1. FOOD ANALYSIS
Metals &
Heavy Metals

3. Nutrient metals
Macronutrients
Micronutrients
Calcium, magnesium, potassium, sodium
Chromium, cobalt, copper, molybdenum, nickle, selenium
Possibly essential micronutrients
Arsenic, boron, vanadium
Toxic metals
Berrylium, cadmium, lead, mercury
Non toxic, non-essential metals
Aluminium, tin

4. Heavy metals Heavy metals  one of inorganic pollutants
Lustrous appearance, good conductor of electricity & enter chemicals reactions as cations
Natural substances, present since the earth’s formation
Become pollutants when human activities (i.e. mining & smelting) release them from the rocks

5. Heavy metals in food Undesirable – Pb, Cd, As, Hg high toxicity level
adverse health effect & no benefit for human
Expected in certain amount – Fe, Cu, Zn, Al
medium toxicity level
have important roles in human metabolism

6. Heavy metals PTWI (mg/kg BW) Equivalent (mg/day for 60 kg adult)
Table. PTWI and MAC for several heavy metals
Heavy metals
PTWI
(mg/kg BW)
Equivalent
(mg/day for 60 kg adult)
Lead (Pb)
0.025
0.21
Cadmium (Cd)
0.007
0.06
Arsenic (Ar)
0.015
0.12
Mercury (Hg)
0.005
0.043
Copper (Cu)
3.5 30
Aluminium (Al) 7 60
Zinc (Zn)
* PTWI = Provisional Tolerable Weekly Intake
* According to JECFA (Joint Expert Committee on Food Additives) FAO

7. Heavy Metals Analysis

8. Principle of metal analysis:
1. Size reduction  for solid samples
2. Destruction (using strong acid solution & high
temperature)  removal of non metal compound
3. Filtration  removal of undesirable residue
4. Measurement using instrument, such as:
Atomic Absorption Spectrophotometry (AAS)- Inductively Couple
Plasma-Mass Spectrometry (ICP-MS)
etc

9. Sample Preparation Obtaining a representative sample
The bulk sample must be sufficiently homogenized to ensure that the subsample which is selected is representative of the whole. The same must be done for less bulky samples (animal/plant tissues) random/representative sampling
Prevention of contamination
Contamination sources especially come from laboratory equipments, such as homogenizer blade, knives, corers, grinders, sample containers, blenders.
A high degree of contamination of samples may result unless they have been given a thorough chemical clean up before use.
Contamination from airborne dust and particles coming from clothing, skin, cosmetics and cigarette smoking.

10. Purity of chemical reagents and water
Drying
If samples have to be stored before analysis, or if dry weight results are required, drying to constant weight is necessary
Vacuum oven or freeze dryer can be alternatives.
Freeze dryer is suitable for trace element analysis  followed by storing samples in polyethylene bag.
Purity of chemical reagents and water
The water used to dilute samples and reagents can be a potential source of contamination  deionized water which has a very low conductivity is highly recommended.
All chemical reagents used must be the highest quality.

11. Glassware and other equipments
All glassware must be thoroughly cleaned  soaking all glassware overnight in an alkaline detergent, then rinsing with deionised water, with a further soaking in 2% hydrochloric acid. The items should be then washed in deionised water, followed by two more rinsing in water. They should then be left overnight for drying.
Similar care in washing and drying should be used for other containers and vessels made of plastic.

12. Digestion of organic matters
In most other foods, organic matter has to be removed as this would interfere with the analytical process.
Exceptions are liquids such as beverages, including water, which may only require dilution before analysis.
Organic matter is usually removed from food samples by some form of oxidation, either by the use of oxidizing acids in a wet digestion or by dry ashing in the presence of air or pure oxygen.
The method used will depend on the metals to be analyzed and the nature of the food.

