BACKGROUND OF THE INVENTION

Speciation is one of the major challenges present in analytical chemistry ¹ . Of

particular interest is metal speciation. for example, while Cr(lll) is essential to

our bodies and part of our daily diet, Cr(VI), i.e., chromate, is highly toxic to

human causing gastrointestinal disorders, dermatitis, uncertain of skin and is

a suspected carcinogenic agent. Thus, the determination of trace levels of

Cr(VI), that are often below 1 ppb in natural waters and in biological fluids in

the presence of relatively high concentrations of Cr(lll) is of particular importance. Although the redox speciation of chromium has been

accomplished by separate preconcentration of Cr(lll) and Cr(VI) fractions

using chelating resins, coprecipitation, ion chromatography and solent

extraction, such procedures are obviously complicated ² . Electro-analytical

methods that are potentially sensitive for redox-specification have also been

used to determine chromate ³ . Although Cr(VI) has been preconcentrated and

determined on mercury and chemically modified solid electrodes, most of

these interfaces exhibit moderate stability and selectivity when employed in

natural samples.

SUMMARY OF THE INVENTION

According to the present invention there is provided an assay of 'extremely high sensitivity for the selective determination of Chromium (VI). There is also provided a selective electrode for such determinations based on the molecular design of the solid-liquid interface, using a self-assembled mono¬ layer. By structuring the solid liquid interface using a self-assembled monolayer (SAM), a highly sensitive electrode exhibiting speciation capabilities toward Cr(VI), was developed. Application of SAMs in electroanalytical chemistry presents a very attractive approach as a means of assembling selective electrodes. SAMs offer highly organised systems in which the solid-liquid interface can be pre-designed at the molecular level in order to acquire a modified surface with desired properties. For example, Rubinstein and co-workers ⁴ demonstrated that a mixed functionalised SAM recognised selectively Cu ²⁺ ions in the presence of other ions. More recently, the inventor presented a highly sensitive electrode for cadmium ions using ω- mercapto-carboxylic acid monolayers ⁵ .

Thus, according to one aspect, the invention relates to a highly sensitive electrode for the selective quantitative detection and determination of Cr(VI)

which comprises a gold electrode provided with a self-assembled monolayer

(SAM) of a compound of the formula

where n is zero or an integer from 1 to 18, preferred are electrodes where the

SAM is one of positively charged 4-(2-ethanethiol) pyridinium. The invention

also relates to a process for the preparation of Cr(VI)-selective gold

electrodes which comprises polishing a gold surface of an electrode and

contacting it with a dilute solution of a compound defined above, in the presence a mineral acid. A preferred mineral acid is sulfuric acid, and it is

preferred the use of a solution of the pyridinium compound of about 5 mM and

that of the sulfuric acid of about 0.1 M. Another aspect is a highly sensitive

selective assay for Cr(VI), in the presence of Cr(lll) and other cations, which

is carried out by voltammetry using an electrode as defined above. Preferably

there is used cyclic or square wave voltammetry. The assay is suitable for

determinations in the range as low as the order of 1 ppt (part per trillion, 10 ¹² ). There may be prepared a calibration curve of concentrations in the 1 ppt

range is prepared, and actual results are read with the aid of such calibration

curve. The can be carried out in the presence of Cr(lll), Cu ²⁺ , Ag\ and Fe ³⁺ ,

of a concentration greater than up to about 10 ⁷ as large than the Cr(VI)

concentration, which ions do not interfere with the accuracy of the assay.

The invention is illustrated in the following with reference to the enclosed

Figures, in which:

Figure 1 : A- Square wave voltammetry of a 4-(2-ethanethiol)pyridine modified

gold electrode (scan rate 90 mV.s' ¹ ) after a preconcentration step in solutions

Figure 2: Reflection-absorption FTIR spectrum of 4-(2-ethanethiol)pyridine

adsorbed on gold (1) and transmission spectrum of liquid 4-(2-

ethanethioDpyridine (2).

Electrodes of the invention for Cr(VI) are based on a positively charged 4-

thiopyridinium or 4-(n-alkylthio)pyridinium, (I) monolayer on a gold surface.

Pyridinium derivatives form strong and stable complexes with chromate,

suggesting that a pyridinium-based SAM would effectively extract Cr(VI),

while repelling cations, e.g., Cr(lll). Figure 1 shows a calibration curve for

Cr(VI) using a gold electrode covered with a 4-(2-ethanethiol)pyridinium, l _n-2 , monolayer ⁶ after optimizing all the parameters that control the performance of

the electrode. The monolayer was assembled upon immersing a polished Au

surface in 5 mM solution of thiol and 0.1 M H S0 ₄ for 10 min.

