- Email: [email protected]
Applications of polarographic and voltammetric analysis in the fields of agriculture and alimentation
155
trends in analyiical chemistry, vol. 4, no. 6, I985
Chem., 55 (1983) 1987. 4 P. Coffey, D. R. Mattson and J. C. Wright, Am. Lab., 10 (1978) 126. 5 J. A. De Haseth and T. L. Isenhour, Anal. Chem., 49 (1977)
H. Muller and Y. Moschetto, Analusis, 13 (1985) 133. 11 M. Deveaux, J. P. Huvenne, G. Fleury and P. H. Muller,
1977. 6 S. R. Lowry and D. A. Huppler,
Marc Deveaux obtained his degree in Chemistry at the University of Lilli in 1978. Since 1981, he is Assistant in Toxicology and Forensic Mkdicin, Lilli, France and Assistant in Nuclear Medicin at the University Hospital of Lilli. His current research interest is the detection of drugs of abuse in biological extracts.
Anal. Chem., 53 (1981)
889. 7 J. P. Huvenne, M. Deveaux, G. Fleury, Ph. Tran Van KY, and P. H. Muller, Xth Meeting of Z.A. F.S., Oxford, September 18-25, 1984. (Abstract in J. For. SC. Sot., 24 (1984) 328). 8 G. H. Draffan, R. A. Clare, D. L. Davies, G. Hawfsworth, S. Murray andD. S. Davies, J. Chromatogr., 139 (1977) 311. 9 M. T. Romon, B. Lacroix, J. P. Huvenne and G. Fleury, Z.T.B.M.,
3 (1982) 704.
10 M. M. Idilbi, J. P. Huvenne,
G. Fleury, Ph. Tran Van Ky, P.
Xth Meeting of I.A.F.S., Oxford, September (Abstract in J. For. SC. Sot., 24 (1984) 328).
18-25,
1984.
Jean-Pierre Huvenne is Ma&e Assistant at the Faculty of Pharmacology in Lille. He obtained his Ph. D in 1979 at the University of Science in Lille. Since 1980, he works in the Centre Universitaire de Mesure et d’Analyse of the Vniveristy of Lille II. His research interests are in the transferring of physical technologies to the biology.
Applications of polarographic and voltammetric analysis in the fields of agriculture and alimentation P. Nangniot Gembloux, Belgium
About 1970, the appearance on the market of inexpensive differential-pulse polarographic instruments brought about a considerable revival in the electrochemical analysis of the elements that effect the growth of plants and animals. Mineralisation techniques, however, still play an essential role which is often neglected. A great number of organic compounds is polaroactive, e.g. pesticides, vitamins, mycotoxins, antioxidants, alkaloids etc., and can be determined by electrochemical analysis. In addition, since the development of high-performance liquid chromatography (HPLC), electrochemical detectors (especially amperometrics) have proved to be by far the most sensitive, and among the most selective, detectors. Determination of trace elements Mineralisation
Except in the case of certain fertilizers, natural waters and beverages, it is first necessary to mineralize the organic matter present in the sample subjected to analysis (e.g. animal and plant tissues, soil, biological material). Mineralisation can take place in three different ways: (1) Calcination in a muffle furnace at 500-550°C for 12 h (the dry way). (2) Mineralisation by a mixture of 65% (w/v) nitric 0165-9936/85/$02,00.
acid-70% (w/v) perchloric acid (l:l, v/v) (the wet way). (3) Calcination in a cold plasma: low-temperature asher (LTA). We have compared these three methods of mineralisation over many years, in order to determine the trace elements. A statistical analysis of the results of these experiments shows that the least favourable process is method 1 (dry calcination). Method 2, wet mineralisation by strong acids, is a very good choice in most cases. The ideal solution is given by the lowtemperature asher (method 3). This method uses a ‘cold’ plasma, formed by a mixture of molecular, atomic and ionized oxygen produced by a high-frequency generator (13.56 MHz, 250 W) in vacua (1 mmHg). The temperature in the calcination chamber is always lower than 130°C. This is the preferred method for the determination of volatile elements, especially arsenic, antimony, bismuth and selenium, providing that the treatment is not longer than 1-2 h, which is only possible when the sample is small (0.1-0.2 g) and easily mineralized. Determination
of trace elements
in plants and foods
Elements which are easily determined using differential pulse polarography (DPP) are: Cu, Pb, Cd, Ni, Co and Zn. The procedure is as follows. Dry material (1 g) is ground and mineralized by 10 ml of the nitric acid-perchloric acid mixture. The temperature must not rise above 200°C. After mineralisation, the sample is evaporated to dryness. The whole operation is schematically depicted in Fig. 1. OElsevier
Science Publishers B.V.
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trends in analytical chemistry, vol. 4, no. Q, 1985
In polarogram B, the peak of cobalt overlaps with that of zinc, but the interference is negligible, at least in the biological environment. This mode of operation is simple and permits a rapid determination of the above-mentioned elements. The limit of detection is cu. 0.01 ppm. Molybdenum is determined in the form MOO:-, making use of its catalytic properties in the presence
Dry
matter
lg HNO3 - HClO4 (IOml)
t Dry
residue
I
HCI
3N
(IOO~C)
Trace
elements
Precipitate Na2 tartrate NH40H -
10%
(pH9)
Dlthizone
0.02%
(CHCl3)
Dithlzonates
of 0.05 M hydrogen peroxide. This results in the following reaction mechanism. O+neGR R+Z=O
(I) (2)
In reaction 2, Z represents hydrogen peroxide. The chemical reoxidation of R to 0 is expressed by a considerable increase in the classical wave 0 G R. With 0.5 M sulphuric acid, the limit of detection is 0.02pglml. Molybdenum is extracted by dithiol. The green precipitate, corresponding to the formation of the molybdenum-dithiol complex, is extracted by chloroform. The chloroform is evaporated and the residue is mineralised by the nitric acid-perchloric acid mixture. The residue is dissolved in 1 ml 4.5 M sulphuric acid. Hydrogen peroxide is then added (2.5 ml in 5 portions). The sample is transferred into a 25ml volumetric flask and water is added to volume. Polarograms are recorded by classical polarography, between 0.0 and 0.40 V. Mineralisation of selenium is realised (without loss) by the nitric acid-perchloric acid mixture. Selenium (in the form of Se4+) is reduced by ascorbic acid. A precipitate of elementary selenium is formed, which is redissolved in a solution of bromine-hydrobromic acid. The excess bromine is removed by passing nitrogen gas through the solution. The peak obtained by DPP is situated at cu. -0.35 V (vs. a saturated calomel electrode, SCE). The limit of detection is 0.02pglml of selenium. More recently, methods based on the formation of a piaselenol have been developed; a piaselenol is
Glossary HN03-
E p (S.C.E.) (1) (2) (3) (4):
(2-3ml)
: - 0,ll v : - 0,34 v : - 0.55 v - 0.94
v
(5). - I,22
v
(6):
V
-1,17
H’S104
DPP
differential pulse polarography. Since 1970 the most used polarographic method. For the principles of the method see ref. 5.
Catalytic wave
in this kind of wave the chemical reaction (2) proceeds parallel to the electrochemical reaction (l), see Determination of trace elements in plants and foods.
