Agriculture Reference
In-Depth Information
to scratch them. When using cuvettes that have not been used before, they
should be tested to make sure that they are all the same. This is accomplished
by inserting them into the spectrophotometer and noting their absorbence. All
should be the same. Keep in mind that empty cuvettes will have a higher
absorbence than when filled with water. This is because light is refracted at
each surface and when filled with water there is less refraction at the surfaces.
8.8.1.
Zeroing and Blanks
When using a spectrophotometer for a colorimetric analysis, both the 0 (
A)
and 100% (0A) readings must be set. Once the instrument has warmed up,
with nothing in the sample compartment, the readout is set to 0. A blank, a
solution containing all the components used in the analysis except the analyte
being measured, is placed in a cuvette and placed in the sample compartment,
and the instrument is adjusted to 100%. This procedure is intended to account
for all interferences that may be introduced into the measurement by com-
ponents other than the analyte of interest. Once the instrument is adjusted,
determination of the absorbence of standards and samples can be made.
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8.8.2.
Relating Component Concentration to the Original Sample
Performing an analysis requires the preparation of a standard or calibration
curve. A series of standard solutions, a minimum of three, containing known
amounts of the component of interest are prepared. There are two primary
restrictions on these solutions: (1) they should be over the range of the
expected concentrations of the component of interest and (2) all results for
the extracted component must be higher than the lowest standard solution and
lower than the highest standard. If a result is beyond these limits, its concen-
tration cannot be determined. In the table of data for Figure 8.9, Unk1
(unknown 1) is 0.05, which is below the lowest standard, specifically, 0.15, and
so its value cannot be determined. Likewise, the Unk2 value is 0.97, which is
above the highest standard and also cannot be determined.
If, as with Unk 2, the value is above the highest standard, then the sample
may be diluted and retested. In this case the concentration must be corrected
for the amount of dilution. Similarly, in some cases when the concentration of
an unknown is lower than the lowest standard, the solution can be concen-
trated and reanalyzed.
Furthermore, standards must be over a range where there is a direct or
straight-line relationship between the amount of color produced and the
amount of component present. It is common for data points beyond the stan-
dard curve to be part of a different straight line or simply not measurable
because the solution is too light or too dark. In Figure 8.9 the distance between
points 0,0 and 10,0.15 may form a line very different from the one shown in
the graph. We know nothing about this region. The same can be said about the
region above points 90,0.95. With many instruments very low absorbances are
