Eppendorf BioSpectrometer fluorescence - Manual (Page 29)

29
Operation
Eppendorf BioSpectrometer ® fluorescence
English (EN)
5.3.3 Important measurement instructions
Check for each measurement:
• For plastic cuvettes: How many consecutive measurements can be reliably carried out in
the cuvette?
• Measure the cuvette blank value before the sample or standard measurements in order to
compensate the cuvette blank value in addition to the reagent blank.
• Blank results remain saved for one measuring series, but a new blank result measurement
can be performed at any time, even between sample measurements.
• The displayed absorbance and RFU values always correspond to the directly measured
values. The dilution or cuvette factor as well as background absorbances only will be
incorporated for the following result calculation (see Absorbance values on p. 87).
• The measuring result is typically displayed 2 to 3 seconds after a measurement has been
started. If a small amount of light reaches the receiver (high absorbance values or low RFU
values), the measuring time can be automatically extended by up to 9 seconds
(photometry) or 6 seconds (fluorimetry) in order to increase the precision of the
measurement.
• Observe that the measured absorbance values do not exceed the upper limit of the
photometric measuring range. In this case, reject the measuring result. The upper limit of
the photometric measuring range does not only depend on the wavelength (see
Photometric properties on p. 84) but also on the cuvette blank. Ultra-micro cuvettes with a
small diaphragm, such as
TrayCell (Hellma), may have a cuvette blank of approx. A = 1.
The available photometric measuring range is reduced by this amount. You can estimate
the cuvette blank by measuring the cuvette filled with demineralized water as a sample in
comparison with the empty cuvette shaft as a blank. The cuvette blank of the Eppendorf
μCuvette G1.0 is negligible (approximately A = 0).
Fluorimetry: An increased autofluorescence of the cuvette (typical for plastic cuvettes) may
restrict the available measuring range.
• After the measurement, remove the measuring solution completely before filling in the
next measuring solution in order to minimize carry-over. If a carry-over from one sample to
the next sample can be expected due to a high concentration difference, rinse the cuvette
between the measurements.
• If the temperature between the lamp and the ambience differs, photometric drift may
occur. Therefore a device from a colder ambience first has to be adjusted to the ambient
temperature.
Avoid quick changes of temperature. Carry out a new blank measurement for a long series
of measurements or measurements over a long period of time.