#  Circular Dichroism (CD) 

 



 ##  

  expand\_more  

 
  

 

[**CMI CD resources and user guide**](/node/750971#cd-resources)**⬇︎**

Circular Dichroism (CD) Spectroscopy is used to determine the optical isomerism and **secondary structure of molecules**. Circular dichroism (measured in molar ellipticity) is the difference in absorption of left-handed and right-handed circularly polarized light and can be observed in optically active molecules with chiral centers. Proteins have many chiral centers. CD spectra in the Far-UV region (185 – 250 nm) can be used to determine protein secondary structure. Thermal stability (Tm) can be measured by following changes in molar ellipticity with increasing temperature at characteristic peaks for folded proteins.

   ![CD Example Data. Adapted from Greenfield 1969.](/sites/g/files/omnuum4016/files/styles/hwp_1_1__360x360_scale/public/2025-07/CD_data_examples.png?itok=Vg-r77Ta) 

 

Far-uv region of the CD spectra of Poly-L-lysine in three conformations: α-helix, β-sheet and random coil. Adapted from N.Greenfield, 1969.**Features**

- CD scans are easy, rapid, and non-destructive (~10 min/sample)
- Label-free
- Far-UV CD can predict secondary structure
- Near-UV CD can reflect changes in tertiary structure
- Thermal stability experiments can measure reversible unfolding

**Limitations**

- Requires concentrated, purified samples
- Some common solvents absorb in the region of the CD
    - Chloride (e.g. NaCl), DMSO

   ![Jasco J-1500 image](/sites/g/files/omnuum4016/files/styles/hwp_1_1__360x360_scale/public/2025-01/j-1500new.png?itok=h-jf9yGH) 

 

The CMI has a [Jasco J-1500](https://jascoinc.com/products/spectroscopy/circular-dichroism/j-1000-series-models/j-1500-circular-dichroism-spectrophotometer/) CD Spectropolarimeter with a Peltier temperature controller and single cuvette holder.



 



###    Improvements in J-1500  expand\_more  

In April 2023, the Jasco J-815 was retired, after serving the BCMP department and HMS for almost 2 decades.

**Key** [**J-1500**](https://jascoinc.com/products/spectroscopy/circular-dichroism/j-1000-series-models/j-1500-circular-dichroism-spectrophotometer/) **improvements:**

- Higher Sensitivity
    - lower minimal recommended protein concentration
    - Faster scanning speeds
- Improved Peltier Cell Holder - PTC-517
    - Faster temperature gradients
    - No separate power supply -Peltier turns on when the J-1500 is on
- Multivariate Secondary Structure Estimation (SSE) Software 
    - Uses several models with data ranges varying from 176-260 to 200-260
    - Includes optical constant calculator to convert mdeg to molecular ellipticity

 

 



###    CD Sample Data  expand\_more  

 

####  CD spectrum of alpha-helical protein, BSA.

####  ![CD Sample Data - BSA](/sites/g/files/omnuum4016/files/cmi/files/cd_example_-_bsa.png)

 





 

 

 



 

 

 

 

 CD Resources Required Supplies Sample Preparation λ Cutoff For Solvents Data Analysis 

## CD Resources

[CMI Jasco J-1500 CD Getting Started Guide](/sites/g/files/omnuum4016/files/2026-02/CMI%20Jasco%20J-1500%20CD%20Getting%20Started%20Guide.pdf "CMI Jasco J-1500 CD Getting Started Guide"), guide to experimental design and standard Far-UV CD protocols.

Additional resources are available at the instrument. Instrument and software manuals are located in the “Jasco Manuals” desktop folder on the instrument computer.

[Jasco Training Videos](https://jascoinc.com/training-video/video-category/spectra-manager-for-cd/) provide more information on various topics related to CD measurements and the Spectra Manager program.

\*J-815 RETIRED APRIL 2023 - [CMI Jasco J-815 CD Getting Started Guide](/sites/g/files/omnuum4016/files/2025-03/CMI%20Jasco%20J-815%20CD%20Getting%20Started%20Guide.pdf "CMI Jasco J-815 CD Getting Started Guide"). Replaced with newer J-1500 instrument.

 

 

 

## Required Supplies

- High Quality Quartz cuvette
    - 1 mm pathlength cuvette
        - Hellma 110-1-40, style 110-QS (available from Sigma-Aldrich)
        - Starna 21-Q-1
    - 1 cm pathlength cuvette
        - Hellma 100-10-40, style 100-QS (available from Sigma-Aldrich)
        - Starna 1-Q-10 or 21-Q-10
- your sample
- buffer blank

**For Far-UV measurements (protein secondary structure)**: 1 mm  
**For Near-UV measurements**: 1 cm or 1 mm, depending on application and concentration

 

 

 

## Sample Preparation

### Assay Buffers

Buffer selection is critical for accurate CD measurements. Solvent absorbance can severely interfere with the CD signal. Many commonly used buffers and additives absorb in the far UV region used for CD measurements.

