Circular Dichroism



General Guidelines

This is not intended to teach you how to use the instrument.
See TurnOn procedure on wall next to instrument.
Important: do NOT run instrument without nitrogen (make sure there is enough before starting) or you will damage the optics.

General Scan Parameters (typical)
  • Use cylindrical cell whenever possible
  • Check all parameter values - instrument defaults to last values used
  • Collect data for 1 point per nm wavelength
  • Use averaging time of 5 to 10 seconds and do not use repeat scans
  • Use Peltier temperature control (e.g., 20°C) for cylindrical cells

Baseline
  • use water, not buffer
  • use same data collection scheme as used for samples

Protein Samples
  • wavelength range is 260 to 190nm for 0.1 cm cell; to 200 for 1.0 cm
  • 0.15 mg/ml protein for 0.1 cm (1.0 mm) cell
  • scale concentrations to match pathlength if not using 0.1 cm cell

DNA Samples
  • wavelength range 330 to 200 (for 1cm, maybe lower for short pathlengths)
  • concentration to give A260 = 0.5 in chosen pathlength

Exporting CD data to text file from GlobalWorks

Export only a few CD spectra
  • click on scan name so that scan appears on graphing window
  • right-click in graph window and select “Export as ASCII”
  • repeat for each scan

Export many spectra
  • right-click on scan name and “Select” the scan (a check mark will appear next to it)
  • repeat for all scans, both baseline and sample scans
  • then right-click on any scan name and select “Process Multiple Data” -> “Convert into 3D Matrix”
  • a 3D view of the data will appear in the graphing window
  • click icon toward top of window to convert 3D into 2D slice mode (hover over icons to view)
  • you will now see overlays of your scans in the graph window (all may not by visible)
  • right-click in graph window and select “Export as ASCII”
  • the ordering of the scans in the multicolumn data set is the same order as listed in the GlobalWorks scan window

Temperature Melts

You can only measure spectra. You cannot measure the CD at one wavelength as a function of temperature. If you want to measure a high resolution melting curve, i.e., at every degree increment, you will have to finagle the instrument as described below.

Temperature Correction
Due to thermal loss in the system, the actual temperature in the cell is not what is displayed by the temperature controller. A calibration curve has been determined using a temperature probe inserted in a 1cm cell containing water. The actual temperature (Tactual) is calculated from measured temperature (Tmeas) using the equation
Tactual = 3.07 * Tmeas + 0.867

Temperature Equilibration
The temperature of the sample in the cell lags behind the controller. Enough time must be allowed for the system to come to proper equilibrium. The time required for equilibration depends on current temperature and the temperature step. These have been empirically determined for several different melting schemes. See temperature scripts in Edmondson’s directory.

High resolution melting curves
  1. first set temperature to 20°C and collect baseline and sample scans as usual
  2. on temperature page, set temperature to starting temperature of your melt
  3. now copy Edmondson’s temperature script to your directory and edit the file to encompass the temperature range you want to use. Make sure you set the number of temperatures correctly. See comments in scripts (and heed them).
  4. do NOT change any of the equilibration times
  5. set instrument to collect data with one (1) increment (this will be 2 wavelengths)
  6. set starting wavelength at 260nm
  7. set ending at wavelength you want to measure (e.g., 220 nm)
  8. set averaging time to 30 seconds
  9. on temperature page, select temperature script that you made in above steps
  10. after temperature has equilibrated from step #2, start data collection

Spectra measured at different temperatures (low resolution)
  • you can collect the entire CD spectrum, but you will have to limit temperatures to 10 or 5 degree increments
  • follow guide above for high resolution melts but use entire wavelength range
  • you may want to keep averaging time toward short side (5 seconds)
  • use temperature script appropriate for your temperature step size (from Edmondson’s directory) to ensure proper equilibration

Exporting Melt
use procedure above for exporting many spectra
you already have a 3D data set so skip first part of procedure

Analyzing Melting Curves
The following method uses Edmondson’s IgorCD procedures with IgorPro.
  • First import the baseline and sample scan and process as usual (although this is not really needed for melt)
  • Using Igor’s built-in “Smooth” function (Analysis Menu), apply a 9 or 11 point Savitzky-Golay smooth to the baseline
  • Plot the smoothed baseline and determine the value of the baseline at melting wavelength (e.g., 220 nm)
  • Load the multicolumn ASCII text file of the melt data into IgorPro using the LoadCD menu item
  • In the Process CD control panel, select 1st scan of melt for sample and set baseline to “none”
  • Insert baseline value determined above into “Subtract Constant” field.
  • Uncheck “SmoothData” and “ZeroBaseline” (ZeroBaseline checked may also work if first point(s) of scans are in baseline region (260nm)).
  • Check “ProcessListOfSpectra”
  • Fill in “Concentration” etc. and then “DoIt”
  • Move to Process subfolder in Igor databrowser
  • Select all relevant spectra with mouse
  • Now select under Macro menu OperateOnSpectralData -> ExtractRowFromMultipleSpectra and fill in with melt wavelength (e.g., 220nm)
  • Give resulting wave a name with prefix of prg_
  • Create a wave of temperature values with same root name using a prefix of tmp_
  • You can now graph the melting curve and analyze using IgorDenat routines.

Processing and Analyzing CD Spectra