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Operation
Diode Array/Multiple-Wavelength Detectors (VC-D11, VC-D12, VF-D11)
Operating Manual
Page 105
Reference Wavelength
Interfering substances can absorb in addition to the absorption of the
analyte in the sample. The absorption of the interferences adds to the
measured signal from the analyte. As a result, this can lead to errors in
the measurement of the analyte concentration. Interfering absorption
frequently results from a changing absorption of the eluent or refractive
index effects. Refractive index effects can particularly occur with
separations with gradients.
If the interfering substance absorbs over a wide spectrum range, the
effect of the interfering absorption on the measured signal can be
reduced mathematically. For this purpose, the absorption is measured at
the measurement wavelength, and at the same time at a reference
wavelength. To correct the signal, the absorbance measured at the
reference wavelength is subtracted from the absorption at the
measurement wavelength.
TIP
The use of a reference wavelength can result in additional
interference in the chromatogram.
Use the reference wavelength carefully and only in special cases. In most
cases, measurement without a reference will provide better results.
You can set the reference wavelength separately for each signal channel
and the 3D field (only available for the VC‑D11 and VF‑D11 detectors).
Guidelines
To reduce the interference effectively, select the reference wavelength
so that it meets the following requirements:
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The absorption of the interfering substance is approximately the
same at the measurement wavelength and the reference
wavelength.
If the height of the interfering absorption differs between the
measurement wavelength and the reference wavelength, the
interference is insufficient or overcompensated.
•
The sample does not absorb at the reference wavelength.
If the sample also absorbs in the spectrum range of the reference
wavelength, the measured peak height and peak area are reduced.
•
No other substances, such as co-eluting sample components, absorb
at the reference wavelength.
The absorption of additional substances at the reference wavelength
can lead to negative peaks in the signal channel.