Kinetic wave
in this kind of wave, the chemical reaction precedes the electrochemical reaction (e.g. acetaldehyde).
I
I
Evaporation
HCI
to dry
0,lN
(5ml) (Polarographlc
cell)
i Cu-Pb-Cd (1) (2)(3)
Polarogram
A
NH40H I NI -Zn-Co (4) (5)
(0,5ml) Polarogram Dlmethylglyoxlme
B. 1%
(0,lml) 1 Ni-Co (6)
Fig.
1. Scheme
lead, cadmium,
of the polarogruphic nickel,
zinc and cobalt.
Polarogram
determination
C.
of copper,
peak potential vs. SCE.
trenakin analytical chemistry,
vol.
4,
no.
6,
formed by the reaction of Se4+ with 3,3’-diaminobenzidine. The complex is examined by cathodic stripping voltammetry, and the second peak is then measured (E,, = -0.55 V vs. SCE in 0.2 M hydrochloric acid). The sensitivity is excellent: 3-4 ppb. It is also relatively easy to determine arsenic and antimony, and very easy to determine iron and manganese. The latter two are determined in a solution consisting of 0.2 M triethanolamine and 2 M sodium hydroxide. If the proportion of the above elements is very small, it is first necessary to form an amalgam with mercury, and then use the technique of anodic stripping voltammetry. Determination of trace elements in water
For drinking water there is no problem of mineralisation. The elements most easily detected, and among the most toxic, are lead and cadmium. In 0.1 M hydrochloric acid the Ep of cadmium is -0.55 V. Usually, anodic stripping voltammetry is used. Copper (EP = -0.38 V), nickel (EP = -0.94 V) and zinc (F,, = -1.22 V) can be determined equally easily by this technique, in 0.1 M ammonium hydroxide-ammonium chloride. Iron (as Fe3+) and manganese (Mn2+) are determined in a solution of triethanolamine-sodium hydroxide
157
I985
.
For beverages, there is only one problem, which is easily solved: the mineralisation of organic compounds. The liquid (juices, wine, beer, etc.) is evaporated to dryness and the residue treated with the nitric acid-perchloric acid mixture. The sample is again evaporated to dryness and the rest of the procedure is the same as that for drinking water. Other water samples are often contaminated by sludges. These are removed by decanting in an Imhoff cone. If the resulting water sample contains organic matter, either in solution or as a colloidal suspension (particle size less than 1 pm), the water is evaporated to dryness in the presence of a little concentrated nitric acid. The residue is mineralised by the nitric acid-perchloric acid mixture. Chromium, as chromate, is examined in those effluents arising from the galvanic industry. Chromates are easily determined by DPP in a basic solution of 0.1 M sodium hydroxide (EP = -0.85 V). It should be noted that atomic absorption spectrometry cannot be used to determine chromate, since this not only detects chromate but also all other forms of chromium present in water, especially Cr3+, which is not toxic at all. Determination of trace elements in soils and fertilizers
These two kinds of samples are the most difficult to analyse. In soils, usually more or less significant
quantities of mineral silicates are encountered (mostly clays), which adsorb the trace elements. In these cases, a special kind of mineralisation is necessary: first nitric acid, followed by hydrogen fluoride and perchloric acid (NFP mineralisation). Copper and zinc can be determined after extraction with dithizone at pH 9-9.5. Molybdenum, important for its antagonistic effect on copper, is extracted selectively by dithiol in perchloric acid solution, which enables the separation from tungsten. TABLE I. Influence of the mineralisation termination of copper and zinc in soil.
technique on the de-
Sample
Total amount of copper after mineralization (ppm)
Total amount of zinc after mineralization (ppm)
NPa
NFPb
NPa
NFPb
21.4 12.4
26.7 27.8
89.9 57.2
93.1 76.1
1 2
a Mineralisation by 65% nitric acid-70% perchloric acid (1:l). b Pretreated by 65% nitric acid followed by mineralisation by 70% perchloric acid-40% hydrofluoric acid (1: 1).
Table I shows that absorption of trace elements (copper and zinc) by silicates cannot be neglected. In non-phosphate-containing fertilizers, the determination of trace elements poses no particular problems. An aqueous or acidic solution enables the biogenie elements (iron, manganese, zinc, molybdenum, copper, nickel) to be determined, using supporting electrolytes, as described above, and without the risk of post-precipitation. Cobalt is determined via the special solution, described above. The case of the phosphate-containing fertilizers is much more delicate. To avoid precipitation of phosphates it is necessary to use very acidic solutions (i.e. pH < 3). Although the determination of copper, molybdenum, lead and cadmium remains easy, nickel, cobalt, zinc and manganese are extremely difficult to determine. Indeed, the precipitation of phosphates may be prevented or delayed by complexation (by tartrates and citrates in alkaline solution, for exampie), but the procedure cannot be recommended. Determination of trace elements in biological materials
The elements mainly covered in the literature are: copper, lead, zinc, selenium, cadmium, cobalt and manganese. The mineralisation of blood, by the wet procedure 2, can be performed without difficulty. In the case of serum (or whole blood), 2-3 ml of serum (blood) is decomposed by lo-15 ml of the classic nitric acid-perchloric acid mixture. The mineralisation is complete in cu. 10 min, and after evaporation
158
trends in analytical chemistry,
to dryness one is again on familiar ground (see above). For urine samples, the common procedure is to evaporate 50 ml of urine in a porcelain dish in the presence of a few milliliters of nitric acid. This is repeated twice. The mineralisation is completed by means of the nitric acid-perchloric acid mixture. A white precipitate of potassium perchlorate is observed. After filtration the clear filtrate is evaporated to dryness and the element(s) is (are) determined in the usual way. In the cases of lead and cadmium, direct anodic stripping voltammetry can be used on urine acidified by hydrochlorid acid. The concentrations of lead and cadmium are then determined by the standard addition method. Determination of pesticides Although gas-liquid chromatography (GLC) is still the most-used technique for the determination of pesticide residues, polarography is the preferred method for a number of compounds. HPLC with electrochemical detection is gaining in importance in this field, and is already an important method in the biomedical field. We will present here the most important families of pesticides. Q&one
derivatives
Quinones can be reversibly reduced. Determination of traces of tetrachlorobenzoquinone and 2,3dichloro-1,6naphtoquinone is easily achieved with a solution of 0.1 M sodium tartrate in water-dioxane (50:50).
0
1; r:3 N--SCC13
0
I
HgS+2H++2e-
+ 2 H+ + 2 e- -_, 2 R-C-SH
II S
exhibit similar beEthylene-bis-dithiocarbamates haviour to that of dithiocarbamates, but oxidation is less reversible at a more negative potential. Thus it is possible to determine simultaneously, in a strongly alkaline solution, TMTD and ethylene-bis-dithiocarbamates. Derivatives of phtalimide
Phaltan (N-[(trichloromethyl)thio]-phtalimide, structure I) is hydrolysed in alkaline solution.
