trcndc in nnalytid chmistry, vol. 3, no. I, I!?84 25

Sample preparation for the analysis of heavy metals in foods

The analysis of foodstuffs for heavy metals continues to be an area of intense activity for analytical chemists. Methods of sample preparation are changing to allow a rowing number of samples to be handled and to aciiitate tg

‘speciation’ studies.

Coiin Watson Word, Essex, UK

There have been a number of important changes in the approach to the analysis of foodstuffs for heavy metals in recent years, largely as a result of the change from chemical methods of analysis (mainly calorimetric) to spectroscopic methods, predominantly atomic absorp- tion spectrophotometry (AAS). Traditionally, heavy metals specifically implied lead and arsenic, but in recent years the term has come to embrace many of the transition metals, e.g. nickel, copper, zinc, cadmium and mercury as well as other toxic elements, such as antimony and selenium. The common use of tin-plated containers for foodstuffs has also led to a lot of work on the analysis of tin in foods, although tin is generally regarded as non-toxic.

The trend toward widening the use of the term ‘heavy metals’ is reflected in the list of publications from the Metallic Impurities in Organic Matter Sub-Committee of the Analytical Methods Committee (UK). Their first seven reports on the determination of metals (up to 1960) included three reports on methods of analysis for lead and two on arsenic’-‘. However, in the last 20 years the sub-committee has issued a further 14 reports on recommended methods for the analysis of metals in organic matter+“, only two of which have concerned lead and arsenic’2-‘4.

There has also been an almost complete change in the methods used for the analysis ofmetals in foods over this period which is also well illustrated by these published methods. Prior to 1969 all the methods made use of calorimetry in one form or another to assay the element of interest’-“; since 1969, with one excep- tion12, all the methods recommended have used spectroscopic methods other than calorimetry. Atomic absorption methods were recommended for zincl”*13, cadmiumll*l”, lead14, nickel”, antimony”, selenium” and tinl’. As a guide to future trends it may be worth noting that the report on seleniuml* also included the use of atomic fluorescence spectroscopy, while the use of inductively coupled plasma-optical emission spec- troscopy (ICP-OES), was recommended for the first time by the sub-committee for the analysis of tin.

This revolution in analytical methodology has led to some quite marked changes in requirements for sample preparation. When calorimetric methods are used, it is vital that every trace of organic matter is destroyed 0165-9936/84/$02.00.

and, usually, that no extraneous oxidants are left behind at the end of the oxidation. The reasons for these requirements are quite straightforward. Any remaining organic matter may interfere, either by virtue of its colour, or by competing with the calorimetric reagents being used. Similarly, residual oxidants may interfere by changing the oxidation state of the element of interest or by attacking the organic ligand being used. Most calorimetric systems employ buffer solutions, and ionic strength is not often critical. Therefore, relatively large amounts of acids may be present at the end of the sample preparation stage and the concentration of these will not normally be very important. In contrast with this situation, ifone wishes to aspirate the sample solution into an atomic adsorption spectrophotometer, complete removal of organic matter is not normally vital and trace amounts of residual oxidants are of no importance. But as the final quantity of acid may well affect the viscosity ofthe solution and hence its uptake rate, it is important that this should not only be as low as possible but that it should also be consistent. These requirements should be remembered when preparing food samples for analysis, regardless of the method used.

Common methods of sample preparation Wet Oxidation This is a widely used method of preparing food samples for analysis. Its advantages may be summarized as follows: l It is universally applicable, all foodstuffs may be destroyed by wet oxidation methods. l Control of the matrix is relatively simple, usually the residue consists of sulphuric acid or, occasionally, perchloric acid containing the inorganic species derived from the foodstuff. l Reagents used are all available in high purity. l Losses of most elements during the oxidation step are usually minimal and can be completely eliminated by using a Bethge apparatus. Even very volatile elements, such as mercury, may be determined following wet oxidation using this technique, providing the correct conditions are employed’. It is interesting that, although there are many reports of ‘losses’ using wet oxidation methods, studies using radio-tracer methods on zinc”, arsenic12, lead14, antimony” and selenium’* have always failed to show any losses, either due to volatilization or adsorption onto the surfaces of the

0 1984 Elsevicr Science Publishers B.V.

26 trends in analytical chemistry, vol. 3, no. I, 1984

glassware, which are the explanations frequently offered for poor recoveries. In general, failure to recover a trace metal following wet oxidation procedures is not due to it being ‘lost’, more probably it is in a non-reactive form, either by virtue of its valency state or due to the formation of complex sulphates or phosphates. These, while not thermodynamically stable in the presence of the organic ligand being used to determine or extract the metal, have such a slow reaction rate that they fail to react in a reasonable time.

