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M. FE DE LA TORRE Y COLS. AN. R. ACAD. NAC. FARM.

the pH of the assay (AMPSO, NaOH, pH 9.4), centrifugation and
recovery of the supernatant, which was treated as described above.

This study has been carried out using two blanks: one with Cu:PPi
at a ratio of 1:20 and other with Cu:PPi at 1:5. The choice of one or
the other depended on the expected amount of PPi in the problem
solution. Here we report the calculations for the first blank.

Software

Curve fitting by non-linear simple and multiple regression
analyses were accomplished with the QNFIT module from the
SIMFIT, program developed by Prof. W. Bardsley and distributed
freely by the author at http://www.simfit.man.ac.uk.

Statistical treatment

Each experimental condition was performed at least twice, and
all experiments were replicated at least three times independently.
Suspected outliers placed at 1.5 times the interquartile range above
the third quartile or below the first quartile were considered carefully
before being included in the analysis. Differences were considered
significant applying the Kolmogorov-Smirnoff test, and further
validated with Student’s unpaired t-test when the experimental data
were consistent with a normal distribution.

RESULTS

1. PPi forms complexes with Cu(II) in solution,
1. thereby modifying its absorption spectrum.

In aqueous solution, the presence of PPi modified the absorption
spectrum of Cu(II) in a concentration-dependent manner. Changes
in absorptivity were monitored in the interval between 240 and 330
nm, keeping the concentration of Cu(II) constant while varying the
PPi concentration.

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