1: a
cooCO-NH-XC,,
+ Hg + H,S (E, = -0.54 V)
Captan, N-[(trichloromethyl)thio]-4-cyclohexene1,2-dicarboximide, gives an almost similar reaction, its hydrolysis being merely somewhat slower. Difolatan (N-[(1,1,2,2-tetrachloroethyl)thio]4-cyclohexene-1,2_dicarboximide) is resistant to hydrolysis by sodium hydroxide, but can still be determined at the trace level by condensation with glycine in 1 M sodium hydroxide (EP = -0.58 V). Nitroderivatives
All nitro derivatives give excellent reduction peaks in acidic, neutral or alkaline solution. Determination of their residues is very easy, see Table II, as the potentials of the peaks are not far from the maximum electrocapillary conditions favourable for determination at the trace level. TABLE II. Best electrolyte supports for certain nitro derivatives. The figures in parentheses represent the peak potentials. (V). Compound
Pendimethalin Dinoseb
0.05 M hydrochloric acid
Acetic acidsodium acetate (0.025 A4)
Buffer pH 7
Ammonium hydroxideammonium chloride (0.025 M)
(-0.24) (-0.49) (-0.24) (-0.48)
(-0.46) (-0.82) (-0.48) (-0.82)
(-0.48) (-0.90) (-0.48) (-0.84) (-0.53)
(-0.23)
(-0.45)
(-0.43)
(-0.16)
Dinoterb
R-C-S-S-C-R
OH- -
After 50 min, the solution is rendered acidic by the addition of hydrochloric acid. A polarogram is then recorded, corresponding to the reduction of sulphur:
Sulphur derivatives
Among these compounds are bis(dimethylthiocarbamoyl)disulfide (TMTD), dithiocarbamates and ethylene-bis-dithiocarbamates. TMTD produces a reduction-adsorption wave in acidic, neutral or slightly alkaline solution:
+
vol. 4, no. &,I985
Bifenox Nitrothalisopropyl
Phosphoric esters
This group is amply studied by oscillopolarography. The most commonly used solutions are: hydrochloric acid, potassium chloride and sodium hydroxide, all at a concentration of 0.1 M in ethanol-water, methanol-water or acetone-water (50:50). All the procedures described can be transposed to DPP, but without much advantage. Oscillopolarography is faster and gives much better characterization of peaks (very pointed isosceles triangles). Most of the com-
159
trenak ih analytical chemistry, vol. 4, no. f&1985
pounds of this important group have been studied by Nangniot
.
Dipyridiles
Dipyridiles are ionic compounds containing quaternary ammonium, and are used as herbicides. Diquat, paraquat and morfanquat are among the most interesting compounds of this group. They provide excellent reduction peaks in a buffer of 0.25 M acetic acid-sodium acetate. The response is linear between 0.01 and 10 ppm, and is based on the measurement of the first of the two observed peaks (-0.50 V). Applications of polarography in the analysis of beverages Determination of sulphur dioxide
The determination of sulphur dioxide is of great importance, especially in wines. It can be achieved by a simplified amperometric method, based on the principle of dead-stop end point, or by ionometry by means of a gas probe. Using polarography, sulphur dioxide can only be reduced in acidic solution, the optimal pH being around 1. A volume of 25 ml of wine is introduced into the cell, together with 1 ml of sulphuric acid (30%). Deaeration must be prevented. A reduction wave is observed, most likely corresponding to the reaction:
2 SO, + 2 e- + S,O,2(dithionite or hydrosulphite)
The observed wave corresponds to free sulphur dioxide. 35 To obtain the total amount of sulphur dioxide, I_, ml of wine is introduced into the cell and 2 ml of 10 M sodium hydroxide is added. After 10 min the cell is deaerated, 1.5 ml of 30% (w/v) sulphuric acid is added, and the polarogram is recorded. Determination of dissolved oxygen
Polarography is particularly well suited to the determination of dissolved oxygen in beverages. The determinations are always based on measuring the first or the second reduction wave of oxygen. Commercial instruments use a constant potential, generally -0.40 V, between two electrodes of different nature (often a gold cathode and a silver anode) immersed in a potassium chloride gel. This assembly is separated from the sample solution by a thin PTFE membrane, permeable to oxygen.
Determination of ascorbic and dehydroascorbic
acid
The oxidation wave of ascorbic acid was discovered in 1938. In 1953, three Japanese scientists prepared dehydroascorbic acid and showed that this acid ‘can be reduced. The reduction wave of dehydroascorbic acid is only one-thousandth of a normal diffusion wave and is of kinetic nature, i.e. the limiting current is determined by the dehydration rate of dehydroascorbic acid. Ascorbic acid is easily determined in plants and beverages. The recommended electrolyte support is an acetate buffer, pH 5.33. The anodic wave is recorded between 0 and +0.25 V. This vitamin exists almost entirely in the reduced form. On average, only 5% exists in the form of dehydroascorbic acid. In juices contained in tinplate cans, it is possible to determine the amount of vitamin C and the amount of tin simultaneously. The determination is as follows: Into a 50-ml volumetric flask are introduced successively: a 20-ml buffer, consisting of 1 M acetic acid and 1 M sodium acetate, 0.5 ml of a solution of potassium thiocyanate (100 mg/l) and 10 ml of juice. Water is added to volume. The polarogram of ascorbic acid and tin is recorded between +0.20 V and -0.7 V (vs. SCE) Wasa, Takagi and Ono made an important contribution to the problem of the determination of ascorbic and dehydroascorbic acid. Their research focussed on the kinetic nature of the reduction wave of dehydroascorbic acid, on a method of preparing dehydroascorbic acid, and on the determination of dehydroascorbic acid by the reaction with o-phenylenediamine. Depending on the concentration of the diamine, two or three reduction waves are obtained at -0.28 V, -0.45 V and -0.71 V (see Fig. 2). Determination of acetaldehyde in beer and wine Wine. Approximately 25 ml of wine is made slight-
ly alkaline and distilled. The first 5 ml of distillate contain practically all the acetaldehyde present. Lithium hydroxide or lithium chloride is used as the supporting electrolyte. The wave obtained at E+ = -1.89 V is partially kinetic, but much less than in the case of formaldehyde. The average acetaldehyde content is 0.0025% (25 ppm). In young wines the average content is even less (16 ppm). The highest concentrations are found in certain Rieslings, and are probably due to too much ventilation of the wine cellar. These specimens are in fact easily transformed into vinegar. Beer. The sample is neutralised until it is slightly alkaline. Then 0.1 M lithium chloride is added and a polarogram recorded. In this case, preliminary distillation is not necessary, since the same results are
160
trends in analytical chemistry, vol. 4, no.6 1985
in the case of benzaldehyde. Bitter almond oil. A solution of 50 mg oil in 100 ml water-ethanol (9:l) is prepared. The oil is first dissolved in pure ethanol. The solution is then diluted with 0.2 M lithium chloride. Note that benzaldehyde is found in the Japanese alcoholic drinks sake and shoyu. Wine. Benzaldehyde is obtained by steam distillation and measured in a phosphate buffer (pH 7).