In spite of these desirable properties, wet oxidations do have some disadvantages. The procedures are time consuming. In general it is difficult for one operator to wet oxidize more than IO-12 samples at a time and oxidation times are in the order of 30-90 min. The method is therefore labour intensive and although some efforts at automation have been successfulzo, the nature ofthe process makes automatic methods the exception rather than the rule. The reagents used - nitric, perchloric and sulphuric acids and hydrogen peroxide - are corrosive and hazardous. Procedures using nitric and perchloric acid are particularly damaging to the fume cupboards which are obligatory for wet oxidation methods because of the large volumes of acidic fumes that are produced.

The most common procedure for the wet oxidation of samples is to add nitric acid, which for reactive materials may need to be diluted. This destroys the easily oxidized materials. The temperature is then raised usually in the presence of sulphuric acid and more nitric, or perchloric acid is added to complete the oxidation. When perchloric acid is used, care must be taken to ensure that only very small quantities of organic material remain when the temperature of the reaction mixture reaches 2OO”C, otherwise the rapid increase in oxidation potential of perchloric acid around this temperature may lead to an extremely violent explosion! (The author has experience. of an explosion involving about 300 mg of 3-nitrophthalic anhydride, derived from ‘ninhydrin’, which was stable to oxidation by nitric acid but detonated with perchloric acid leading to the destruction of a fume cupboard and considerable other damage to the laboratory.)

For many samples, particularly cellulosic or carbohydrate based materials, a preliminary reflux with nitric acid (1 + 1) prior to the addition ofsulphuric acid may greatly reduce the time taken for the final oxidation.

An alternative procedure using hydrogen peroxide as the oxidant overcomes many of these disadvantages, since the only acidic fumes produced are from sulphuric acid. Care is needed to prevent formation of carbon since hydrogen peroxide does not oxidize carbon readily. Although organic material should never be left for long periods in contact with hydrogen peroxide, samples which are liquid or which can be liquidized may conveniently be oxidized by mixing the sample with the hydrogen peroxide and adding the mixture dropwise to the hot sulphuric acid. This

technique prevents the formation of carbon and is very safe as very little oxidant or organic material is present at any given time.

Information on these methods is generally available, but the reports on the subject from the Analytical Method Committee give a concise summary of the perchloric acid**, mixed acidZ3 and hydrogen perox- ide 24,25 methods.

Dry ashing This method of sample preparation is simple and,

since large numbers of samples can be handled, is often preferred to wet oxidation methods. However, uncertainty over recoveries has meant that, although widely used for routine analysis, dry ashing procedures have not been extensively studied by collaborative trial, with a method for referee analysis as the objective.

There are considerable variations in the results for dry ashing of materials. For example, Gorsuch*l found that dry ashing resulted in complete loss of selenium, while more recently Reamer and Veillon26 obtained satisfactory results for biological materials, when using magnesium nitrate as the ashing aid.

In general, dry ashing may lead to losses, not only due to volatilization, particularly if the temperature exceeds 5OO”C, but also due to adsorption onto the crucible or dish where the analyte may form a glass or refractory which is resistant to solubilization by acids.

Acid extraction The above methods lead to total destruction of the

organic matrix and were obligatory when calorimetry was used. However, as previously mentioned, the presence of organic matter is of little importance for spectroscopic methods and in 1966 Simpson and Blay*’ developed a simple method for determining several elements by heating the sample with diluted hydrochloric acid followed by filtration. The filtrate could then be aspirated directly into the atomic absorption spectrophotometer. Various workers have extended this approach and a method for tin was recently recommended” which was based on extrac- tion with hydrochloric acid.

Comparison of determinations using this rapid procedure with a wet oxidation method showed no significant difference. However, it should be noted that complete recoveries will only occur if the sample is boiled for the correct time. For example, fruits must not be boiled for more than about 10 min. or charring occurs and recoveries diminish, whereas meat products may require up to 45 min. heating for complete extraction. It is therefore recommended that recovery experiments should always be performed with each type of food prior to adoption of such methods.

An extension of this method entails oxidizing the carbohydrates with nitric acid and then extracting the residue with hydrochloric acid. This approach was adopted by Dabeka and McKenzie28 for the determination of tin and was thought to be more robust than using hydrochloric acid alone.

tr& in analytical chemishy, vol. 3, no. 1, 1984 27

Other methods of sample preparation Samples of foods may be destroyed using several

other procedures. However, none of these have attained the importance of the above methods. An improvement on simple dry ashing is to use a furnace, similar to a micro-combustion furnace, in which a stream of oxygen is passed over the sample. However, although quicker than dry ashing and less prone to volatilization loss because the issuing fumes can be trapped, absorption losses remain serious. The microwave oxygen plasma furnace should overcome these problems since it operates a very low temperature, but its very limited oxidation rates (only a few mg per hour) have prevented its widespread adoption.

