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How in the Nanodrop converting A260 to concentration in ng/uL?



When you are measuring the concentration of DNA at 260 nm, the software automatically compensates for the fact that you are measuring your sample at a 1 mm path length instead of the standard 1 cm pathlength. For cell culture at OD 600 nm, the software does not do the same thing. Instead it displays the 1mm absorbance. Why?


This answer was received in an email response from an Application Scientist at Nanodrop Technologies, Inc.

"Yes, the Cell Cultures module is currently displayed at 1 mm and not 10 mm.

Note: absorbance data shown in archive files are represented as displayed on the software screen. For Nucleic Acid, Protein A280 and Proteins and Labels modules, data are normalized to a 1.0 cm (10.0 mm) path. For MicroArray, UV-Vis, Protein BCA, Protein Bradford, Protein Lowry and Cell Culture modules the data are normalized to a 0.1 cm (1.0 mm) path. For high absorbance UV-Vis samples, data are normalized to a 0.1mm path.

Regarding microbial cell cultures in suspension:

The short answer: The ND-1000 can be used to estimate bacterial growth cultures, and any limitations will be similar to limitations of other spectrophotometers.

The long answer: The fundamental issue surrounding bacterial culture growth measurements is that an absorbance spectrophotometer is being asked to determine light scattering caused by particulates in suspension. In this case, transmittance is not related to absorbance in the classical sense. Under normal true absorbance conditions, spectrophotometers can be comparable to one another because the sample actually absorbs electromagnetic energy. In the case of a reduction of transmittance caused by light scattering, readings are very dependent on the optics of a specific spectrophotometer as well as the cell type in suspension. For bacterial growth determination, even 1cm path length systems can vary greatly due to the variability of the optics of each system. The ND-1000 will display an 'absorbance' value approximately 10 fold less than 1cm systems due to the fact that the instrument is utilizing a 1mm path length. However, the difference will not be exactly 10 fold due to the reasons I've described above. The point is that the differences between the ND-1000 and a 'conventional' spec (apart from the 10 fold difference due to path length), will be similar to differences found between spectrophotometers that utilize a 1cm path length."