- The ideal *CD experiment is performed in a buffer:*
    - in which your protein is well behaved and soluble
    - with no buffer absorbance through the range of the CD spectrum.
- **Always take a scan of your buffer blank to determine whether absorbance interferes with your region of analysis.** 
    - Ensure that the buffer blank is well matched to the final dilution of your protein (even trace amounts of some solvents will interfere with the CD measurements).
- Phosphate buffers with little-to-no NaCl are recommended. 10 mM phosphate buffer is ideal.
- **Ideal aqueous solutions contain as little chloride as possible.**
    - NaCl and Tris buffers are not recommended but can be tolerated at low concentrations.
    - If salt is required, SO42- or F- are preferred counter ions, as they are transparent in the far UV. Potassium fluoride may be a good substitute for chloride-containing salts.
    - Chloride ions will result in a loss of signal below about 200±5 nm, which affects secondary structure estimation, but generally will not block detection of the alpha-helical peaks (208 nm and 222 nm) or the beta-sheet peak (218 nm).
- DTT, ß-ME, or EDTA can be present at low concentrations (1 mM).
- Some detergents are fairly transparent in the far UV (e.g. Chaps and octylglucoside). Triton detergents should be avoided, as they can oxidize readily and form UV-absorbing materials.
- DMSO, formamides, and imidazole absorb strongly in the far UV region, and thus should be avoided in a CD experiment.

### Samples

- An accurate protein concentration is required for all CD experiments. 
    - Over-estimating or under-estimating the protein concentration interferes with signal
    - Algorithms that estimate secondary structure depend on correct protein concentrations
- The amount of sample required for a CD experiment depends on the size, cuvette pathlength and type of measurement being performed.
- **1 mm pathlength cuvettes holds 300 µl** and a 1 cm cuvette holds 3 ml.
- Required protein concentration is inversely proportional to the cuvette pathlength, and thus a 1 mm path cuvette requires 10x the concentration of a 1 cm cuvette.
- **Recommended concentrations for Far-UV measurements of protein secondary structure are (Jasco J-1500):**
    - **0.1 mg/mL in 1 mm path cuvette**
    - **0.01 mg/mL in 10 mm patch cuvette**
    - Mol/L=115/(MW\*7000)\*10/pathlength(mm).
    - If possible, make a concentrated stock solution of your protein (at least 2X) and dilute as needed. Protein requirements vary with secondary structure, as alpha-helical proteins have a stronger signal than beta-sheet proteins.
- Protein aggregates can interfere with the CD signal.
    - Assess protein heterogeneity via light scattering.
    - Purify protein samples with soluble aggregates by size-exclusion chromatography.
- Samples can be recovered from the cell. This is not recommended for thermal melts, unless you know your thermal denaturation is reversible.

 

 

 

## λ Cutoff For Solvents

The absorption and CD spectra should be checked for each buffer.

*Low Wavelength Cutoff For Common Solvents and Buffers in 1mm cuvette*

Martin, S. R. Proteins Labfax (Protein, N.C., ed.), Bios Scientific Publishers, Oxford, 1996.

SortSolvent SystemCut off (nm)Water

<185

Trifluoroethanol

<185

Hexafluoroisopropanol

<185

Acetonitrile

185

Methanol

195

Ethanol

196

2-Propanol

196

Cyclohexane

<185

Dimethylsulfoxide

251

Dioxane

232

(NH4)2SO4 0.15 M

191

NaCl 0.15 M

196

NaClO4 0.15 M

<185

NaNO3 0.15 M

245

Phosphate 100 mM

<185

Tris 100 mM

195

Pipes 100 mM

215

Mes 100 mM

205

GdnHCl 4 M

210

Urea 4 M

210





Solvent components not listed here may be compatible but should be tested. EGTA, DTT and some detergents at modest concentrations may be tolerated.

 

 

 

## Data Analysis

[DichroWeb](http://dichroweb.cryst.bbk.ac.uk/html/home.shtml), a free online tool for determining protein secondary structures based on CD spectra.

- Academic users may apply for a username and password.

[CDToolX](http://cdtools.cryst.bbk.ac.uk/index.html), a free, downloadable software program that enables processing and analyses of circular dichroism (CD) spectroscopic data.

[Protein Circular Dichroism Data Bank](http://pcddb.cryst.bbk.ac.uk/home.php), a database of CD data

[BeStSel](https://bestsel.elte.hu/index.php) (Beta Structure Selection), a free online tool based on a novel method for the secondary structure determination, especially beta-sheet rich proteins.

[ValiDichro](http://valispec.cryst.bbk.ac.uk/circularDichroism/ValiDichro/upload.html) tests the quality of CD data based on based on common characteristics of CD protein spectra observed in the literature.

 

 

 

 

 

 [ Data Management and File Formats arrow\_circle\_right ](/data-management) [ CD Services arrow\_circle\_right ](/cd-services) [ About Data Collection Services arrow\_circle\_right ](/services) 

 

 

 

 

 



 

 See also:- [ molecular properties ](/cmi-terms/molecular-properties)
- [ Circular Dichroism ](/cmi-terms/circular-dichroism)