WAVELENGTH ACCURACY OF THE SPECTROPHOTOMETERS
Although the absorbance of a nucleic acid at 260 nm is generally on a plateau, the absorbance curve at 280 nm is quite steeply sloped. A slight shift in wavelength accuracy will have a large effect on 260/280 ratios. For example, a +/- 1 nm shift in wavelength accuracy will result in a +/-0.2 change in the 260/280 ratio. Since many spectrophotometers claim a 1 nm accuracy specification, it is possible to see as much as a 0.4 difference in the 260/280 ratio when measuring thesame nucleic acid sample on two spectrophotometers that are both within wavelength accuracy specification.