n
Determination of cystine in beer
Methionine, cystine and cysteine are the only sulphur-containing amino acids. Methionine is polarographically inactive, but cystine can be easily determined in different solutions, especially in hydrolysates of proteins and in beers. Two waves are obtained at -1.70 V in BrdiEka’s solution (water containing 0.1 M ammonium chloride, 0.1 M ammonium hydroxide and 0.001 M cobalt (II) chloride) and they are catalytic in nature. Their normal height, corresponding to the classical reduction cystine + 2 cysteine, is cu. 500 times as large as the normal diffusion wave. The sensitivity of the method is therefore very high. Determination of formaldehyde Fig. 2. Differential pulse polarogram of 2.10-t M dehydroascorbit acid. Electrolyte support: solution containing 0.1 M citric acid, 0.2 M disodium hydrogen phosphate and 0.02 M o-phenylenediamine. Sensitivity, 500 nA; start potential 0.00 V; scan increment, 4 mVlsec.
in beverages
Formaldehyde produces a kinetic wave, the intensity of which is limited by the rate of dehydration of the hydrated form: H \ /OH C
H’ ‘OH
k 2 k’
H
‘C = 0 + H,O
H’
obtained as for the direct analysis case. Determination of anthocyanes in wine and juices
Similar reduction waves were observed in many fruit juices and red wines. In practice, the juice is diluted with an equal volume of 0.05 M sulphuric acid. The cathodic wave (Ed = -0.60 V) corresponds to the anthocyanes. The more positive anodic wave corresponds to sulphhydrile compounds. In cherry juice, for example, the anthocyane content differs greatly from sample to sample: from less than 1 mg% to 47.5 mg%. Green fruits do not contain any anthocyanes. Determination of benzaldehyde and wine
in bitter almond oil
Let us first recall that the polarographic behaviour of aldehydes is very specific. For aliphatic aldehydes the reduction wave is best developed in basic solutions; for aromatic aldehydes the reduction wave can be observed over the whole pH range, and is often divided into two parts to provide average values, e.g.
The value of k increases with increasing temperature and increasing pH until pH = 13. In the literature, a neutral or weakly alkaline (0.1 M lithium hydroxide) solution is recommended. The temperature must be at least 80°C to ensure that the obtained wave corresponds to the normal diffusion wave. The detection limit of this method is 0.07 pug/ml. The E, value of formaldehyde is clearly more positive than that of other aliphatic aldehydes (cu. -1.5 V in lithium hydroxide solution), and it is therefore possible to determine formaldehyde alone. For the determination in wine, 25 ml is rendered slightly alkaline and distilled. The first 5 ml of distillate contain almost all the formaldehyde present in the sample. Determination of fumaric and maleic acid in beverages
These two acids have been determined in sake. The classical ammonium hydroxide-ammonium
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trendsjn analyticalchemistry, vol. 4, no. 6, I985
chloride buffer is used. The E4 of maleic acid is V (vs. SCE) and that of fumaric acid is-l.55 V (vs. SCE).
. -1.34
Determination of fructose in wine
Only reducible monosaccharides (ketonic), e.g. fructose, give a reduction wave. Under the same conditions (e.g. in 0.1 M lithium hydroxide), glucose (an aldehyde) does not produce a detectable wave. Fructose is determined as follows: 10 ml of wine is evaporated in a water bath to eliminate aliphatic aldehydes. The residue is dissolved in 100 ml 0.1 A4hydroxide lithium (E4 = cu. - 1.8 V). Determination sf glutathion in juices
Certain kinds of fruit contain, in addition to ascorbic acid, other reducible substances that produce an anodic wave at a potential cu. 300 mV more negative than that of ascorbic acid. The more negative wave corresponds to the depolarisation of an -SH (sulphhydryl) group, probably from glutathion, which is seen mostly in tropical fruits, tomatoes and many others. In the presence of large amounts of glutathion, the E4 value is shifted to more positive values and the measurement of the height of the ascorbic acid wave becomes more difficult. In the case of juicy fruits, the determination is easily accomplished by simply squeezing the fruit, followed either directly by polarography, or after mixing with an acetic acid-sodium acetate buffer (pH 4.7). Determination of malic acid in wine and beverages
The dehydration of malic acid, resulting in fumaric acid, can be used to determine malic acid quantitatively. The juice containing malic acid is evaporated to dryness on a water bath. The residue is warmed in an oil bath at 180°C for 1 h. Dehydration then takes place: HOOC-CHOH-CH,-COOH HOOC-CH
= CH-COOH
+ + H,O
The total amount of fumaric and maleic acid formed is determined after dissolving in 0.5 A4 hydrochloric acid (E4 = -0.6 V). In wine, malic acid is determined after conversion to fumaric acid in the presence of an hydroxide, which prevents the sublimation of malic acid. In wines containing large amounts of sugars, which are converted into caramelized products, these sugars must first be eliminated by fermentation.
References 1 M. Brezina and P. Zuman, Polarography in Medicine, Biochemistry and Pharmacy, Interscience, New York, 1958. 2 R. Pointeau and J. Bonastre, Elements de Polarographie,
Masson et Cie, Paris, 1970. 3 P. Nangniot, La Polarographie en Agronomie et en Biologie, Editions J. Duculot, Gembloux, 1970. 4 J. Heyrovskjl and P. Zuman, Einfiihrung in die Praktische Polarographie, Veb Verlag Technik, Berlin, 1959. 5 A. M. Bond, Modern Polarographic Methods in Analytical Chemistry, Marcel Dekker, New York, 1980. 6 P. T. Kissinger and W. R. Heineman, Laboratory Technics in Electroanalytical Chemistry, Marcel Dekker, New York, 1985.
Paul Nangniot has been a professor at the Faculte des Sciences Agronomiques de 1’Etat in Gembloux, Belgium since 1,965. In 1951 he became an engineer in chemistry and agricultural industry, and in 1960 he received his D.Sc. degree. He is specialized in electrochemical analysis techniques and developed several polarographic methods for the determination of pesticides and race elements. He is now involved in the development of selective electrodes and agrochemical applications of HPLC with electrochemical detection.
ANNOUNCEMENT
ANALYTICA 86 Munich, F.R.G. June 3-6,1986 The 10th International
Trade Exhibition ANALYTICA and the 10th International Conference ‘Biochemical Analysis 86’ will be held at the Munich Trade Fair Center from June 3rd to 6th, 1986. ANALYTICA will once again have Exhibition Halls l-16 and the conference rooms at its disposal. The registration deadline for exhibitors is November 30th, 1985. A main feature of the exhibition this time will be bio- and gene technology. The scientific program of the International Conference ‘Biochemical Analysis 86’ (chairman: Prof. Dr. Dr. Helmut Greiling, Aachen) will be sub-divided into three sectors: - 10 symposia at which new developments in analysis will be dealt with, with regard to both methods and the various fields of application. Analytical advances in the field of gene technology will be dealt with in particular detail. - A poster exhibition featuring, as in the past, the latest results and findings in the complete range of analysis in the biological sciences. - The ‘Munich Analytica Forum’ which will provide the exhibiting industry with an opportunity for introducing and discussing their equipment and method innovations in the form of lectures as a supplement to the exhibition. Addresses for further information: For the trade exhibition: Mtinchener Messe- und Ausstelhmgsgesells&aft mbH, Postfach 12 10 09, D-8000 Miincben 12, F.R.G. Tel: (0)89/5107-O, telex: 5 212 086 ameg d. For the International Conference: Generalsekretsr Dr. Rosmarie Vogel, Nymphenburgerstr. 70, D8000 Mtinchen 2, F.R.G. Tel: (0) 89112 34 500, telex: 5 216 018 bird d.
trends in analyiical chemistry, vol. 4, no. 6, I985
Chem., 55 (1983) 1987. 4 P. Coffey, D. R. Mattson and J. C. Wright, Am. Lab., 10 (1978) 126. 5 J. A. De Haseth and T. L. Isenhour, Anal. Chem., 49 (1977)
H. Muller and Y. Moschetto, Analusis, 13 (1985) 133. 11 M. Deveaux, J. P. Huvenne, G. Fleury and P. H. Muller,
1977. 6 S. R. Lowry and D. A. Huppler,
Marc Deveaux obtained his degree in Chemistry at the University of Lilli in 1978. Since 1981, he is Assistant in Toxicology and Forensic Mkdicin, Lilli, France and Assistant in Nuclear Medicin at the University Hospital of Lilli. His current research interest is the detection of drugs of abuse in biological extracts.
