Calibration Correction Factor (CCF) for Laser-Induced Fluorescence (LIF) Calibration


日付: 04/23/2019
カテゴリー: PA 800 Plus Pharmaceutical Analysis System

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For research use only. Not for use in diagnostic procedures.


Answer

The Calibration Correction Factor (CCF)  for laser-induced fluorescence (LIF) calibration is the mathematical relationship between the fluorescence from a known concentration of the analyte and the associated signal detected by the instrument. Subsequent measurements are multiplied by the factor to determine the relative fluorescence unit. 
 
The CCF algorithm is our internal design and proprietary, but the top level logic is:
 
1. Calculate max step height from e-gram data (measured RFU value)
2. CCF = ([Target RFU value] * 105)/( Measured RFU value) * CCF0
 
Where the Target RFU value = Target value for the standard solution used (entered by user into the Wizard)

Measured RFU value = Change in signal between background buffer and standard

CCF0 = Current value of the Calibration Correction Factor (CCF) stored in the firmware (under communication item number 255 & 256). 
 
For bare, fused silica capillaries:
Target RFU values for a 50 nM Fluorescein in buffer test mixture are:

  • 62.2 for a 100 µm ID capillary
  • 35 for a 75 µm ID capillary
  • 15.6 for a 50 µm ID capillary
  • 2.5 for a 20 µm ID capillary
Target RFU values for a 100 nM Cy5 in water test mixture are:
  • 8.9 for a 100 µm ID capillary
  • 5 for a 75 µm ID capillary
  • 2.2 for a 50 µm ID capillary
For nCHO capillaries:
Target RFU value for a 50 nM Fluorescein in water test mixture is:
  • 7 for a 50 µm ID capillary
Once the CCF value is calculated, all subsequent measurements will be multiplied by this value. As long as run conditions, capillary I.D. and chemistries remain unchanged, the CCF calculated should remain valid.
 

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