Golden Rules:
Various conditions must be obeyed in order to achieve valid
and reproducible spectra from Circular Dichroism 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 100
ºC,
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 100
ºC
Solvent Effects, the Lorentz Factor:
Due to its polarisable nature, a solvent will affect the Circular
Dichroism 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/(n
λ
2
+ 2)
where: n λ = refractive index of solvent at wavelength λ nm