I have a question about chloride absorbance in CD spectra of proteins. It is well known that Cl- ions absorb strongly at wavelength less than 195 nm. Suppose I take a solution of 100 mM NaCl and 100 mM KCl and measure their CD spectra separately. If you know about Van't Hoff factors (i), you will guess why I am asking this question. For those who don't: Because of ion pairing in solution, dissociation of salts is rarely 100% and the ratio between the actual concentration of particles produced when the substance is dissolved, and the concentration of a substance as calculated from its mass is called Van't Hoff factor. Now, i for 100 mM NaCl is 1.87 while that of KCl is 1.85. So relatively speaking, the concentration of chloride ions will be more in NaCl.. My question is therefore " Does the dissociation matter in CD spectroscopy or will Chloride ions, whether in dissociated (Cl-) or paired state (Na+Cl-) absorb equally?
Thanks!
P.S: I know it can be tested in an instrument, I need an understanding why :)
CD spectra gives a signal only when the molecule/species is optically active i.e., there is asymmetry in the molecule.
CD uses left circularly polarized light and right circularly polarized and the difference in the extinction coefficients of both are measured. The difference will be seen only if the molecule is asymmetric. All optically inactive molecules don't give any signal. Only optically active molecules give signal. It is not a correct technique to study optically inactive molecules.
I apologize I'm not able to answer your question. However, I hope help you suggesting to ask to:
or in
http://www.cerm.unifi.it/
Good luck
The effect of Cl- absorbance on your CD spectrum will depend on the effect of having Na+ in close proximity on the absorbance wavelength and intensity of Cl-. I don't know enough theory to predict what a positive charge will do (red or blue shift etc) but you might be able to determine this experimentally. As stated above though, the effect will be pretty small.
The asymmetry in electron distribution due to these differences in interaction will be very small and i don't think they can be picked up by CD.
You also need to worry about what is the absorbance of the solution at the concentrations you mentioned i.e., how much light is being transmitted through the sample.
Also note that you are measuring the difference in extinction coefficients.
Thank you everyone. It does clear up some doubts. It all primarily because a colleague reported that KCl was better then NaCl when measuring protein CD spectra. Since NaCl dissociates slightly better than KCl, I was thinking it could explained by that logic that more chloride ions in NaCl than KCl at identical concentrations lead to signal issues. But from what i get, the differences would be very small.
Sodium fluoride, which does not absorb UV light, is a better choice if the protein is not especially sensitive to the choice of anion
@Jeffrey, I know fluorides are a better choice, But between KCl and NaCl, people seem to say that there is not much difference. I was wondering why
You framed a 12 lines question without mentioning the word 'protein'.
@ V.N.RaviKishore V. : Ah now I see why you gave that answer..I was assuming people would get that. Without a chromophore you wouldnt expect a CD signal..
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