To anyone with more experience in the area of CMC determination,
I am working with a small molecule that can exist in 2 states which can both self-assemble, and my goal is to show a difference in their ability to aggregate. The easiest way of showing this would clearly be to just calculate the two CMC values, but I have run into significant issues in doing so.
Via the method of fluorescence using a hydrophobic dye, the commonly used probes I have seen being used are Nile Red and pyrene. Pyrene's emission peaks are seemingly quenched upon increasing the concentration of my molecule via FRET. I have considered using this quenching to determine CMC, but the 2 states have different FRET efficiencies so that has not given sensible data.
Moving outside the range of FRET, Nile Red's emission peak unfortunately almost perfectly overlaps with the emission of State A, and it is completely overshadowed. Additionally, Nile Red and it's sibling Nile Blue (which I have also tried) don't work well at my operating range of pH ~2.5 due to the amines becoming charged, more hydrophilic, and then not being sequestered as efficiently.
So, would anyone be able to suggest a strategy for determining the molecule's CMC? Or possibly suggest a hydrophobic dye that could operate at the pH range of 2-4? Attached are he absorbance spectra of the 2 states for reference.
Thanks for any help that you can offer.
Since the molecule is fluorescent, have you considered monitoring the fluorescence excitation and emission spectra, intensities or the lifetimes? Self-association would probably have an effect on one or more of these properties.
The challenge is the high concentration of the compound that is likely going to be needed, causing a severe inner filter effect. To deal with this problem, you can use a triangular cross-section cuvette in the spectrofluorometer to allow you to measure the surface fluorescence by reflection, or rectangular cuvette with a short pathlength.
Thomas Reardon
The spectra of your two compounds differ little, but there are still differences. Determine the absorption maximum for them. Then, for each molecule, determine how it changes depending on the concentration of these molecules. The concentration of the inflection of the intensity dependence - the concentration will be their CMC.
there are further options to determine the cmc, surface tension measurements, conductivity, DLS, surfactant selective electrodes, density, viscosity etc.
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