13. Dry ashing
This method of sample preparation involves the incineration of food samples in a muffle furnace at a suitable temperature. The resulting ash, free of organic compounds, is dissolved in dilute acid.
Ashing is usually performed at temperatures between 400 and 600oC.
These temperatures can be too high for volatile metals such as mercury, arsenic, selenium and lead. Other metals, such as tin, may form insoluble refractory compounds during dry ashing. The addition of ashing aids (salts of metals; acids) may improve the efficiency of ashing and to assist recovery of certain elements.

14. Dry ashing is a convenient and versatile method for preparing food samples for instrumental analysis. It allows use of relatively large sample sizes and can minimize contamination from reagents, and needs minimal attention from the operator.
Some disadvantages include time consuming operation; problems may be caused by incomplete combustion and absorption on surface of the incineration crucibles; losses through volatilization.

15. Wet digestion
Wet digestion requires the use of strong oxidizing acids, such as nitric; sulphuric and perchloric acids, or, in certain cases, hydrofluoric, phosphoric and hydrochloric acids.
The acids are used either alone or in various combinations, depending on the nature of the sample and the metals to be analyzed.
Nitric and perchloric acids are very strong oxidizing agents.
Generally, sulfuric acid is used with an additional substance such as hydrogen peroxide to yield a cleaner decomposition mixture. Combinations of the above acids are normally recommended for food analyses.

16. Compared to dry ashing, acid digestion gives greater flexibility for digestion of a wide range of organic matter with higher recovery rates for many trace elements.
The disadvantages of the method are that it is only suitable for small sample sizes and relatively large volume of reagents are required. This can lead to higher blank and introduce contamination. The use of strong acids also makes it potentially hazardous method that requires constant attention by the operator.
Wet digestions of biological samples commonly were carried out in open vessels, such as Kjehdahl flasks.
Heating was carried out on a sandbath, or in a specially designed device such as an aluminium block digester.
Until relatively recently, microwave heating methods became available and closed digestion system were introduced.

17. Nitric acid digestion
Nitric acid is recommended in the case of food samples with high chloride content.
It is good for analyzing Cd, Pb and Zn.
In the case of some metals, such as Cr, additional steps such like addition of potassium permanganate may be required.
Hydrogen peroxide can also increase the oxidizing power of the nitric acid and is specially efficient for determination of Zn, Cu, Pb, and Cd.

18. Nitric-sulphuric acids digestion
Nitric and sulfuric acid mixtures are widely used for the decomposition of samples with low organic matter content
Sulphuric acid, which ha a boiling point of 330oC improves oxidizing power.
This digestion mixture has been used successfully to determine Cu, Fe, Zn and Mn in food.
Addition of H2O2 can improve the determination of As, Fe, Al, Zn and Cr in plant tissue.
The addition of hydroflouric acid can increase the efficiency of recovery of mercury after nitric-sulphuric acid digestion of seafood.
Nitric-sulphuric acid digestion has been less successful for the determination of Se and unsuitable for the determination of Pb, due to the formation of insoluble lead sulphate.

19. Perchloric acid
Perchloric acid is a very powerful oxidizing agent and its addition to nitric acid or nitric-sulphuric acid mixture increases the speed of digestion.
Perchloric acid is explosive  problem.
Under certain conditions the presence of this acid in a digestion mixture may result in the loss of volatile elements, such as Cr and Se  can be overcome by using combination acid mixtures and by monitoring of the digestion conditions.
Nitric-sulphuric-perchloric acids
Mixtures nitric–sulfuric–perchloric acids are employed for samples with high fat content.

20. Nitric–perchloric acids
Nitric–perchloric acidic mixtures are generally used in the case of samples rich in proteins and carbohydrates, but in the absence of fat.
Hydrofluoric acid
Hydrofluoric acid has no oxidizing power and finds unique application in the decomposition of organic samples containing silicon or silica in varying amounts, such as corn leaves.
Only in this case, because hydrofluoric acid attacks silicon bonds, it is essential to use non-glass apparatus for the sample treatment (generally Teflon apparatus is employed).