The oxygen flask may be used to destroy most samples, but again sample size is limited and considerable operator skill is required if a reasonable rate of sample processing is to be achieved.

A novel approach used by Greenfield (communica- tion to Metallic Impurities in Organic Matter Sub-committee) was to aspirate the sample as a solution or slurry into an oxyhydrogen flame on a Beckman ‘Total Consumption’ burner and to trap the issuing gases. Reasonable results were obtained for selenium using this method.

An alternative to wet oxidation is catalytic oxidation with hydrogen peroxide in the presence of iron (II) sulphate. However, other metals in the latter reagent make the method unsuitable for many determinations.

Special considerations In the routine analysis of foods an important

consideration is the time taken for the analysis. For this reason simple metliods, such as dry ashing, are often preferred. Similarly, the use ofextraction methods such as concentration using ammonium pyrolidine dithio- carbamate (APDC) (Ref. 30) are often avoided because of the time they take. For low levels of metals it is often necessary to resort to electrothermal atomization (ETA) methods in spite of their well known shortcomings. Fortunately, recent advances in techniques for obtaining isothermal atomization by the use of ‘platforms’, have reduced the tedious matching of standards that was previously essential. Improved sample introduction methods with autosamplers have improved precision to acceptable levels, although ETA is still inferior to measurements made in the flame.

It should, however, be remembered that in some cases rapid routine analysis is not the requirement, and reliability is all important. In these cases it is always safer to use an established - and preferably collaboratively tested - method, although it may be slower than one in common use. This requirement for reliable, tested methods, which can be applied with confidence will lead to the continuing development of methods based on wet oxidation, followed by solvent extraction when pre-concentration is required, with the final measurement being made by flame spectroscopy or ICP-OES.

Future trends In the immediate future, ‘referee’ methods are likely

to evolve as mentioned above. However, for routine analysis the emphasis will be on more rapid methods to cope with increasing sample loads. It is reasonable to speculate that acid extraction methods will generally replace wet oxidation and dry-ashing methods for routine analysis.

The increasing interest in metals in nutritional studies will lead to increasing demands for analysis at low levels, particularly as some workers feel that most, if not all, metallic elements may be essential at very low levels30. These requirements will be met mainly by methods based on acid extraction followed by ETA with minimum sample handling. Such methods have not been widely evaluated and this will be a task for validating bodies, such as the Analytical Methods Committee, in the near future.

A further area of growing interest is speciation of metals to supplement the total metal figure. This is a complicated field, for which methodology is not available except for simple cases, such as arsenic which is readily fractionated into A.+**), As(“), ‘methyl arsenic’ and ‘dimethyl arsenic’, using hydride evolution AAS (Ref. 3 1). A more general solution to this problem is likely to arise by using selective solvent extraction, possibly following enzymolysis. These extracts would then be analysed using either conventional AAS, with ETA for low levels, or possibly using a hybrid technique, such as HPLC-ICP-OES.

Although the analysis of heavy metals in foods has been extensively studied, current interest in the subject still appears to be on the increase and many developments can be expected over the next few years.

References 1 Analytical Methods Committee (1930) Anllryst (London) 55, 102 2 Analytical Methods Committee (1935) Analyst (London) 60,541 3 Analytical Methods Committee (1954) Anulyst (London) 79,397 4 Analytical Methods Committee (1959) An@st (London) 84, 127 5 Analytical Methods Committee (1960) Anulyst (London) 85,629 6 Analytical Methods Committee (1963) An&t (London) 88,253 7 Analyticai Methods Committee (1965) Analyst (London) 90,5 15 8 Analytical Methods Committee (1967) AnalyJt (London) 92,320 9 Analytical Methods Committee (1968) Analyst (London) 93,414

10 Analytical Methods Committee (1967) AnulyJt (London) 92,324 11 Analytical Methods Committee (1969) An&t (London) 94,1153 12 Analytical Methods Committee (1975) Analyst (London) 100,54 13 Analytical Methods Committee (1973) Analyst (London) 98,458 14 Analytical Methods Committee (1975) Analyst (London) 100,899 15 Analytical MethodsCommittee (1975)Analyst (London) 100,761 16 Analytical Methods Committee (1979) Analyst (London) 104,

1070 17 Analytical Methods Committee (1979) Ann&t (London) 105,66 18 Analytical MethodsCommittee (1979)Anuyst (London) 104,778 19 Analytical MethodsCommittee (1983)Analyst (London) 108,109 20 Jackson, C. J., Porter, D. G., Dennis, A. L. and Stockwell, P. B.