Anal. Chem., 53 (1981)
889. 7 J. P. Huvenne, M. Deveaux, G. Fleury, Ph. Tran Van KY, and P. H. Muller, Xth Meeting of Z.A. F.S., Oxford, September 18-25, 1984. (Abstract in J. For. SC. Sot., 24 (1984) 328). 8 G. H. Draffan, R. A. Clare, D. L. Davies, G. Hawfsworth, S. Murray andD. S. Davies, J. Chromatogr., 139 (1977) 311. 9 M. T. Romon, B. Lacroix, J. P. Huvenne and G. Fleury, Z.T.B.M.,
3 (1982) 704.
10 M. M. Idilbi, J. P. Huvenne,
G. Fleury, Ph. Tran Van Ky, P.
Xth Meeting of I.A.F.S., Oxford, September (Abstract in J. For. SC. Sot., 24 (1984) 328).
18-25,
1984.
Jean-Pierre Huvenne is Ma&e Assistant at the Faculty of Pharmacology in Lille. He obtained his Ph. D in 1979 at the University of Science in Lille. Since 1980, he works in the Centre Universitaire de Mesure et d’Analyse of the Vniveristy of Lille II. His research interests are in the transferring of physical technologies to the biology.
Applications of polarographic and voltammetric analysis in the fields of agriculture and alimentation P. Nangniot Gembloux, Belgium
About 1970, the appearance on the market of inexpensive differential-pulse polarographic instruments brought about a considerable revival in the electrochemical analysis of the elements that effect the growth of plants and animals. Mineralisation techniques, however, still play an essential role which is often neglected. A great number of organic compounds is polaroactive, e.g. pesticides, vitamins, mycotoxins, antioxidants, alkaloids etc., and can be determined by electrochemical analysis. In addition, since the development of high-performance liquid chromatography (HPLC), electrochemical detectors (especially amperometrics) have proved to be by far the most sensitive, and among the most selective, detectors. Determination of trace elements Mineralisation
Except in the case of certain fertilizers, natural waters and beverages, it is first necessary to mineralize the organic matter present in the sample subjected to analysis (e.g. animal and plant tissues, soil, biological material). Mineralisation can take place in three different ways: (1) Calcination in a muffle furnace at 500-550°C for 12 h (the dry way). (2) Mineralisation by a mixture of 65% (w/v) nitric 0165-9936/85/$02,00.
acid-70% (w/v) perchloric acid (l:l, v/v) (the wet way). (3) Calcination in a cold plasma: low-temperature asher (LTA). We have compared these three methods of mineralisation over many years, in order to determine the trace elements. A statistical analysis of the results of these experiments shows that the least favourable process is method 1 (dry calcination). Method 2, wet mineralisation by strong acids, is a very good choice in most cases. The ideal solution is given by the lowtemperature asher (method 3). This method uses a ‘cold’ plasma, formed by a mixture of molecular, atomic and ionized oxygen produced by a high-frequency generator (13.56 MHz, 250 W) in vacua (1 mmHg). The temperature in the calcination chamber is always lower than 130°C. This is the preferred method for the determination of volatile elements, especially arsenic, antimony, bismuth and selenium, providing that the treatment is not longer than 1-2 h, which is only possible when the sample is small (0.1-0.2 g) and easily mineralized. Determination
of trace elements
in plants and foods
Elements which are easily determined using differential pulse polarography (DPP) are: Cu, Pb, Cd, Ni, Co and Zn. The procedure is as follows. Dry material (1 g) is ground and mineralized by 10 ml of the nitric acid-perchloric acid mixture. The temperature must not rise above 200°C. After mineralisation, the sample is evaporated to dryness. The whole operation is schematically depicted in Fig. 1. OElsevier
Science Publishers B.V.
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trends in analytical chemistry, vol. 4, no. Q, 1985
In polarogram B, the peak of cobalt overlaps with that of zinc, but the interference is negligible, at least in the biological environment. This mode of operation is simple and permits a rapid determination of the above-mentioned elements. The limit of detection is cu. 0.01 ppm. Molybdenum is determined in the form MOO:-, making use of its catalytic properties in the presence
Dry
matter
lg HNO3 - HClO4 (IOml)
t Dry
residue
I
HCI
3N
(IOO~C)
Trace
elements
Precipitate Na2 tartrate NH40H -
10%
(pH9)
Dlthizone
0.02%
(CHCl3)
Dithlzonates
of 0.05 M hydrogen peroxide. This results in the following reaction mechanism. O+neGR R+Z=O
(I) (2)
In reaction 2, Z represents hydrogen peroxide. The chemical reoxidation of R to 0 is expressed by a considerable increase in the classical wave 0 G R. With 0.5 M sulphuric acid, the limit of detection is 0.02pglml. Molybdenum is extracted by dithiol. The green precipitate, corresponding to the formation of the molybdenum-dithiol complex, is extracted by chloroform. The chloroform is evaporated and the residue is mineralised by the nitric acid-perchloric acid mixture. The residue is dissolved in 1 ml 4.5 M sulphuric acid. Hydrogen peroxide is then added (2.5 ml in 5 portions). The sample is transferred into a 25ml volumetric flask and water is added to volume. Polarograms are recorded by classical polarography, between 0.0 and 0.40 V. Mineralisation of selenium is realised (without loss) by the nitric acid-perchloric acid mixture. Selenium (in the form of Se4+) is reduced by ascorbic acid. A precipitate of elementary selenium is formed, which is redissolved in a solution of bromine-hydrobromic acid. The excess bromine is removed by passing nitrogen gas through the solution. The peak obtained by DPP is situated at cu. -0.35 V (vs. a saturated calomel electrode, SCE). The limit of detection is 0.02pglml of selenium. More recently, methods based on the formation of a piaselenol have been developed; a piaselenol is
Glossary HN03-
E p (S.C.E.) (1) (2) (3) (4):
(2-3ml)
: - 0,ll v : - 0,34 v : - 0.55 v - 0.94
v
(5). - I,22
v
(6):
V
-1,17
H’S104
DPP
differential pulse polarography. Since 1970 the most used polarographic method. For the principles of the method see ref. 5.
Catalytic wave
in this kind of wave the chemical reaction (2) proceeds parallel to the electrochemical reaction (l), see Determination of trace elements in plants and foods.
