Beckman Coulter UniCel DxC 600, Instructions For Use Manual

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A13914AF
8-16
User Defined Reagents
Determination of Extinction Coefficients
• The shortest distance between the two selected wavelengths (without overlapping the spectral
curve) optimizes the flash characteristics of the lamp, providing better precision of the
absorbance data. It is strongly recommended that precision and correlation data be
accumulated to verify proper selection of the wavelengths as well as all of the defined
parameters.
Summary
•
A secondary wavelength closer to the primary wavelength reduces noise and increases
accuracy.
•
Obtaining the maximum absorbance difference increases sensitivity.
•
Avoid secondary wavelengths with peak spectral interferences within the primary spectral
curve.
•
Select a secondary wavelength that produces a net absorbance that is close to the net
absorbance of the primary wavelength.
Determination of Extinction Coefficients
Introduction
In general, there are two ways to determine the molar absorptivity or extinction coefficient "e" for
a given chemistry at each of the wavelengths required for analysis.
Option 1
Prepare a stock, standard solution of the chromophore. From this stock solution, prepare a working
standard solution at the same molar concentration recommended by the reagent manufacturer.
Obtain absorbance values on this working standard solution within a narrow-bandpass, manual
spectrophotometer which uses 1 cm pathlength cuvettes. Calculate the extinction coefficient for
the appropriate wavelength as follows:
This method may only be used when the chromophore is measurable in a stable form. When the
chromophore is not attainable in a stable form and may only be measured in a dynamic state, the
second method of determining extinction coefficients is suggested. For additional information,
refer to
Textbook of Clinical Chemistry , Norbert W. Tietz, 1999.
E014427L.EPS
e =
Concentration of standard sol u tion (mmol / L)
×
pathlength
Absorbance of chromophore