Circular Dichroism Spectroscopy
Circular dichroism (CD) spectroscopy is an optical technique that allows the detection and quantitation of the chirality of molecular structures. It also provides information about the secondary and tertiary structures of proteins. Circular dichroism is the difference in the absorption of left and right circularly polarized light. A periodic variation in the polarization of the light beam is induced by the polarization modulator through all ellipticities from left circular through elliptical, unchanged linear and elliptical to right circular. This polarized light passes through the sample to a photomultiplier detector. If the sample is not optically active, the light beam does not vary through this cycle. With the introduction of an optically active sample, a preferential absorption is seen during one of the polarization periods and the intensity of the transmitted light now varies during the modulation cycle. The variation is directly related to the circular dichroism of the sample at that wavelength. Successive detection is performed at various wavelengths leads to the generation of the full CD spectrum. For CD to be exhibited for a sample, the sample must be optically active and not superposable on its mirror image.
Information CD can provide
- Protein secondary structure
- Used to estimate the secondary structure (conformation) of the polypeptide chain (a-helix, b-sheet)
- Nucleic acid conformation
- Enhanced CD that is representative of the relative base-base orientation
- Interaction studies
- Direct, noninvasive means of monitoring binding of molecules to proteins or enzymes
From The Protein Protocols CD-ROM, J.M. Walker, ed., Humana Press, Totowa, NJ, 1998
(Jasco Instruction Manual, 1993)
| Solvent | Usable short wavelength limit (nm) | Remarks | ||
| 1 cm cell |
1 mm cell |
0.1
mm cell |
||
| n~hexane | ~210 | ~185 | ~180 | Nonpolar. small solubility |
| Cyclohexane | ~210 | ~185 | ~180 | Nonpolar, small solubility |
| Isooctane | ~210 | ~185 | ~180 | Nonpolar. small solubility |
| Dioxane | ~220 | ~210 | ~202 | Nonpolar, commonly used for organic compounds |
| Benzene | ~280 | ~275 | ~270 | Nonpolar, sometimes used in the measurement of symthetic polymers |
| Carbon tetrachloride | ~250 | ~240 | ~230 | Nonpolar, special in ORD/CD |
| Chloroform | ~240 | ~230 | ~220 | Intermediate polarity, used in comparison with NMR data |
| 1,2-dichloroethane | ~220 | ~210 | ~200 | Nonpolar, high solubility |
| Methanol | ~210 | ~195 | ~185 | Polar, commonly used for organic compounds |
| Ethanol | ~220 | ~200 | ~190 | Polar, frequently used for organic compounds |
| Trifluoroacetic acid | ~260 | ~250 | ~240 | Measurement of synthetic polymers; corrosive |
| Dimethylsulfoxide | ~264 | ~252 | ~245 | Used in the measurement of synthetic polymers |
| Tetrahydrofuran | ~265 | ~230 | ~204 | Used in the measurement of synthetic polymers |
| t-decalin | ~220 | Solvent for high temperature measurement (bp + 194.6°C) | ||
| P5-Ml | ~220 | ~210 | Isopentane/methylcyclohexane (5:1), mixed solvent, nonpolar low temperature solvent (-196°C) | |
| EPA | 220 | 210 | Ethylether/isopentane/ethanol (5:5:2), mixed solvent, commonly used tor low temperature measurement (-196°C) | |
| Ethanol/methanol 4:1 | ~220 | ~200 | Polar low temperature solvent (-160°C) | |
| Water Solvent | ||||
| Distilled water | ~185 | ~180 | ~175 | |
| 10 mM Sodium phosphate | ~182 | |||
| 0.1 M Sodium phosphate | ~190 | |||
| 0.1 M Sodium chloride | ~195 | |||
| 0.1 M Tris-HCl | ~200 | |||
| 0.1 M Ammonium citrate | ~220 | |||
- Information on CD from Alliance Protein Laboratories
- CD Tutorial from Bernhard Rupp, including the program CDFit
- Circular Dichroism Submission Form (PDF)
