Spectrophotometric determination of a nanomolar amount of ascorbic acid using its catalytic effect on copper(II) porphyrin formation

A simple, fast and sensitive flow-injection method is proposed for the determination of nanomolar amounts of ascorbic acid in tea, urine and blood. The procedure is based on the accelerating effect of a nanomolar level of ascorbic acid on the reaction of cooper(II) with 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin, H(2)tmpyp(4+). Ascorbic acid reduces Cu(II) to Cu(I) which catalyzes the incorporation of Cu(II) into H(2)tmpyp(4+) to form Cu(II)(tmpyp)(4+). In this method two solutions, one containing ascorbic acid and H(2)tmpyp(4+) and the other containing copper(II) and acetate buffer (pH 5.0), were injected into two flowing streams of water through two sample injectors of 120 mu1 sample volume. The mixture was allowed to react in a 2 m reaction coil and the colored solution of Cu(II)(tmpyp)(4+) was monitored at 550 nm (epsilon = 2.01 x 10(4)M(-1)cm(-1)). The present method was applied to the determination of ascorbic acid in tea, urea and blood. Reducing agents such as sugars and vitamins B(1), B(2), B(6) and B(12) did not give serious errors at a concentration of 10(-6) M for the determination of 1.0 x 10(-8)M ascrobic acid. The relative standard deviation of the present method was 2.8% for the determination of 1.0 x 10(-8)M ascorbic acid. The reaction mechanism was clarified from the kinetic results of the formation of Cu(II)(tmpyp)(4+) in the presence of various concentrations of ascorbic acid, copper(II) and hydrogen ion.