution of dithizone is added to a solution containing lead, a red lead-dithizone complex soluble in chloroform or carbon tetrachloride is formed; the red colour is measured at 510 nm. Dithizone is not specific for lead — it forms coloured compounds with 14 other metals — so lead is first separated: by extraction with dithizone from alkaline citrate-hexametaphosphate solution (Method I, for fruits and vegetables not rich in calcium, magnesium and phosphates), or, for samples very rich in calcium, magnesium and phosphate, by preliminary extraction from acid solution with diethylammonium diethyldithiocarbamate in chloroform (Method II). Method II suits lead contents up to 5 ppm; bismuth interferes and is handled by the modified procedure given.
Apparatus required
- Separating funnels, 100 ml
- Spectrophotometer or colorimeter (510 nm; also 490 and 520 nm for the bismuth check)
- Cotton-wool plugs
- Cuvettes
Reagents
a. 1% dilute nitric acid
b. 5 M hydrochloric acid: dilute 442.25 ml of concentrated HCl (35% by weight, sp. gr. 1.1778 at 15 °C) to 1 litre with water
c. Ammonium hydroxide, sp. gr. 0.880
d. 25% ammonium citrate [(NH₄)₂HC₆H₅O₇] solution w/v in water
e. 10% sodium hexametaphosphate solution w/v in water
f. 10% potassium cyanide (KCN) solution w/v in water: keep for at least 2 days before use to oxidize traces of sulphides that may be present
g. 20% hydroxylamine hydrochloride (NH₂OH·HCl) solution w/v in water
h. Chloroform (CHCl₃): shake 250 ml of CHCl₃ with 25 ml of water containing 1 ml of 10% KCN solution and about 20 drops of 5 M NH₄OH; separate and reject the aqueous layer; wash the CHCl₃ with water and filter
i. 0.10% dithizone (diphenylthiocarbazone) w/v stock solution in CHCl₃: filter and store in a refrigerator
j. Dithizone working solution: shake 6 ml of the dithizone stock solution with 9 ml of water and 1 ml of 5 M NH₄OH; separate and reject the lower CHCl₃ layer; centrifuge the aqueous layer until clear; prepare freshly on the day of use
k. Ammoniacal sulphite-cyanide solution: mix 340 ml of NH₄OH (sp. gr. 0.880), 75 ml of 2% w/v sodium sulphite solution, 30 ml of 10% w/v KCN solution and 605 ml of water (the concentrations of these reagents are critical)
l. Standard lead solution: dissolve 1.60 g of lead nitrate [Pb(NO₃)₂] in water, add 10 ml of concentrated HNO₃ and dilute to 1 litre; to prepare the working standard, dilute 1 ml of the lead solution to 100 ml with water as required (1 ml = 10 μg of lead)
m. 0.04% thymol blue w/v indicator: warm 0.1 g of thymol blue with 4.3 ml of 0.05 N NaOH and 5 ml of 90% ethanol; when dissolution is complete, dilute with 20% ethanol to 250 ml
Note: use glass-distilled water and analytical-grade lead-free reagents throughout.
Additional reagents for Method II: dilute H₂SO₄ (1+1); perchloric acid, sp. gr. 1.54; 20% sodium iodide w/v solution in water; 1.25% sodium metabisulphite w/v solution in water (prepare fresh as required and filter before use); 1% diethylammonium diethyldithiocarbamate (carbamate reagent) solution: dissolve 1 g of the pure crystalline reagent in 100 ml of redistilled chloroform and store in an amber bottle (not stable — discard after 1 week); 0.01% methyl red indicator: warm 25 mg of methyl red with 0.95 ml of 0.05 N NaOH and 5 ml of 90% ethanol; when dissolution is complete, make up to 250 ml with 50% ethanol.
Sample preparation
Destroy the organic matter either by wet digestion or by dry ashing. If wet digestion is adopted, use not more than 5 ml of concentrated H₂SO₄ (if much calcium is present, use perchloric acid in place of H₂SO₄); after digestion is complete, cool, add water, transfer to a 100-ml volumetric flask, rinsing the digestion flask twice with minimum (1–2 ml) quantities of water; then add 10 ml of 5 M HCl to a Kjeldahl flask, boil, and if insoluble matter is present filter through ashless filter paper into the volumetric flask; rinse the digestion flask with water, pass through the filter paper and make up to volume. Dry ashing at a temperature not exceeding 500 °C is usually convenient for foods relatively chloride-free; if necessary, continue ashing overnight; for Method I use 2 ml of a solution containing 15% ammonium nitrate and 15% potassium sulphate as ash aid; for Method II use 10 ml of 10% magnesium nitrate solution; when a clean ash is obtained, add 5 ml of water and 10 ml of 5 M HCl and boil gently for 5 min; filter through ashless filter paper into a 100-ml volumetric flask; if residue is present, boil with acid, pass through the filter paper, wash with water, cool and make up to volume.
