The Stern-Volmer plot is used to study the interaction dynamics between a fluorescent emitter and a quencher (a substance that reduces the fluorescence intensity). The plot shows the ratio of the fluorescence intensity as a function of the quencher’s concentration [Q], described by Eq. 1:

Where I₀ and I are the fluorescence intensity in the absence and presence of a quencher respectively and K_SV is the Stern-Volmer constant. For systems showing only dynamic quenching, K_SV can be determined from the slope of the Stern-Volmer plot, as shown in Figure 1. 

Figure 1. Example of an intensity Stern-Volmer plot of the system fac-Ir(ppy)₃ and the quencher 2,4dinitrotoluene. The slope provides the Stern-Volmer constant. 

The Stern-Volmer constant provides information about the sensitivity of the quenching system and, for dynamic quenching, is given by Eq. 2: Where τ₀ is the fluorescence lifetime of the emitter in the absence of a quencher, and k_q is the bimolecular quenching constant. The constant k_q describes how efficiently the quencher suppresses the emission intensity of the system, and its value is typically in the range of 10⁹ – 10¹⁰ M⁻¹ s^−1.1,2  

Since the relative change in intensity I₀/I is proportional to a corresponding change in fluorescence lifetime, Eq. 1 can also be written as a function of lifetime (Eq. 3). This allows a dynamic quenching interaction to also be characterised from changes in the fluorescence lifetime. The Stern-Volmer constant K_SV and bimolecular quenching constant, k_q, can be determined as a function of changes in lifetime with quencher concentration (Figure 2).  

Figure 2. Example of a lifetime Stern-Volmer plot of the system fac-Ir(ppy)₃ and the quencher 2,4dinitrotoluene. The slope provides the Stern-Volmer constant. 

What Causes Non-Linear Deviations in the Stern-Volmer Plot? 

A linear relationship in the Stern-Volmer (SV) plot is the most common behaviour of fluorescence quenching. However, upward, or downward deviation behaviour is also possible (Figure 3). 

Figure 3. Stern-Volmer plot for quenching systems showing linear relationship (red curve); upward deviation relationship (blue curve); and downward deviation (green curve). 

A linear relationship is most associated with dynamic quenching, where the quenching is mainly due to collisions between the emitter and quencher molecules. However, the linear relationship on the Stern-Volmer plot can also be associated with a purely static quenching system. Since static quenching does not show changes in the emission lifetime as a function of the quencher concentration lifetime measurements can be used to distinguish between the two.¹  

The upward/positive deviation is found in systems in which both static and dynamic quenching are contributing to the decrease in fluorescence intensity. A downward/negative deviation is generally associated with the presence of two emitters with different Stern-Volmer constants, e.g., for the same concentration of quencher, the emitters will be quenched at different rates. In most cases, at lower concentrations of quencher, a linear relationship is evident; however, as the quencher concentration increases, the relationship becomes non-linear.