Kinetic wave
in this kind of wave, the chemical reaction precedes the electrochemical reaction (e.g. acetaldehyde).
I
I
Evaporation
HCI
to dry
0,lN
(5ml) (Polarographlc
cell)
i Cu-Pb-Cd (1) (2)(3)
Polarogram
A
NH40H I NI -Zn-Co (4) (5)
(0,5ml) Polarogram Dlmethylglyoxlme
B. 1%
(0,lml) 1 Ni-Co (6)
Fig.
1. Scheme
lead, cadmium,
of the polarogruphic nickel,
zinc and cobalt.
Polarogram
determination
C.
of copper,
peak potential vs. SCE.
trenakin analytical chemistry,
vol.
4,
no.
6,
formed by the reaction of Se4+ with 3,3’-diaminobenzidine. The complex is examined by cathodic stripping voltammetry, and the second peak is then measured (E,, = -0.55 V vs. SCE in 0.2 M hydrochloric acid). The sensitivity is excellent: 3-4 ppb. It is also relatively easy to determine arsenic and antimony, and very easy to determine iron and manganese. The latter two are determined in a solution consisting of 0.2 M triethanolamine and 2 M sodium hydroxide. If the proportion of the above elements is very small, it is first necessary to form an amalgam with mercury, and then use the technique of anodic stripping voltammetry. Determination of trace elements in water
For drinking water there is no problem of mineralisation. The elements most easily detected, and among the most toxic, are lead and cadmium. In 0.1 M hydrochloric acid the Ep of cadmium is -0.55 V. Usually, anodic stripping voltammetry is used. Copper (EP = -0.38 V), nickel (EP = -0.94 V) and zinc (F,, = -1.22 V) can be determined equally easily by this technique, in 0.1 M ammonium hydroxide-ammonium chloride. Iron (as Fe3+) and manganese (Mn2+) are determined in a solution of triethanolamine-sodium hydroxide
157
I985
.
For beverages, there is only one problem, which is easily solved: the mineralisation of organic compounds. The liquid (juices, wine, beer, etc.) is evaporated to dryness and the residue treated with the nitric acid-perchloric acid mixture. The sample is again evaporated to dryness and the rest of the procedure is the same as that for drinking water. Other water samples are often contaminated by sludges. These are removed by decanting in an Imhoff cone. If the resulting water sample contains organic matter, either in solution or as a colloidal suspension (particle size less than 1 pm), the water is evaporated to dryness in the presence of a little concentrated nitric acid. The residue is mineralised by the nitric acid-perchloric acid mixture. Chromium, as chromate, is examined in those effluents arising from the galvanic industry. Chromates are easily determined by DPP in a basic solution of 0.1 M sodium hydroxide (EP = -0.85 V). It should be noted that atomic absorption spectrometry cannot be used to determine chromate, since this not only detects chromate but also all other forms of chromium present in water, especially Cr3+, which is not toxic at all. Determination of trace elements in soils and fertilizers
These two kinds of samples are the most difficult to analyse. In soils, usually more or less significant
quantities of mineral silicates are encountered (mostly clays), which adsorb the trace elements. In these cases, a special kind of mineralisation is necessary: first nitric acid, followed by hydrogen fluoride and perchloric acid (NFP mineralisation). Copper and zinc can be determined after extraction with dithizone at pH 9-9.5. Molybdenum, important for its antagonistic effect on copper, is extracted selectively by dithiol in perchloric acid solution, which enables the separation from tungsten. TABLE I. Influence of the mineralisation termination of copper and zinc in soil.
technique on the de-
Sample
Total amount of copper after mineralization (ppm)
Total amount of zinc after mineralization (ppm)
NPa
NFPb
NPa
NFPb
21.4 12.4
26.7 27.8
89.9 57.2
93.1 76.1
1 2
a Mineralisation by 65% nitric acid-70% perchloric acid (1:l). b Pretreated by 65% nitric acid followed by mineralisation by 70% perchloric acid-40% hydrofluoric acid (1: 1).
Table I shows that absorption of trace elements (copper and zinc) by silicates cannot be neglected. In non-phosphate-containing fertilizers, the determination of trace elements poses no particular problems. An aqueous or acidic solution enables the biogenie elements (iron, manganese, zinc, molybdenum, copper, nickel) to be determined, using supporting electrolytes, as described above, and without the risk of post-precipitation. Cobalt is determined via the special solution, described above. The case of the phosphate-containing fertilizers is much more delicate. To avoid precipitation of phosphates it is necessary to use very acidic solutions (i.e. pH < 3). Although the determination of copper, molybdenum, lead and cadmium remains easy, nickel, cobalt, zinc and manganese are extremely difficult to determine. Indeed, the precipitation of phosphates may be prevented or delayed by complexation (by tartrates and citrates in alkaline solution, for exampie), but the procedure cannot be recommended. Determination of trace elements in biological materials
The elements mainly covered in the literature are: copper, lead, zinc, selenium, cadmium, cobalt and manganese. The mineralisation of blood, by the wet procedure 2, can be performed without difficulty. In the case of serum (or whole blood), 2-3 ml of serum (blood) is decomposed by lo-15 ml of the classic nitric acid-perchloric acid mixture. The mineralisation is complete in cu. 10 min, and after evaporation
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trends in analytical chemistry,
to dryness one is again on familiar ground (see above). For urine samples, the common procedure is to evaporate 50 ml of urine in a porcelain dish in the presence of a few milliliters of nitric acid. This is repeated twice. The mineralisation is completed by means of the nitric acid-perchloric acid mixture. A white precipitate of potassium perchlorate is observed. After filtration the clear filtrate is evaporated to dryness and the element(s) is (are) determined in the usual way. In the cases of lead and cadmium, direct anodic stripping voltammetry can be used on urine acidified by hydrochlorid acid. The concentrations of lead and cadmium are then determined by the standard addition method. Determination of pesticides Although gas-liquid chromatography (GLC) is still the most-used technique for the determination of pesticide residues, polarography is the preferred method for a number of compounds. HPLC with electrochemical detection is gaining in importance in this field, and is already an important method in the biomedical field. We will present here the most important families of pesticides. Q&one
derivatives
Quinones can be reversibly reduced. Determination of traces of tetrachlorobenzoquinone and 2,3dichloro-1,6naphtoquinone is easily achieved with a solution of 0.1 M sodium tartrate in water-dioxane (50:50).
0
1; r:3 N--SCC13
0
I
HgS+2H++2e-
+ 2 H+ + 2 e- -_, 2 R-C-SH
II S
exhibit similar beEthylene-bis-dithiocarbamates haviour to that of dithiocarbamates, but oxidation is less reversible at a more negative potential. Thus it is possible to determine simultaneously, in a strongly alkaline solution, TMTD and ethylene-bis-dithiocarbamates. Derivatives of phtalimide
Phaltan (N-[(trichloromethyl)thio]-phtalimide, structure I) is hydrolysed in alkaline solution.
