Eppendorf BioPhotometer - Manual (Page 46)

88
12 Calculation
Sample dilution, optical light path of the cuvette and correction A 320
See Section 12.1.
12.3 Protein with addition of reagent
Methods: Bradford, Bradford micro, BCA, BCA micro, Lowry, Lowry micro
Selection for the calculation of results:
– Absorbance
– Calculation of the concentration via factor
– Calculation of the concentration via standard
Selection for the calculation procedures via standard:
– One-point calibration
– Multiple-point calibration (standard line)
– Multiple-point calibration (standard curve)
Calculating the concentration via factor and calculating the concentration via standard
(one-point calibration)
See Section 12.2; Measuring wavelength: 595 nm (Bradford; Lowry) or 562 nm (BCA)
Calculating the concentration via standard (multiple-point calibration; calibration line)
A calibration line (concentration as a function of the absorbance) is calculated from 2 to 10 standards, which
are measured in single, double or triple determination. The equation of the line is calculated via linear
regression.
C = a
o
+ a
1
A
a
1
= Slope of the calibration line (Factor)
a
o
= Intersection point of the calibration lines with the concentration axis
(concentration of a sample with the absorbance "0" [Offset])
After the calibration has been calculated, the CV value (coefficient of variation in "%") is calculated (exception:
two-point calibration with single determination of the two standards). The CV value is a measure for the
scattering of the measured values around the calculated calibration line. If the value is greater than 10 %, the
calibration is not stored automatically; it must first be confirmed by the user. In the case of more than two
standards, the CV value always appears in the display (even when the value is lower than 10 %).
The calculated parameters ("a
o
" and "a
1
") of the stored calibration line can be printed out by calling up the
functions list by pressing the key.
Calculating the concentration via standard (multiple-point calibration; calibration curve)
A calibration curve (concentration as function of the absorbance) is calculated from 5 to 10 standards
measured in single determination or from 4 to 10 standards measured in double or triple determination. The
non-linear regression is calculated via a third-grade polynomial.
C = a
o
+ a
1
A + a
2
A 2 + a
3
A 3 + . . .
a = Coefficients (The coefficients are determined using the least square method).
CV value: see above (linear regression).
The calculated parameters of the stored calibration line can be printed via the key.
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Function
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Function
12_Auswertg_e.fm Seite 88 Montag, 20. Februar 2006 11:34 Uhr