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Short answer: Circular dichroism (CD) is the small difference in how a chiral sample absorbs left- versus right-circularly polarized light. CD spectroscopy measures that difference across wavelength; for proteins, far-UV spectra report on secondary structure and near-UV spectra on the environment of aromatic side chains.
What does CD mean in chemistry?
In chemistry, CD is the differential absorption of left and right circularly polarized light by optically active (chiral) matter. For isotropic solutions it is seen for chiral solutes; D- and L-tryptophan, for example, give mirror-image CD spectra (NCI OSTR Biophysics Facility). The effect is tiny: the absorbance difference is on the order of 10-3 to 10-5 absorbance units, which is why a dedicated CD spectrometer (spectropolarimeter) is required rather than an ordinary UV-visible instrument (NCI OSTR; Rupp tutorial).
What is circular birefringence?
Circular birefringence is the difference in refractive index of the sample for left- and right-circularly polarized light. It is what produces optical rotation, whereas CD (reported as ellipticity) comes from the difference in absorption (Rupp tutorial).
CD versus ORD
Optical rotatory dispersion (ORD) is optical rotation plotted against wavelength. CD and ORD are two views of the same underlying optical activity: where CD exists, optical rotation must exist, and the two are related mathematically by the Kronig-Kramers transformation. Ellipticity (CD) is not the same quantity as optical rotation (Rupp tutorial).
| Quantity | Physical origin | What is plotted |
|---|---|---|
| Circular dichroism | Different absorption of left and right circularly polarized light | Ellipticity versus wavelength |
| Circular birefringence | Different refractive index for the two circular components | Gives rise to optical rotation |
| ORD | Consequence of circular birefringence | Optical rotation versus wavelength |
What instrumentation is used in CD spectroscopy?
A CD spectropolarimeter uses a light source (a xenon lamp, described as 150 W air-cooled in the Rupp tutorial) and a modulator that alternates the polarization; piezo modulators have replaced the older Pockels cells. Below about 190 nm the optical path is purged with nitrogen, and protein work needs to reach into the far-UV, down to 190 to 170 nm (Rupp tutorial).
The NCI OSTR facility describes one such instrument (Applied Photophysics Q100) as covering 163 to 1150 nm with Peltier temperature control. Its cell options illustrate how pathlength is matched to the region measured:
| Cell | Minimum volume (NCI OSTR) | Typical use (NCI OSTR) |
|---|---|---|
| Autosampler flow cell, 0.1 mm | 100 µL (200 µL recommended) | Recommended for far-UV |
| Autosampler flow cell, 1 mm | 200 µL | Far-UV |
| Autosampler flow cell, 10 mm | 900 µL | Near-UV |
| Manual cells, 0.5, 1 and 10 mm | Not stated | Required for thermal unfolding; the autosampler is not for thermal scans |
Buffers and sample preparation
Because the signal is small and the far-UV is crowded with absorbing species, buffer choice matters as much as the protein. The NCI OSTR guidance for far-UV work, in one place:
| Component | Guidance (NCI OSTR) | Reason given |
|---|---|---|
| Acetate, 10 mM phosphate | Preferred | Suited to far-UV work |
| PBS | Usually acceptable | High NaCl or KCl can prevent measurement below 200 nm |
| Tris | Acceptable if pH is set with phosphoric acid, not HCl | Chloride absorbs strongly in the far-UV |
| NaCl / KCl | Use Na2SO4 or NaF instead | Chloride absorbance |
| Imidazole, MOPS, PIPES | Avoid | Not recommended for far-UV measurements |
| DTT, EDTA | Only at sub-millimolar levels | Limit to keep far-UV data usable |
Pre-run checklist
- Measure the absorbance of both the sample and the buffer against water across the CD wavelength range first (NCI OSTR).
- Aim for a total absorbance near 0.9 OD; 0.4 to 1.6 OD is described as good, and 2 should never be exceeded (NCI OSTR).
- For far-UV protein work in a 1 mm cell, roughly 0.1 to 0.2 mg/mL is the guide concentration (NCI OSTR).
- Collect in the order buffer, sample, buffer, sample so the baseline is bracketed (NCI OSTR).
- Use manual cuvettes for thermal unfolding experiments (NCI OSTR).
For the surrounding bench work, see protein purification, biological buffers and protein refolding.
Reading far-UV and near-UV spectra, and the limits
Near-UV (about 250 to 300 nm) signals come from aromatic residues (Phe, Tyr, Trp). Far-UV (below 250 nm) is dominated by backbone transitions, which is what allowed spectral characteristics of secondary structure to be identified (NCI OSTR). Helix, sheet and coil each have characteristic spectra, and a protein spectrum can be deconvoluted into those components (Rupp tutorial). Typical applications listed by NCI OSTR are secondary structure, interaction-induced conformational change, effects of mutations, and thermal or chemical stability.
The Rupp tutorial is explicit about the limits of deconvolution:
- It ignores chromophore interactions between regions and other structural elements such as 3-10 helices.
- Fit accuracy is around ±10%, and results vary with the wavelength region used for fitting.
- CD is more reliable for monitoring conformational change than for absolute secondary-structure values.
- Buffer and additive absorbance limits usable data below about 200 nm.
Common misunderstandings
- “CD gives the structure.” It gives a low-resolution estimate of secondary-structure content, with the ±10% caveat above; it does not locate individual residues. For sequence-level identification see protein sequencing by mass spectrometry.
- “CD is the same as optical rotation.” They are related through the Kronig-Kramers transformation but are different measured quantities.
- “PBS is always fine.” High chloride can limit data below 200 nm.
- “Any UV spectrometer will do.” The differential absorbance is about 10-3 to 10-5, so a CD spectropolarimeter is needed. For general spectroscopy background see spectrograph.
FAQ
What does CD mean in chemistry?
Circular dichroism: the difference in absorption of left- and right-circularly polarized light by a chiral sample, measured across wavelength.
What kind of instrumentation is used in circular dichroism spectroscopy?
A CD spectropolarimeter with a xenon lamp, a polarization modulator, nitrogen purging for the far-UV, cuvettes of 0.1 to 10 mm pathlength and often Peltier temperature control.
What is circular birefringence?
The difference in refractive index for left- and right-circularly polarized light, which gives rise to optical rotation.
What is the difference between CD and ORD spectroscopy?
CD measures differential absorption (ellipticity); ORD measures optical rotation against wavelength. Both reflect the same optical activity and are linked by the Kronig-Kramers transformation.
Sources
- NCI OSTR Biophysics Facility, Circular Dichroism: ostr.ccr.cancer.gov/cd-2 and the facility CD guide PDF (read 2026-11-07).
- Rupp B., Circular dichroism tutorial: ruppweb.org/cd/cdtutorial.htm (read 2026-11-07).
This page is general educational information about a laboratory technique, not instrument-specific or clinical advice.
