if i keep the same concentration for 4 complexes (All the complexes are cobalt schiff base complexes containing different substituent ) it is easy to compare the their interaction with DNA. but the problem is one of the complex give the intensity "0.8" at 1x10-5 but another complex give 0.2 only. at 4x10-5 only it give around "0.8". So could i take different concentrations. is there any paper related to that kindly let me know.
Yes you should keep concentration same for all the complexes in study for Binding interaction this will help you to evaluate the binding propensity of complexes towards DNA and shall also make your binding constant value comparable for the series.
regarding your different absorbance related problem i think with a very good spectrophotophotmeter 0.2 absorbance should be easy to monitor in fact for good spectrophotometeric work aborbances less than 0.5 are fine higher absorbance at higher concentrations leads to deviation from Beer law moreover absorbance is only of one method of DNA binding interaction other like emission, CD spectroscopy, viscosity, epr etc can also be used(see my paper attached PDF) .
In summary now, for good data keep concentration of all the complexes fixed and equal to the concentration of complex with lowest concentration for spectrophotometeric study.
for other methods(emission, CD, EPR, Ethidium bromide replacement you can choose a different concentration than spectrophotometer case but keep that fixed as well.
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If you want to determine the binding constants by spectroscopic titration, it is not necessary to have the same concentration of each complex species. For determination of DNA-complex binding constant, it is important to keep constant concentration of tested complex in the way that successive adding of μL volumes of DNA solution does not change the volume of tested complex.
you can find binding affinity and comparison between interactions only when you are keeping the concentration same. You need extinction coefficient for calculating kb , thats why you have to keep the concentration same. In case, if you are changing the concentration , it wont be comparative study.
and sometimes it depends on the solvent and as well as ligand system when you get diff absorbance values at same concentration.
good luck
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