1: a
cooCO-NH-XC,,
+ Hg + H,S (E, = -0.54 V)
Captan, N-[(trichloromethyl)thio]-4-cyclohexene1,2-dicarboximide, gives an almost similar reaction, its hydrolysis being merely somewhat slower. Difolatan (N-[(1,1,2,2-tetrachloroethyl)thio]4-cyclohexene-1,2_dicarboximide) is resistant to hydrolysis by sodium hydroxide, but can still be determined at the trace level by condensation with glycine in 1 M sodium hydroxide (EP = -0.58 V). Nitroderivatives
All nitro derivatives give excellent reduction peaks in acidic, neutral or alkaline solution. Determination of their residues is very easy, see Table II, as the potentials of the peaks are not far from the maximum electrocapillary conditions favourable for determination at the trace level. TABLE II. Best electrolyte supports for certain nitro derivatives. The figures in parentheses represent the peak potentials. (V). Compound
Pendimethalin Dinoseb
0.05 M hydrochloric acid
Acetic acidsodium acetate (0.025 A4)
Buffer pH 7
Ammonium hydroxideammonium chloride (0.025 M)
(-0.24) (-0.49) (-0.24) (-0.48)
(-0.46) (-0.82) (-0.48) (-0.82)
(-0.48) (-0.90) (-0.48) (-0.84) (-0.53)
(-0.23)
(-0.45)
(-0.43)
(-0.16)
Dinoterb
R-C-S-S-C-R
OH- -
After 50 min, the solution is rendered acidic by the addition of hydrochloric acid. A polarogram is then recorded, corresponding to the reduction of sulphur:
Sulphur derivatives
Among these compounds are bis(dimethylthiocarbamoyl)disulfide (TMTD), dithiocarbamates and ethylene-bis-dithiocarbamates. TMTD produces a reduction-adsorption wave in acidic, neutral or slightly alkaline solution:
+
vol. 4, no. &,I985
Bifenox Nitrothalisopropyl
Phosphoric esters
This group is amply studied by oscillopolarography. The most commonly used solutions are: hydrochloric acid, potassium chloride and sodium hydroxide, all at a concentration of 0.1 M in ethanol-water, methanol-water or acetone-water (50:50). All the procedures described can be transposed to DPP, but without much advantage. Oscillopolarography is faster and gives much better characterization of peaks (very pointed isosceles triangles). Most of the com-
159
trenak ih analytical chemistry, vol. 4, no. f&1985
pounds of this important group have been studied by Nangniot
.
Dipyridiles
Dipyridiles are ionic compounds containing quaternary ammonium, and are used as herbicides. Diquat, paraquat and morfanquat are among the most interesting compounds of this group. They provide excellent reduction peaks in a buffer of 0.25 M acetic acid-sodium acetate. The response is linear between 0.01 and 10 ppm, and is based on the measurement of the first of the two observed peaks (-0.50 V). Applications of polarography in the analysis of beverages Determination of sulphur dioxide
The determination of sulphur dioxide is of great importance, especially in wines. It can be achieved by a simplified amperometric method, based on the principle of dead-stop end point, or by ionometry by means of a gas probe. Using polarography, sulphur dioxide can only be reduced in acidic solution, the optimal pH being around 1. A volume of 25 ml of wine is introduced into the cell, together with 1 ml of sulphuric acid (30%). Deaeration must be prevented. A reduction wave is observed, most likely corresponding to the reaction:
2 SO, + 2 e- + S,O,2(dithionite or hydrosulphite)
The observed wave corresponds to free sulphur dioxide. 35 To obtain the total amount of sulphur dioxide, I_, ml of wine is introduced into the cell and 2 ml of 10 M sodium hydroxide is added. After 10 min the cell is deaerated, 1.5 ml of 30% (w/v) sulphuric acid is added, and the polarogram is recorded. Determination of dissolved oxygen
Polarography is particularly well suited to the determination of dissolved oxygen in beverages. The determinations are always based on measuring the first or the second reduction wave of oxygen. Commercial instruments use a constant potential, generally -0.40 V, between two electrodes of different nature (often a gold cathode and a silver anode) immersed in a potassium chloride gel. This assembly is separated from the sample solution by a thin PTFE membrane, permeable to oxygen.
Determination of ascorbic and dehydroascorbic
acid
The oxidation wave of ascorbic acid was discovered in 1938. In 1953, three Japanese scientists prepared dehydroascorbic acid and showed that this acid ‘can be reduced. The reduction wave of dehydroascorbic acid is only one-thousandth of a normal diffusion wave and is of kinetic nature, i.e. the limiting current is determined by the dehydration rate of dehydroascorbic acid. Ascorbic acid is easily determined in plants and beverages. The recommended electrolyte support is an acetate buffer, pH 5.33. The anodic wave is recorded between 0 and +0.25 V. This vitamin exists almost entirely in the reduced form. On average, only 5% exists in the form of dehydroascorbic acid. In juices contained in tinplate cans, it is possible to determine the amount of vitamin C and the amount of tin simultaneously. The determination is as follows: Into a 50-ml volumetric flask are introduced successively: a 20-ml buffer, consisting of 1 M acetic acid and 1 M sodium acetate, 0.5 ml of a solution of potassium thiocyanate (100 mg/l) and 10 ml of juice. Water is added to volume. The polarogram of ascorbic acid and tin is recorded between +0.20 V and -0.7 V (vs. SCE) Wasa, Takagi and Ono made an important contribution to the problem of the determination of ascorbic and dehydroascorbic acid. Their research focussed on the kinetic nature of the reduction wave of dehydroascorbic acid, on a method of preparing dehydroascorbic acid, and on the determination of dehydroascorbic acid by the reaction with o-phenylenediamine. Depending on the concentration of the diamine, two or three reduction waves are obtained at -0.28 V, -0.45 V and -0.71 V (see Fig. 2). Determination of acetaldehyde in beer and wine Wine. Approximately 25 ml of wine is made slight-
ly alkaline and distilled. The first 5 ml of distillate contain practically all the acetaldehyde present. Lithium hydroxide or lithium chloride is used as the supporting electrolyte. The wave obtained at E+ = -1.89 V is partially kinetic, but much less than in the case of formaldehyde. The average acetaldehyde content is 0.0025% (25 ppm). In young wines the average content is even less (16 ppm). The highest concentrations are found in certain Rieslings, and are probably due to too much ventilation of the wine cellar. These specimens are in fact easily transformed into vinegar. Beer. The sample is neutralised until it is slightly alkaline. Then 0.1 M lithium chloride is added and a polarogram recorded. In this case, preliminary distillation is not necessary, since the same results are
160
trends in analytical chemistry, vol. 4, no.6 1985
in the case of benzaldehyde. Bitter almond oil. A solution of 50 mg oil in 100 ml water-ethanol (9:l) is prepared. The oil is first dissolved in pure ethanol. The solution is then diluted with 0.2 M lithium chloride. Note that benzaldehyde is found in the Japanese alcoholic drinks sake and shoyu. Wine. Benzaldehyde is obtained by steam distillation and measured in a phosphate buffer (pH 7).
