658 M. Dubernet
12.3.3.1 Sampling
A pre-determined volume of sample is removed by the robotic arm from the vial in
the sampling tray and placedin the reaction cuvette.
12.3.3.2 Addition of the First Reagent
A pre-determined volume of the first reagent (R1) is taken from the reagent tray and
placed in the reaction cuvette containingthe sample. The sample and reagent are
generally mixed by a stirring mechanism.
12.3.3.3 Delay 1
The sample and reagent are allowed to react for a specified period – usually from 4
to6min.
12.3.3.4 Absorbance Measurement
The absorbance is first measured at the selected wavelength. This is the point zero
of the reaction and may be used to take the colour of the sample into account.
12.3.3.5 Addition of the Second Reagent
The second reagent (R2) is added to the reaction cuvette and this starts the reaction
leading to the formation of the coloured product.
12.3.3.6 Delay 2
The sample and reagent are allowed to react for a specified period – usually from 4
to6min.
12.3.3.7 Absorbance Measurement
A second absorbance reading measures the variation in absorbance due to the reac-
tion itself. This basic scheme may vary and thus with more sophisticated machines
readings are taken more frequently, for example every 12s. Thus the reaction curve
may be established and the initial reaction rate measured which, under precise ana-
lytical conditions, may be proportional to the concentration of the compound being
measured.
12.3.3.8 Calculations
For oenological applications, calibrations always involve the preparation of syn-
thetic wine standards containing known concentrations of the analyte. The number
of calibration standards may be variable and a greater number of calibration points
is required if the reaction curve is not linear.