I am having an issue regarding circular dichroism spectra while measuring blanks. We are studing DNA secondary structure by CD using oligonucleotides in Tris-HCl pH 7.5 10mM and KCl 1mM, so that would be my blank.
At some point blanks started giving negative ellipticity (we measure between 220-320nm), before that it was always close to 0 no matter which buffer we used for the experiment. So I performed various measures (shown in the attached image) with three different cuvettes we use regularly (two self-masking quartz with different volume, and a regular quartz one, all three 1cm path) and with miliQ water and the Tris buffer.
The issue here is not the weird shape spectra I get (as you can see it is always the same), but for the same blank measured at the time I start the experiments and the same blank measured last I get the same shape but with different CD values so resting them to my sample would result in different results.
edit1: I also did a measure with no cuvette and still got the same spectra (not shown in the graphic)
Thanks in advance for the help.
Have you tried thoroughly cleaning your cuvettes? I recommend a soak in chromic acid to destroy organic residues on the inner surfaces.
Wash the cuvettes by freshly prepared chromic acid and then by water. After that rinse with acetone( spectroscopy grade) and dry it. Meanwhile, run a CD spectra of air (no cuvettes). If I assume the cuvettes are clean, then this pattern may come from the instrument noise. Also make sure that you turn on the nitrogen flow earlier (around 30 min) than instrument.
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