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3.5.2 Absorbance Versus Concentration Calibration
3.6 Adapting Procedures to Other Spectrophotometers
3.6.1 Selecting the Best Wavelength
3.6.1.1
General steps to select the best wavelength on a spectrophotometer:
Note: Sample color
provides a good indication
of what wavelength region
to use. A yellow solution
absorbs light in the
400–500 nm region. A red
solution absorbs light
between 500–600 nm. A
blue solution absorbs light
in the 600–700 nm range.
5940022ChemAnal.fm
If absorbance values are measured, plot the results on linear graph paper. Plot
the absorbance value on the vertical axis and the concentration on the horizontal
axis.
Plot increasing absorbance values from bottom to top. Plot increasing
concentration values from left to right. Values of 0.000 absorbance units and 0
concentration will begin at the bottom left corner of the graph. A calibration
table can be extrapolated from the curve or the concentration values can be read
directly from the graph. Or determine an equation for the line using the slope
and y-intercept.
Hach procedures may be used with other spectrophotometers, if calibration
curves are made that convert absorbance to concentration. Regardless of the
spectrophotometer used, prepare the sample and calibration standards
following the Hach procedure and use the optimum wavelength used in the
Hach procedure.
To calibrate for a given analyte, a series of standards are prepared and measured
to establish the calibration curve. The absorbance vs. concentration is plotted on
linear graph paper (as in Figure 10 and Figure 11). Points on the graph are
connected with a smooth line (curved or straight). If necessary, use the curve to
make a calibration table.
When developing a new procedure, or using procedures that are sensitive to
wavelength, it is normal to select the wavelength where the instrument gives the
greatest absorbance (see Figure 13). Because Hach chemists have selected the best
wavelength for the procedures in this manual, selecting the wavelength is not
necessary in most cases.
1. Refer to the instrument manual for specific instructions for wavelength
adjustments.
2. Select single wavelength adjustment.
3. Enter a wavelength in the range of interest.
4. Prepare the sample and blank for analysis. Fill the appropriate sample cells
with the blank and the sample solutions.
5. Place the blank in the cell holder. Zero the instrument
6. Place the prepared sample into the cell holder. Read the absorbance level.
7. Increase the wavelength so it is at least 100 nm greater than the range of
interest. Re-zero as in step 5. Measure and record the absorbance of
the sample.
8. Repeat, decreasing the wavelength by 50 nm. Re-zero, then measure and
record the absorbance at each increment. Continue this process throughout
the wavelength range of interest. Note the wavelength of greatest absorbance.
(See Table 10.)
Chemical Analysis
.
Chemical Analysis
Page 47
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