Although the method for the determination of OD260 is simple and convenient, it is recommended that the validity of the graph or slope is checked once in a while. This is especially important when HPLC conditions such as column, eluents, gradient, flow rate and temperature are changed. In addition, if the flow cell of the UV detector is replaced or cleaned, a new graph should be generated. Gratifyingly, in most labs, HPLC is typically performed under consistent conditions and UV detector flow cell can last for many years, there is no need to validate the graph frequently. Another thing we wish to point out is that when the area of the peak of an ODN is out of the areas used to generate the correlation graph, caution is needed to use the graph to calculate its OD260 because the correlation may not be linear when the concentration of the ODN in the eluent is too high.