n
Determination of cystine in beer
Methionine, cystine and cysteine are the only sulphur-containing amino acids. Methionine is polarographically inactive, but cystine can be easily determined in different solutions, especially in hydrolysates of proteins and in beers. Two waves are obtained at -1.70 V in BrdiEka’s solution (water containing 0.1 M ammonium chloride, 0.1 M ammonium hydroxide and 0.001 M cobalt (II) chloride) and they are catalytic in nature. Their normal height, corresponding to the classical reduction cystine + 2 cysteine, is cu. 500 times as large as the normal diffusion wave. The sensitivity of the method is therefore very high. Determination of formaldehyde Fig. 2. Differential pulse polarogram of 2.10-t M dehydroascorbit acid. Electrolyte support: solution containing 0.1 M citric acid, 0.2 M disodium hydrogen phosphate and 0.02 M o-phenylenediamine. Sensitivity, 500 nA; start potential 0.00 V; scan increment, 4 mVlsec.
in beverages
Formaldehyde produces a kinetic wave, the intensity of which is limited by the rate of dehydration of the hydrated form: H \ /OH C
H’ ‘OH
k 2 k’
H
‘C = 0 + H,O
H’
obtained as for the direct analysis case. Determination of anthocyanes in wine and juices
Similar reduction waves were observed in many fruit juices and red wines. In practice, the juice is diluted with an equal volume of 0.05 M sulphuric acid. The cathodic wave (Ed = -0.60 V) corresponds to the anthocyanes. The more positive anodic wave corresponds to sulphhydrile compounds. In cherry juice, for example, the anthocyane content differs greatly from sample to sample: from less than 1 mg% to 47.5 mg%. Green fruits do not contain any anthocyanes. Determination of benzaldehyde and wine
in bitter almond oil
Let us first recall that the polarographic behaviour of aldehydes is very specific. For aliphatic aldehydes the reduction wave is best developed in basic solutions; for aromatic aldehydes the reduction wave can be observed over the whole pH range, and is often divided into two parts to provide average values, e.g.
The value of k increases with increasing temperature and increasing pH until pH = 13. In the literature, a neutral or weakly alkaline (0.1 M lithium hydroxide) solution is recommended. The temperature must be at least 80°C to ensure that the obtained wave corresponds to the normal diffusion wave. The detection limit of this method is 0.07 pug/ml. The E, value of formaldehyde is clearly more positive than that of other aliphatic aldehydes (cu. -1.5 V in lithium hydroxide solution), and it is therefore possible to determine formaldehyde alone. For the determination in wine, 25 ml is rendered slightly alkaline and distilled. The first 5 ml of distillate contain almost all the formaldehyde present in the sample. Determination of fumaric and maleic acid in beverages
These two acids have been determined in sake. The classical ammonium hydroxide-ammonium
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trendsjn analyticalchemistry, vol. 4, no. 6, I985
chloride buffer is used. The E4 of maleic acid is V (vs. SCE) and that of fumaric acid is-l.55 V (vs. SCE).
. -1.34
Determination of fructose in wine
Only reducible monosaccharides (ketonic), e.g. fructose, give a reduction wave. Under the same conditions (e.g. in 0.1 M lithium hydroxide), glucose (an aldehyde) does not produce a detectable wave. Fructose is determined as follows: 10 ml of wine is evaporated in a water bath to eliminate aliphatic aldehydes. The residue is dissolved in 100 ml 0.1 A4hydroxide lithium (E4 = cu. - 1.8 V). Determination sf glutathion in juices
Certain kinds of fruit contain, in addition to ascorbic acid, other reducible substances that produce an anodic wave at a potential cu. 300 mV more negative than that of ascorbic acid. The more negative wave corresponds to the depolarisation of an -SH (sulphhydryl) group, probably from glutathion, which is seen mostly in tropical fruits, tomatoes and many others. In the presence of large amounts of glutathion, the E4 value is shifted to more positive values and the measurement of the height of the ascorbic acid wave becomes more difficult. In the case of juicy fruits, the determination is easily accomplished by simply squeezing the fruit, followed either directly by polarography, or after mixing with an acetic acid-sodium acetate buffer (pH 4.7). Determination of malic acid in wine and beverages
The dehydration of malic acid, resulting in fumaric acid, can be used to determine malic acid quantitatively. The juice containing malic acid is evaporated to dryness on a water bath. The residue is warmed in an oil bath at 180°C for 1 h. Dehydration then takes place: HOOC-CHOH-CH,-COOH HOOC-CH
= CH-COOH
+ + H,O
The total amount of fumaric and maleic acid formed is determined after dissolving in 0.5 A4 hydrochloric acid (E4 = -0.6 V). In wine, malic acid is determined after conversion to fumaric acid in the presence of an hydroxide, which prevents the sublimation of malic acid. In wines containing large amounts of sugars, which are converted into caramelized products, these sugars must first be eliminated by fermentation.
References 1 M. Brezina and P. Zuman, Polarography in Medicine, Biochemistry and Pharmacy, Interscience, New York, 1958. 2 R. Pointeau and J. Bonastre, Elements de Polarographie,
Masson et Cie, Paris, 1970. 3 P. Nangniot, La Polarographie en Agronomie et en Biologie, Editions J. Duculot, Gembloux, 1970. 4 J. Heyrovskjl and P. Zuman, Einfiihrung in die Praktische Polarographie, Veb Verlag Technik, Berlin, 1959. 5 A. M. Bond, Modern Polarographic Methods in Analytical Chemistry, Marcel Dekker, New York, 1980. 6 P. T. Kissinger and W. R. Heineman, Laboratory Technics in Electroanalytical Chemistry, Marcel Dekker, New York, 1985.
Paul Nangniot has been a professor at the Faculte des Sciences Agronomiques de 1’Etat in Gembloux, Belgium since 1,965. In 1951 he became an engineer in chemistry and agricultural industry, and in 1960 he received his D.Sc. degree. He is specialized in electrochemical analysis techniques and developed several polarographic methods for the determination of pesticides and race elements. He is now involved in the development of selective electrodes and agrochemical applications of HPLC with electrochemical detection.
ANNOUNCEMENT
ANALYTICA 86 Munich, F.R.G. June 3-6,1986 The 10th International
Trade Exhibition ANALYTICA and the 10th International Conference ‘Biochemical Analysis 86’ will be held at the Munich Trade Fair Center from June 3rd to 6th, 1986. ANALYTICA will once again have Exhibition Halls l-16 and the conference rooms at its disposal. The registration deadline for exhibitors is November 30th, 1985. A main feature of the exhibition this time will be bio- and gene technology. The scientific program of the International Conference ‘Biochemical Analysis 86’ (chairman: Prof. Dr. Dr. Helmut Greiling, Aachen) will be sub-divided into three sectors: - 10 symposia at which new developments in analysis will be dealt with, with regard to both methods and the various fields of application. Analytical advances in the field of gene technology will be dealt with in particular detail. - A poster exhibition featuring, as in the past, the latest results and findings in the complete range of analysis in the biological sciences. - The ‘Munich Analytica Forum’ which will provide the exhibiting industry with an opportunity for introducing and discussing their equipment and method innovations in the form of lectures as a supplement to the exhibition. Addresses for further information: For the trade exhibition: Mtinchener Messe- und Ausstelhmgsgesells&aft mbH, Postfach 12 10 09, D-8000 Miincben 12, F.R.G. Tel: (0)89/5107-O, telex: 5 212 086 ameg d. For the International Conference: Generalsekretsr Dr. Rosmarie Vogel, Nymphenburgerstr. 70, D8000 Mtinchen 2, F.R.G. Tel: (0) 89112 34 500, telex: 5 216 018 bird d.