Golden Rules:
Various conditions must be obeyed in order to achieve valid and reproducible spectra from CD spectrometers. Not least:
• To avoid stray-light effects & poor S/N
0.4 < A < 1.4
where: A = Absorption (AU)
• For continuous scanning instruments
S.t / 60 < B < W / 10
where: S = scan speed (nm/min)
t = response (sec)
B = spectral bandwidth (nm)
W = width of spectral features (nm)
Thermal Effects:
Changing the temperature of a solution will affect a variety of things, which should be corrected for:
• Concentration, through expansion
H2O ca. 2% from 0 to 100ºC, but can be 10-fold greater for organic solvents
• pH, through buffer temperature coefficient
e.g. Tris varies ca. -2.8 pH units from 0 to 100ºC
Solvent Effects, the Lorentz Factor:
Due to its polarisable nature, a solvent will affect the CD spectrum observed irrespective of whether it directly interacts with or changes the molecules under study. This effect is wavelength dependent and should be accounted for through the Lorentz Factor in studies with varying solvents:
• Lorentz Factor for CD
CDcorrected = CDobserved . 3/(nl2 + 2)
where: nl = refractive index of solvent at wavelength l nm