The choice of the two wavelengths used in a UV-VIS assay depends on the specific molecule being analyzed and the properties of its absorbance spectrum. The two wavelengths are typically chosen based on the following factors:

1. Absorbance maxima: The two wavelengths are often chosen to be the maximum absorbances of the molecule, which correspond to the wavelengths at which the molecule absorbs the most light. These wavelengths provide the most sensitive and specific detection of the molecule.

2. Linear range: The two wavelengths should be within the linear range of the detector to ensure accurate and precise measurements.

3. Background absorbance: The two wavelengths should be chosen in a region where the background absorbance is low to avoid interference with the molecule's absorbance signal.

The ratio of absorbances is often used in UV-VIS assays to correct for any interference from background absorbance or other substances in the sample. The ratio can also be used to correct for any variability in the sample concentration or path length.

The choice of the specific ratio depends on the specific molecule being analyzed and the properties of its absorbance spectrum. For example, in some cases, the ratio of absorbances at two specific wavelengths may be related to the molecular structure or concentration of the molecule.

To determine the values of the two absorbances used in the ratio, the specific wavelengths are measured for the sample and the blank (i.e., a sample without the molecule of interest). The ratio is then calculated as the absorbance of the sample at one wavelength divided by the absorbance of the sample at the other wavelength, minus the absorbance of the blank at one wavelength divided by the absorbance of the blank at the other wavelength.

Overall, the choice of wavelengths and the specific ratio used in a UV-VIS assay depend on the specific molecule being analyzed and the properties of its absorbance spectrum, as well as the desired accuracy and precision of the measurement.

the ratio is calculating just simply dividing the absorbance at wavelenght #1 by the absorbance at wavelenght #2. In some molecules, such as DNA, this range between two wavelenghts (260/280 nm for DNA) is supposed to be kind of constant, because of the chromophore groups presents. Then, the ratio is used as indicative of purity. If you have some other impurities absorbing at one of those wavelenghts, then the ratio will change.

For some other molecules, the ratio can have other applications. You can provide more details in order to help you.