For samples that are inhomogeneous such that the chromophores are not uniformly distributed, a phenomenon called Differential Absorbance Flattening can distort CD spectra.
This occurs, for example, when working with one of the following:
- Proteoliposomes, i.e., membrane proteins reconstituted in the bilayer membranes of lipid vesicles
- Molecules that are covalently attached to nanoparticles, e.g., for drug delivery systems
- Concentration-dependent aggregation
- Immobilised molecules, e.g., in the form of fibers
In all of these cases, the chromophore concentration is locally increased whereas the remainder of the sample is depleted of chromophores.
As a consequence, the Beer-Lambert law breaks down and the absorbance is decreased or 'flattened' relative to the absorbance one would obtain if the chromophores were uniformly distributed in solution.
Absorbance flattening is wavelength-dependent and thus cannot just be accounted for by a simple scaling factor. Generally, the effect is larger in spectral regions of higher absorbance, which is why the tops of absorbance peaks become 'flattened'. The phenomenon is even more complicated for CD signals because the absorbances for left- and right-handed circularly polarised light are flattened to different extents. Hence, absorbance flattening of CD data is differential and resulting spectra can be distorted.
Note that, in addition to Differential Absorbance Flattening, CD spectra for samples that contain particles the size of which is comparable to the wavelength range of interest can be further distorted because of Differential Light Scattering.
The schematic below illustrates the phenomenon: light (yellow) passes from left to right through the sample volume (blue box) and is either absorbed by chromophores or is transmitted without being attenuated at all.
Compare how the relative absorbance differs between uniformly distributed chromophores and chromophores bound to particles (click the button to toggle between the two situations).
The phenomenon of Differential Absorbance Flattening is particularly pronounced (i.e., more light passes through the sample without any attenuation) if the number of chromophores per particle is large and both particle size and number (i.e., concentration) are reasonably low (move the sliders to change these parameters).
Note that this applet is greatly simplified and goes without claim to provide accurate quantitative results.
Differential Absorbance Flattening cannot always be circumvented, but there are different approaches to mitigating the problem.
When working with liposomes, for example, Differential Absorbance Flattening can be somewhat mitigated by reducing the number of chromophores per particle by
- Reducing the protein-to-lipid ratio
- Reducing particle size
where the latter also helps with Differential Light Scattering. If the issue cannot be resolved upon sample preparation, approaches to data correction have been proposed.