Nectar proposes a protocol to help understanding how to optimise a fluorescence titration between a small fluorescent molecule and a biosubstrate such as DNA. This protocol offers some general rules that may be applied to any fluorescence titration and even to absorbance titrations. Please find the protocol here. An additional document on tips/check to be done on the reagents before to start and data analysis may be downloaded here.

The stability constants can be found in the scientific literature or in databases. The databases can be compiled with or without a critical evaluation of the stability constants collected values. Below you can find some links to databases:


Before using a stability constant to draw a species distribution diagram, it is essential to verify the chemical equilibrium to which it refers and ensure that it is compatible with the equilibrium formulation used by the software employed to calculate the concentration of the species.

To draw a species distribution diagram you can use some software (i.e PyES; Hyperquad) able to calculate the species concentration as a function of the concentration of one of the solution components. Some input data have to be entered: type and concentration of the solution components, chemical species formed by the components and stability constants of each species considered.

PhD students represent a large fraction of people working in the scientific world. Differently from the other workers in the research field, PhD students are at the begin of their career and often they can count on few people belonging to their research area (as supervisor or some colleagues).

Becoming a member of a COST ACTION (like NECTAR) can efficiently boost their research work and expand the view of an early-career scientist. The COST ACTION is intended to create a highly specialized scientific network. This connection permits experienced people and young researchers from all around Europe to get closer, favoring knowledge exchange and scientific growth.

PhD students have also the chance to travel and meet other scientists working on similar topics (see STSM). This opens the possibility to experience different scientific approaches and increase own skills. Being part of the ACTION is also a great chance for a PhD student to discuss with colleagues, participate international meetings and directly see how the scientific world works.

Investigating the interaction of a metal ion and a ligand in solution is typically a hard task. However, the speciation of these systems could represent an essential information for a large variety of research studies and applications. Biological and environmental studies are often based on the knowledge brought from the speciation studies of the considered systems. Therefore, speciation studies must be accurate and reliable. When a researcher investigates a metal-ligand system needs to clarify all the aspects related to that particular interaction.

Potentiometry and spectrophotometry represents the election techniques to conduct speciation study, but sometimes are not enough. Other techniques, such as EPR, NMR, calorimetry, mass spectrometry and voltammetry, could be very useful to elucidate hidden aspects. These approaches are usually more expensive and time consuming but can bring to a higher degree of knowledge about the studied system. A multi-technique approach is not always required but can often solve doubts and give a more complete characterization. It is important for a scientist doing speciation studies to have clear in mind the different instrumental possibilities: to know their strengths and weaknesses and the right moment to use them.

A comparison among the overall metal binding ability of different ligands can be performed in different ways, using thermodynamic and chemical equilibrium data. One common approach is based on the construction of competition diagrams, which can be obtained by softwares like Hyss, using the obtained stability constants.

Competition diagrams provide a qualitative comparison of the metal binding affinity of a ligand in presence of other competitive systems, in a chosen pH range. More in detail, they represent simulations of solutions containing the metal and the chosen ligands in equimolar amount and are based on the determined speciation models, admitting that all the components compete to form the respective binary complexes without mixed species formation.

Alternatively, many other parameters can be employed to give an overall estimation of the metal binding affinity for each ligand at a fixed pH value, for example: the Kd (dissociation constant for the generic equilibrium ML = M + L) that corresponds to the concentration of the free metal ion when the half ligand is in a complexed form and half is free; or the negative logarithm of the free metal concentration under given experimental conditions (pM).