Appendices; Instrument Specifications; Blanking And Absorbance Calculations; Concentration Calculation (Beer's Law) - Thermo Scientific NanoDrop 1000 V3.3 User Manual

17. Appendices

Instrument Specifications

•
Sample Size: 1microliter
•
Path Length: 1 mm (with auto-ranging to 0.2 mm)
•
Light Source: Xenon flash lamp
•
Detector Type: 2048-element linear silicon CCD array
•
Wavelength Range: 220-750 nm
•
Wavelength Accuracy: 1 nm
•
Wavelength Resolution: 3 nm (FWHM at Hg 546 nm)
•
Absorbance Precision: 0.003 absorbance ( 1mm path)
•
Absorbance Accuracy: 2% (at 0.76 absorbance at 257 nm)
•
Absorbance Range: 0.02-75 (10 mm equivalent absorbance)
•
Detection Limit: 2 ng/microliter (dsDNA)
•
Maximum Concentration: 3700 ng/microliter (dsDNA)
•
Measurement Cycle Time: 10 seconds
•
Dimensions: 20 cm X 15 cm x 12 cm
•
Weight: 3 kg
•
Sample Pedestal Material of Construction: 303 stainless steel and quartz fiber
•
Operating Voltage: 12 Vdc
•
Operating Power Consumption: 6 W
•
Standby Power Consumption: 1.5 W
•
CE Approval: Units sold in Europe, Australia and New Zealand
•
UL/CSA Approval: Units sold in N. America, S. America, Asia and Africa
•
Included in system: software, compatible with Windows 2000 or XP

Blanking and Absorbance Calculations

®
When the NanoDrop ND-1000 Spectrophotometer is "blanked", a spectrum is taken of a reference material (blank) and stored in
memory as an array of light intensities by wavelength. When a measurement of a sample is taken, the intensity of light that has
transmitted through the sample is recorded. The sample intensities along with the blank intensities are used to calculate the sample
absorbance according to the following equation:
Absorbance = -log (Intensity
Thus, the measured light intensity of both the sample and of the blank are required to calculate the absorbance at a given wavelength.

Concentration Calculation (Beer's Law)

General
The Beer-Lambert equation is used to correlate the calculated absorbance with concentration:
A = E * b * c
Where A is the absorbance represented in absorbance units (A), E is the wavelength-dependent molar absorptivity coefficient (or
extinction coefficient) with units of liter/mol-cm, b is the path length in cm, and c is the analyte concentration in moles/liter or molarity
(M).
Fluorescent Dyes (Microarray Measurement)
The NanoDrop software uses the general form of the Beer-Lambert equation to calculate fluorescent dye concentrations in the
Microarray module. The table of extinction coefficients for each dye is below:
Extinction
Dye Type Coefficient
(liter/mol-cm)
Cy3 150000
Cy5 250000
Alexa Fluor 488 71000
Alexa Fluor 546 104000
Alexa Fluor 555 150000
Alexa Fluor 594 73000
Alexa Fluor 647 239000
Alexa Fluor 660 132000
Cy3.5 150000
Cy5.5 250000
Nucleic Acids
/Intensity
sample blank
Measurement
Wavelength
(nm)
550
650
495
556
555
590
650
663
581
675
)
17-1
Section 17- Appendices