Eppendorf BioSpectrometer kinetic - Manual (Page 89)

89
Evaluation procedure
Eppendorf BioSpectrometer ® kinetic
English (EN)
12 Evaluation procedure
This chapter describes the evaluation procedures available in the method programs as well as the
calculation of a dilution using the device software.
12.1 Absorbance values
Absorbance values are displayed as A
XXX
(XXX represents the wavelength). These displays always match
the directly measured values, i.e., without corrections, which are incorporated in the final evaluation, e.g.,
corrections for optical path lengths of the cuvette, or background corrections.
12.1.1 Blank
All absorbance values are always related to the last measured blank (blank). Therefore, a blank
measurement is compulsory at the start of every series of measurements and can be completed at any time
during a series of measurements. Ideally, the blank measurement should be able to compensate for any
influences on the absorbance value of the measuring solution. The blank should therefore be measured
with the same buffer that was used for the sample measurement and the same cuvette that was used to
measure the sample value – unless the cuvettes used for the blank and sample measurements are optically
aligned and thus have the same absorbance value at the measuring wavelength.
12.1.2 Background correction
Main application: Partial correction of distortions of the absorbance for nucleic acid measurements due to
turbidity in the measuring solution. For example, the absorbance at 320 nm, which should be approx. 0 A
with pure nucleic acids, is subtracted from the absorbance at 260 nm, (the measuring wavelength for
nucleic acids).
A
XXX, corrBkgr
= calculated corrected absorbance at a wavelength of XXX nm.
A
XXX
= measured absorbance at a wavelength of XXX nm.
A
Bkgr
= measured absorbance at the background wavelength.
When comparing the measuring results to the results of other photometers/
spectrophotometers, note that the values may be dependent on the bandwidth of the devices.
In the following cases the differences may be significant:
• The absorbance spectrum shows a narrow peak in the measurement wavelength.
• The measurement is carried out not at the maximum but at the edge of a peak.
Therefore, check the accuracy of the methods by measuring standards.
Bkgr XXX corrBkgr XXX
A A A
,