(1978) Analyst (London) 103, 314 21 Gorsuch, T. T. (1959) Analyst (London) 84, 135 22 Analytical Methods Committee (1959) Anu&st (hndon) 84,2 15 23 Analytical Methods Committee (1960) Analyst (London) 85,643 24 Analytical Methods Committee (1967) Analyst (London) 92,403 25 Analytical Methods Committee (1976) AnalyJt (London) 101,62 26 Reamer, D. C. and Veillon, D. C. (1981) Anal. Chem. 53, 1182

28 trends in analytical chemistry, vol. 3, no. 1, 1984

27 Simpson, G. R. and Blay, R. A. (1966) Food Trade Rev. p. 35 28 Dabeka, R. W. and McKenzie, A. D. (1981)J. Assoc. Off Anal.

Chem. 64, 1297 29 Watson, C. A. (1969) Monograph No. 73, Hopkin and Williams

Ltd 30 Schwarz, K. (1977) Clin. Chem. Chem. Tech. Metals 3 3 1 Howard, A. G. and Arbab-Zavor, M. H. (1981) Analyst 106,2 13

Colin Watson is currently selfemployedfollowing over ZOyears’work, mainly in the field of trace metal analysis, at Hopkin and Williams, where he was

De/rug Manager of the laboratories. He has been active in the analysis of organic materials for trace metals for

many years, being a member of the Metallic Impurities in Organic Matter sub-committee of the UK Analytical Methods Committee, since 1966 and Chairman since 1978. He has been a member of the ‘Trace Metals Speciution ’ sub-committee, since its inception in 1980 and has contributed to the work of various other organizations, which have published methods for the determination of many elements in waters and chemicals as well as in

foodstuffs.

Application of ion-selective electrodes in flow analysis

Continuous flow techniques offer one of the most effective approaches to obtaining analytical data on a large scale and monitoring individual corn

p” nents

in industrial effluents. in biological systems or

he segmented flow methods developed by Skeggsl and the continuous flow methods, including liquid chromatography, which have been developed in the last 10 years are especially noteworthy.

E. Pungor, K. T&h and A. Hrabeczy-Pall Budapest, Hungary

Continuous flow analysers have a number of advantages over batch analysers, especially as they incorporate almost no moving mechanical parts, valves or dilutors and thus need less maintenance. Moreover, the uniform handling of the analyte and standards in the flow-through channel, and the controlled disper- sion ensure high sample throughput and good precision.

Currently available methods of continuous flow analysis (CFA) can be classified as follows: l Segmented CFA l Non-segmented CFA

- flow-injection*; - flow-titration3*4.5; - flow-dilution6

The operating principles of the non-segmented CFA systems are illustrated in Fig. 1.

In the flow-injection system (a), a ‘plug’ of the analyte or reagent solution is injected into a continuous flow of solution, which is transported to the flow-through detector cell where the transient detector signal v. time curve is recorded. The signal v. time curves can correspond to the concentration of the analyte, reagent, or reaction product.

In flow-titration (b) the sample or reagent is introduced continuously, or according to a predeter- mined program, into a stream of reagent or sample, thus ensuring either a constant reagent to sample ratio (‘single point titration’ or ‘titration to the end point’), or continuous variation of the ratios with time, which produces a complete titration curve. The addition program applied may be linear, an isosceles triangle, or an exponential.

01659936/84/$02.00.

In the flow-dilution system (c) a constant volume reactor is included. The liquid in the reactor is diluted continuously and the concentration of the solution leaving the reactor is measured with the help of a flow-through detector. As the concentration of the solution leaving the reactor changes exponentially with the volume of the diluting solution passing through it, the method is suited to the direct recording of EMF v.

p I

I FD

0 C

W

P

(4

R @I_ Pr FD

s 8---+- W

I I (b)

FD

Fig. 1. General pattern of non-segmented CFA devices (a) Flow-injection system: C, carrier solution; P, pump; I, injection point; FD, flow-through detector; W, waste. (6) Flow-titration system: R, reagent; S, sample; P, pump; Pr, programmer unit; FD, ftow-through detector; W, waste. (c) Flow-dilution system: S, sample; D, dilutent; P, pump; V, valve; G, gradient chamber; FD, flow-through detector; W, waste.

0 1984 Else&r Science Publishers B.V.