Circular Dichroism - Experimental Limitations

Experimental Limitations

CD has also been studied in carbohydrates, but with limited success due to the experimental difficulties associated with measurement of CD spectra in the vacuum ultraviolet (VUV) region of the spectrum (100–200 nm), where the corresponding CD bands of unsubstituted carbohydrates lie. Substituted carbohydrates with bands above the VUV region have been successfully measured.

Measurement of CD is also complicated by the fact that typical aqueous buffer systems often absorb in the range where structural features exhibit differential absorption of circularly polarized light. Phosphate, sulfate, carbonate, and acetate buffers are generally incompatible with CD unless made extremely dilute e.g. in the 10–50 mM range. The TRIS buffer system should be completely avoided when performing far-UV CD. Borate and Onium compounds are often used to establish the appropriate pH range for CD experiments. Some experimenters have substituted fluoride for chloride ion because fluoride absorbs less in the far UV, and some have worked in pure water. Another, almost universal, technique is to minimize solvent absorption by using shorter path length cells when working in the far UV, 0.1 mm path lengths are not uncommon in this work.

In addition to measuring in aqueous systems, CD, particularly far-UV CD, can be measured in organic solvents e.g. ethanol, methanol, trifluoroethanol (TFE). The latter has the advantage to induce structure formation of proteins, inducing beta-sheets in some and alpha helices in others, which they would not show under normal aqueous conditions. Most common organic solvents such as acetonitrile, THF, chloroform, dichloromethane are however, incompatible with far-UV CD.

It may be of interest to note that the protein CD spectra used in secondary structure estimation are related to the π to π* orbital absorptions of the amide bonds linking the amino acids. These absorption bands lie partly in the so-called vacuum ultraviolet (wavelengths less than about 200 nm). The wavelength region of interest is actually inaccessible in air because of the strong absorption of light by oxygen at these wavelengths. In practice these spectra are measured not in vacuum but in an oxygen-free instrument (filled with pure nitrogen gas).

Once oxygen has been eliminated, perhaps the second most important technical factor in working below 200 nm is to design the rest of the optical system to have low losses in this region. Critical in this regard is the use of aluminized mirrors whose coatings have been optimized for low loss in this region of the spectrum.

The usual light source in these instruments is a high pressure, short-arc xenon lamp. Ordinary xenon arc lamps are unsuitable for use in the low UV. Instead, specially constructed lamps with envelopes made from high-purity synthetic fused silica must be used.

Light from synchrotron sources has a much higher flux at short wavelengths, and has been used to record CD down to 160 nm. Recently the CD spectrometer at the electron storage ring facility ISA at the University of Aarhus in Denmark was used to record solid state CD spectra down to 120 nm. At the quantum mechanical level, the information content of circular dichroism and optical rotation are identical.