Procedure
Separation of lead — Method I (samples not high in calcium, magnesium and phosphate)
Pipette an aliquot of the ash solution; add 5 ml of ammonium citrate solution and 10 ml of sodium hexametaphosphate solution
↓
Add a few drops of thymol blue indicator and sufficient NH₄OH to give a blue-green colour (pH 9.0 to 9.5); cool and add 1 ml of potassium cyanide solution; if much iron is present, add 1 ml of hydroxylamine hydrochloride solution
↓
Transfer to a 100-ml separating funnel containing 10 ml of chloroform and rinse with a few millilitres of water (the aqueous layer at this stage should be about 50 ml); add 0.5 ml of dithizone working solution, shake vigorously for 1 min and allow to separate; run the chloroform layer into a second separating funnel
↓
To the first funnel add 3 ml of chloroform and 0.2 ml of dithizone working solution; shake vigorously for 30 sec, allow to separate and add the chloroform layer to the main extract; the last chloroform extract should be green — if not, extract further with chloroform and dithizone until the green colour of the final extract shows all the lead is extracted; reject the aqueous layer
↓
Add 10 ml of dilute HNO₃ to the combined chloroform extracts and shake vigorously for 1 min; allow to separate and reject the chloroform layer as completely as possible.
Separation of lead — Method II (samples high in calcium, magnesium and phosphate)
Pipette an aliquot of the ash solution; add 2 drops of methyl red indicator and make just alkaline with NH₄OH; make just acid with 5 M HCl and add a further 10 ml
↓
Warm the solution (50 to 70 °C), add 2 ml of sodium iodide solution and reduce any liberated iodine with 2 ml of sodium metabisulphite solution; cool, transfer to a separating funnel and adjust the volume to 50 to 75 ml so the solution is 1 N with respect to HCl
↓
Add 50 ml of carbamate reagent by pipette and shake vigorously for 50 sec; allow to separate and transfer the chloroform layer to a 100-ml flask; wash the aqueous layer twice with small amounts of chloroform without mixing and add the washings to the flask; repeat the extraction with 10 ml of carbamate reagent and add the second extract to the main extract; reject the aqueous layer
↓
To the combined extracts add 2.0 ml of dilute H₂SO₄ and evaporate the chloroform; add 0.5 ml of perchloric acid to the residual solution and heat until fumes are evolved and the fuming solution is clear and colourless; cool; add 10 ml of water and 5 ml of 5 M HCl; boil for 1 min; cool and add 2 ml of ammonium citrate solution
↓
Add a few drops of thymol blue indicator and continue the separation of lead as in Method I.
Determination of lead
To the HNO₃ layer left in the separating funnel, add 30 ml of ammoniacal sulphite-cyanide solution, exactly 10 ml of chloroform and 0.5 ml of dithizone working solution
↓
Shake vigorously for 1 min and allow to settle; run off a little of the chloroform layer, insert a plug of cotton-wool into the dry stem of the funnel and, after rejecting the first few drops, collect in a cuvette
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Measure the colour at 510 nm in a spectrophotometer or colorimeter, measuring test and blank solutions against chloroform
↓
Prepare the standard curve: measure 0, 1.0, 2.0, 3.0 and 4.0 ml of working standard lead solution (1 ml = 10 μg of Pb) into separating funnels and dilute each to 10 ml with dilute HNO₃; proceed as under determination; measure the colour using chloroform as blank; plot optical density against concentration of lead.
Interference of bismuth
Bismuth is liable to interfere; measure the optical density at 490 nm and at 520 nm — with pure bismuth dithizonate the OD at 490 nm is about 1.20 times that at 520 nm; when considerable bismuth interference is indicated, use the modified procedure below.
Additional reagents: HCl, sp. gr. 1.18; 1% diethylammonium diethyldithiocarbamate (carbamate reagent) solution, prepared as under Method II.
Prepare the digest by wet decomposition or by dry ashing. If the organic matter was destroyed by wet decomposition, add 6.0 ml of HCl to the flask contents and transfer to a 50-ml separating funnel; rinse the conical flask with several 1-ml portions of water and add the rinsings; the volume must not exceed 35 ml so that the HCl concentration is not less than 3 N. Extract the acid solution directly in the cold, first with 10 ml and then with 5 ml of carbamate reagent, shaking 30 sec each time; separate and discard the lower (chloroform) layer; finally shake the acid layer with 5 ml of chloroform for 10 to 15 sec and discard it. Transfer the acid layer to a 100-ml conical flask, rinse the funnel with a few millilitres of water and add the rinsings; proceed as in Method I or II. If the organic matter was destroyed by dry ashing, add 15 ml of HCl (sp. gr. 1.18), transfer to a 50-ml separating funnel and adjust the volume to a maximum of 35 ml so the HCl concentration is about 6 N; continue as above.
Note: after wet digestion the extraction solution consists of residual H₂SO₄ to which HCl has been added; the acidity should not be less than 3 N in either H₂SO₄ or HCl. After dry ashing the extraction solution is HCl alone, and the acidity must be raised to about 6 N in HCl to effect quantitative separation of bismuth and other elements from lead.
Calculation
Lead as Pb, mg/100 g = (mg of lead as read from standard curve × Volume of digest × 100) ÷ (ml of digest taken × Wt of sample × 1000)
Lead, ppm = mg of lead per 100 g × 10
Where,
- mg of lead as read from standard curve = lead read from the curve (mg)
- Volume of digest = total volume of the digest (ml)
- ml of digest taken = aliquot taken for the determination (ml)
- Wt of sample = weight of sample taken (g)
References
- Analytical Methods Committee, Analyst, 84, 127 (1959).
- Hibbard, P.L., Ind. Eng. Chem., Anal. Edn., 9, 127 (1937).