Assessing concentration and purity of nucleic acid solutions via photometric measurements constitutes a routine method in the molecular biology laboratory. Whether a complete absorbance spectrum is obtained from a sample, or whether absorbance is determined at defined wavelengths only, it makes sense to subject the results to quick check. This step not only helps determine the purity of the sample, but it also serves the purpose of evaluating the quality of the measurement itself.
1. Does the absorbance value A260 fall within the linear range of the photometer (0.1 – 1.0 A*)?
2. Is background absorbance (wavelengths ≥ 320 nm) absent or reduced to a minimum, and is background correction activated?
3. Does the ratio A260/A280 fall within the range of approximately 1.8 – 2.0?
4. Does the ratio A260/A230 fall within the range of approximately 2.0 – 2.5?If one or more of the values deviate from the expected range, it may be necessary to analyze the measurement process, or the sample, more closely and, if applicable, follow one of the approaches to solution listed below.
The technical specifications of a photometer often include the photometric measurement range. It specifies the absorbance range that is read by the instrument, but it reveals little about the quality of these data. Absorbance values follow a linear curve only within a limited range of measurement – generally between 0.1 A and 1.0 A, depending on the optical construction and the quality of the instrument; this range may extend beyond these values*.
1. Linear measurement range
Absorbance at 260 nm < 0.1*: In the case of a sample of low concentration, the impact of all inaccuracies during sample preparation and measurement will be magnified and thus compromise the accuracy and precision of the results. Sample concentration can be increased through either lower dilution factors or through concentration of the sample. If possible, the light path may be increased by employing a cuvette with a longer path length, or, alternatively, the sample may be quantified using fluorescence. The emergence of negative values is often a consequence of incorrect sample preparation or blank measurement. In such cases, the possibility of a mix-up of blank and sample prior to measurement should be investigated, as well as suitability of the blank solution used.
Absorbance at 260 nm > 1.0*: A highly concentrated sample, too, may lead to measurement inaccuracies, as values are influenced by light scatter (figure 1). In this case, approaches to solutions include sample dilution or the use of a shorter light path, for example, through the use of a cuvette with shorter path length.