21. Metal species determination

22. Atomic absorption spectroscopy (AAS)
AAS is one of the most widely used instruments for the determination of major and minor inorganic elements.
It includes:
Flame atomic absorption spectrophotometry (FAAS) –rapid and sufficiently sensitive to permit determination of most of the trace elements in food at the μg/g range.
Graphite furnace AAS (GFAAS) – allows determination of a wider range of elements, down to the μg/kg range.
Hydride generation AAS (HGAAS)
Electrothermally heated graphite tube (ETAAS) – can be used to analyse gaseous compounds of volatile element, such As or Se.
Cold vapour AAS (CV AAS) – for determination of Hg (based on the generation of elemental mercury vapour at room temperature).

23. Atomic absorption spectroscopy
Over 70 elements can be determined by the use of this technique, including most of those of interest in food analysis.
AAS method is based on the fact that when a metal is introduced into a flame, an atomic vapour is produced and light, of a wavelength characteristic of the metal, is emitted.

24. AAS There are 4 primary parts: the light source, the flame apparatus,
the detector, and
the data system.
UV Lamp
Burn head assembly
UV Detector

25. FAAS
GFAAS

26. In atomic absorption, there are two methods of adding thermal energy to a sample:
Graphite furnace AAS , it uses a graphite tube with a strong electric current to heat the sample.
Flame AAS, we aspirate a sample into a flame using a nebulizer.
The flame is lined up in a beam of light of the appropriate wavelength. The flame (thermal energy)  the atom to undergo a transition from the ground state to the first excited state  they absorb some of the light from the beam.
The more concentrated the solution, the more light energy is absorbed!

27. The light beam is generated by a lamp that is specific for a target metal. The lamp must be perfectly aligned so the beam crosses the hottest part of the flame and travels into the detector. The detector measures the intensity of the beam of light. When some of the light is absorbed by a metal, the beam's intensity is reduced. The detector records that reduction as an absorption. That absorption is shown on a readout by the data system. Fortunately, we do not need to separate solutions containing different metals. No chromatography is required for this instrument. We merely change lamps and adjust the detector wavelength.
We can find the concentrations of metals in a sample running a series of calibration standards through the instrument. The instrument will record the absorption generated by a given concentration. By plotting the absorption versus the concentrations of the standards, a calibration curve can be plotted.

28. FAAS
FAAS is employed in the determination of major and minor elements (concentrations in the solution introduced in the flame higher than 0.5–1.0mg/L).
FAAS employed especially in the major elements determination in food, recently is also employed in the determination of minor elements like Cd, Co and Ni using a new functionalized resin.

29. The technique is based on the fact that ground state metals absorb light at specific wavelength.
Metal ions in a solution are converted to atomic state using flame. Light of the appropriate wavelength is supplied and the amount of light absorbed can be measured against a standard curve.
The technique of FAAS requires a liquid sample to be aspirated, aerosolized, and mixed with combustible gases, such as acetylene and air, or acetylene and nitrous oxide.
During combustion, atoms of the element of interest in the sample are reduced to free, unexcited ground state atoms, which absorb light at characteristic wavelenghts.

30. FAAS

31. GFAAS
In GFAAS, the sample is introduced into a graphite tube as a small volume of liquid or in solid form.
The tube is heated due to its resistance by passing a controlled current through it.
The sample (usually ~20 µL but less than 100 µL) is added to the graphite furnace either manually or automatically and evaporated at a low temperature, then ashed at a higher temperature. After ashing the current is increased causing the temperature to rise to 2000 – 3000°C and the sample atomises in a few milliseconds.
Obviously, graphite furnace atomic absorption spectroscopy (GF-AAS) is more utilized, considering also the very low element concentrations in food.

33. Reference
Reilly, Connor Metal Contamination of Food. Its Significance for Food Quality and Human Health Third Edition. Blackwell Science Ltd, Oxford.
Barcelo, D Comprehensive Analytical Chemistry. Food Contaminants and Residues Analysis Volume 51. Elsevier, Oxford.