Preconcentration of Cr(VI) was carried out in 0.15 M NaF (pH 4.5) solution for

5 min under open-circuit potential followed by the electrochemical

determination in a chromium-free solution (0.15 M NaF, pH 7.8) by square

wave voltammetry (Figure 1). The remarkable detection limit of this electrode is Iower than 1 ppt (part per trillion, 10 ¹² ) with a relative standard deviation of

10 % (5 ppt Cr(VQ),

where n is an integer from 1 to 18,

The electroanalytical performance of such modified electrodes was studied in

detail and reveals that other cations, e.g. Cu ²⁺ , Ag ⁺ and Fe ³⁺ OO ^"5 M of each),

as well as anions such as Cl ^" . SCN ^" and Mn0 ₄ ^" do not interfere with the

determination of 8 ppt of Cr(VI). Moreover, the analysis of a sample consisting

of 0.1 ppb of Cr)VI) was not affected at all by the presence of 1000 fold

excess of Cr(lll). Finally, the analysis of two samples consisting of 10.1 ppb of

Cr(VI) and 5.05 ppb of Cr(VI) with 15 ppb of Cr(lll) by graphite fumace atomic absorption (total chromium equals 10.1 ppb and 21.0 ppb, respectively) and

by our electrode (10.03 ppb and 5.1 ppt of Cr(VI), respectively) were in

excellent agreement.

The analytical performance of the electrode seems to be a result of the

organisation of the interface as supported by its analysis using

electrochemistry, FTIR, wettability and atomic force microscopy (AFM).

Determination of the excess of surface coverage, r, by cyclic voltammetry of

monolayers composed of 2 and 4-mercaptopyridine, as well as of 1 _n=2 , shows

that 4-substituted pyridines form more densely packed arrays. Specifically, r ₍₄ - _mercaptoPyr i _d i _ne) =1 _* 04±0.09 nmol.cm ^"2 as compared to

^r ₍₂ . _mercaptopyr i _d i _ne) =0 _* 71+0.07 nmol.cm ^'2 . It should be noted that the analytical signal recorded with a gold electrode modified with 2-mercaptopyridine was

much smaller than the value that would have been obtained if the signal had

been dependent only on the excess of surface coverage. This implies that the orientation of the pyridine ring plays a significant role in complexation.

A more detailed characterization was accomplished by FTIR and AFM. Figure

2 shows the IR spectra of the pure l _n . ₂ and its monolayer. These bands are

assigned to the C-H stretching of the rings, whereas the bands at 2852 and

2923 cm ^"1 correspond to the symmetric and assymetric alkyl C-H stretching,

respectively. This indicates that the chain is oriented similarly to the chain of

alkane thiols on gold, and the ring is oriented toward the solution. In this

orientation the four C-H bands of the pyridine have only a minor dipole

moment contribution normal to the surface, and therefore are not detected

using a p-polarized incident beam with incident angle of 80°. AFM also

provides evidence of the relatively high organization of the layer. Parallel rows

with 0.42 nm spacing can be clearly seen in the unfiltered image. The AFM image as well as the IR results, match the structure (LEED pattern of (3 3x3

3)R30°) and orientation (perpendicular to the surface) which was suggested

by Hubbard ⁷ for a 4-mercaptopyridine monolayer on Ag(111).

The high selectivity of the interface toward Cr(VI) cannot be attributed only to

the positive charge of the pyridinium moiety as is indicated by the fact that a

2-aminoethanethio monolayer extracted Cr(VI) very poorly. The extraction of chromate was followed also by wettability and capacitive measurements.

Clear changes in the advancing contact angles (Δ0 = 12 ± 3) of aqueous

buffered solutions on films of l _n=2 on gold, as well as in the differential capacity

of the double layer (ΔC _di = 11.3 ± 1.8 μF.cm _-2 ) were observed upon

introducing chromate ions.

In conclusion, all these results suggest that the high sensitivity and selectivity

toward Cr(VI) are governed by the chemical and physical structure of the

monolayer. The ability to fine tune and probe in depth the interface structure

by microscopic and macroscopic tools is crucial to designing highly sensitive

and species-selective probes.

Figure Captions

Figure 1: A- Square wave voltammetry of a 4-(2-ethanethiol)pyridine modified

gold electrode (scan rate 90 mV-s ^"1 ) after a preconcentration step in solutions

containing different concentrations of Cr(VI): (1)- 0 ppt; (2)- 1.60 ppt; (3)- 4.30

ppt; (4)- 18.36 ppt and (5)- 28.28 ppt. B- Calibration curve for chromium(VI).

Figure 2: Reflection FTIR spectrum of 4-(2-ethanethiol)pyridine adsorbed on

gold (a) and transmission spectrum of liquid 4-(2-ethanethiol)pyridine (b).

SUBSTTTUTE SHEET (RULE 26)

REFERENCES

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