(information below summarized from the ND-1000 user's manual)
1. Change in sample acidity
Small changes in solution pH will cause the 260:280 to vary [reference 1]. Acidic solutions will under-represent the 260:280 ratio by 0.2-0.3, while a basic solution will over-represent the ratio by 0.2-0.3. If comparing the NanoDrop ND-1000 Spectrophotometer to other spectrophotometers, it is important to ensure that the pH of an undiluted sample measured on the ND-1000 is at the same pH as the diluted sample measured on the second spectrophotometer.
2. Wavelength accuracy of the spectrophotometers
Although the absorbance of a nucleic acid at 260nm is generally on a plateau, the absorbance curve at 280nm 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 the same nucleic acid sample on two spectrophotometers that are both within wavelength accuracy specification.
Nucleotide mix in your sample
The five nucleotides that comprise DNA and RNA exhibit widely varying 260:280 ratios [reference 2]. The following represent the 260:280 ratios estimated for each nucleotide if measured independently:
- Guanine: 1.15
- Adenine: 4.50
- Cytosine: 1.51
- Uracil: 4.00
- Thymine: 1.47
The resultant 260:280 ratio for the nucleic acid being studied will be approximately equal to the weighted average of the 260:280 ratios for the four nucleotides present. It is important to note that the generally accepted ratios of 1.8 and 2.0 for DNA and RNA are "rules of thumb." The actual ratio will depend on the composition of the nucleic acid.
Note: RNA will typically have a higher 260:280 ratio due to the higher ratio of Uracil compared to that of Thymine.
References
1. William W. Wilfinger, Karol Mackey, and Piotr Chomczynski, Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity: BioTechniques 22:474-481 (March 1997).
2. Leninger, A. L. Biochemistry, 2nd ed., Worth Publishers, New York, 1975
