A bis-azo dye as a chromogenic reagent for determining traces of copper in foodstuffs, blood sera and body tissues

: A newly synthesized bis-azo dye, 2,6-bis(l-hydroxy-2-naphthylazo)pyridine has been found to be a very sen sitive and highly selective reagent for determining copper. Beer's law is followed upto 1.6 ppm with an optimum con centration range of 0.25-1.0 ppm of Cu 11• Sandell's sensitivity was calculated to be 0.0012 Jlg cm· 2 witb molar absorp tivity of 5.2 x 10 4 dm mol-1 cm-1 at 570 nm. Copper has been determined with the reagent in milk, food-grains, tea leaves, human blood sera and body tissues.


Results and discussion
The reagent and its colour reactions : It is evident from the literature that an azo dye of anaphthol can be obtained by reacting 1,2-naphthoquinone with an aromatic hydrazine 3 • 4 . Thus a bis-azo dye was obtained by reacting 2 moles of 1,2-naphthoquinone with 1 mol of 2,6-dihydrazinopyridine. Its infra-red spectrum confirmed that the compound obtained has an enol-form. The dye showed a light orange colour upto pH 9.0. Below pH 1.5, a hypsochromic shift of 5 nm was observed but a bathochromic shift of 43 nm was seen above pH 9.0. The molar extinction coefficients (E, dm mol-1 cm-1 ) of the dye at different pH values are 1.8 x 10 4 (480 nm, pH 0.9), 1.9 x 1o4 (485 nm, pH 6.0) and 2.7 X 10 4 (528 nm, pH 11.0) The pKNH and PKou values are 2.2 ± 0.2 and 19.16 as determined spectrophotometrically.
The ethanolic solution of PBN gave very deep colour with a number of metal ions at different pH values i.e. deep blue to violet colour with zinc(n), cadmium(n), mercury(n), copper(n), silver(I), cobalt(II), nickel(n), manganese(II) in neutral to alkaline media; violet colour with iron(II), vanadium(v) and thalium(I) in alkaline media; green colour with palladium(II) in neutral to alkaline media and a pink colour with chromium(m) in alkaline media. In all these colour reactions the ethanol concentration was kept above 50% to avoid precipitation.
Preliminary studies on colour reactions of PBN with metal ions also showed that in a phosphate buffered medium, only copper(II), iron(II), cobalt(II), nickel(n) and mercury(II) gave coloured complexes. The composition of the complex formed was 1 : 1 as determined by Job's method of continuous variations. The following tentative structure of the complex of bivalent metals with the bisazo dye may be proposed as : Further investigations revealed that addition of 2% solution of sodium citrate in phosphate buffered medium decomposed all the complexes except that of copper(II), thus making the method highly selective for copper.

Spectroplwtometr.ic studies on copper(ll)-PBN complex :
Ethanolic solution of PBN gave a dark blue complex witll copper(II) in neutral to alkaline medium (containing not less than 50% ethanol to avoid precipitation). The maximum and stable absorbance was found at pH 7.0 to 8.5 and phosphate buffer (2 ml, pH 7.6) was found to be suitable to determine copper selectively. The maximum colour intensity was found for Cu 11 : reagent :: 1 : 5 in molar concentration.
Samples of milk, food-grains, tea leaves, body tissues, eye lenses and blood sera were prepared by using the reported procedure 1 -3 . Milk was procured from the local dairies and biological samples were procured from the Medical College & Hospital, Rohtak.

Procedure:
An aliquot containing 6-25 J..Lg of copper(II) was taken in a 25 ml standard flask and 2 ml of 5 x w-4 M PBN solution, 2 ml of phosphate buffer solution (pH 7 .6) and 1 ml of 2% trisodium citrate solution were added to it. The total volume was made upto 25 ml keeping 50% (v/ v) ethanol. The absorbance was recorded after 2 min at 570 nm against a reagent blank.
Copper(ll) in foodstuffs, blood sera and body tissues etc. : The procured samples were analyzed for copper following the procedure under study. The samples were also analyzed for copper using an atomic absorption spectrophotometer. The results (Table 1) reveal that PBN can successfully be used to determine copper(II) ions in diverse samples.