Operating the Jasco J-815 Circular Dichroism Spectrometer
A. Turning the Instrument on:
1. Turn the nitrogen tank valve on (silver knob on top of the tank) and check the pressure
regulator to ensure there is nitrogen left in the tank. On the flow meter on the instrument
(right side), use the black knob to adjust the flow of nitrogen to ~20 ft3/hr (the measurement
should be read at the top of the silver cylinder). Allow the nitrogen to purge the CD for 15
minutes. IMPORTANT: Running the instrument without nitrogen will result in
permanent damage to the optics of the instrument. Before using the instrument, verify
that there is sufficient nitrogen in the tank, especially before performing long runs.
Switch to a back up tank if necessary.
2. Going from right to left, turn on the water circulator, the Peltier device, followed by the CD.
 Allow the CD lamp to warm for 30 minutes before taking data.
 The circulator must be on for the Peltier temperature controller to function properly.
Attempting to cool the Peltier device to near or below room temperature without the
circulator on can damage it permanently. There should be a small amount of water
flowing through the left tube connected to the front of the instrument.
B. Starting the Spectra Manager Software:
1. Select the Spectra Manager 2 icon from the desktop by double clicking.
 For best results, the Spectra Manager Software suite should be closed when the
instruments are turned on.
2. Select the desired measurement function from the left panel of the Spectra Manager window.
This will prompt the software to “initialize instrument.” If this does not initialize the
instrument, restart the computer and reopen the software. NOTE: For general CD analysis
applications (i.e. protein structure), select “Spectra Measurement” and see pgs. 3-4 of this
manual for more detail.
C. Turning the Instrument off:
1. Close the Spectra Manager software. Leave the PC on.
2. Going from left to right, turn off the CD, the Peltier Device and the Water Circulator.
3. Wait 5-10 minutes, and then turn off the nitrogen gas.
D. General Sample Preparation Notes:
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As a general rule, the absorbance of the sample should be about 1 at the wavelength of
interest in a cuvette with the same path length that will be used for CD spectral analysis.
o A ballpark starting concentration would be ~ 0.5 mg/mL protein, or maybe half that
for -helical proteins and twice that for -sheet proteins.
If the HT voltage is > 600 V, the total absorbance of your sample is too high.
o Use a shorter path length cuvette or dilute the sample.
Note that if your buffer/solvent absorbs in the region of interest, it will add to the total
absorbance (HT voltage) without improving signal.
o Avoid buffers, detergents or other molecules that contain aromatic groups, thiol
reducing agents (eg. DTT), imidazole, metal ions, and chloride salts. The best buffer
for CD is usually low concentration (5-50 mM) phosphate or acetate with no salt (<
300 mM). The table below lists the wavelength cutoffs for common buffer
components.
E. Parameters for a Spectra Measurement
 Select “Spectra Measurement” from the left panel of the “Spectra Management” window.
 Click the “Parameters” icon, which is represented by a white hand hovering over a
keyboard.
1. “GENERAL” TAB
Photometric mode (to select the number of channels to view during collection)
General settings: select 2 channels (HT voltage and CD signal). NOTE: Any data collected with
HT voltage 600-800 V is suspect and above 800 V is not valid.
Sensitivity
General setting: Standard. For samples with small differential absorbance of the circularly
polarized light, use high sensitivity.
D.I.T. (amount of time the detector will read the sample before taking the average) General
setting: For most samples 2 seconds is sufficient. Changing integration times can result in a
smoother spectrum if necessary.
Band width
General setting: 1 nm. Increase to 2 nm if the signal is too low.
Slit width
Leave the box unchecked. This will allow the instrument to automatically adjust to the
appropriate slit width at different wavelengths.
Start and End (determine the start and end wavelengths to be collected)
General setting: For proteins, the far-UV range for estimating secondary structure is 190-250 nm
and the near-UV range, which reports on the environment of aromatic amino acids, is 250-350
nm. NOTE: “Start” is the high wavelength of the range, and “End” is the low wavelength.
Data pitch (determines the number of data points taken during the scan)
General setting: 0.5 nm, but depends on the “Scanning speed” (see below under “Scanning
mode” for more detail).
Start mode
General setting: Immediately
Scanning mode
General setting: CONTINUOUS. Collects data as a sliding window while still averaging for
each point in the “Data pitch”. This method is dramatically faster than the other methods and no
less accurate. The rate at which CONTINUOUS mode collects data is determined in the
“Scanning speed” drop box. 100 nm/min is appropriate for most applications. As a general rule,
the “Data pitch” x “Scanning speed” should be below 200.
Accumulation/Repeat
ACCUMULATION mode specifies the number of scans (accumulation), and the software
automatically averages the scans and exports the data as one curve.
REPEAT mode specifies the number of scans (“cycle times”) and each curve is exported
independently. This mode makes it easier to see the reproducibility of the spectra.
2. “CONTROL” TAB
The instrument allows for manual or automatic subtraction of background.
Correction
NONE: One would scan their buffer as a sample, their sample, and manually subtract the blank
using the Spectra Manager Analysis or Excel.
BASELINE: At the start of the run, the software will prompt one to insert the blank and collect
the background spectra. This spectrum will automatically be subtracted from the subsequent
samples.
Option to open and close shutter automatically: Select this option for light sensitive samples.
3. “ACCESSORIES” TAB
The Accessories tab controls external devices like the Peltier device. The temperature can be
adjusted in this window as well as the method for temperature monitoring.
General settings: HOLDER in both. If one is concerned with thermal conductivity and the exact
temperature of the sample, external probes (the thin clear wire) can be placed within the cuvettes
(in this case CELL would be selected in both).
4. “INFORMATION” TAB
Annotate the file with useful information that will be exported to the Analysis software
5. “DATA” TAB
This is the location one enters the directory in which the data will be saved to.
Leave “Auto save” unchecked and “Send to Spectra Analysis” checked. This setting will allow
for you to manually save your data in the Spectra Analysis section.
Leave “Print measured data” unchecked.
F. Running a Wavelength Scan.
For NONE Correction – Press the cyan “S” in the toolbar. Selecting MEASURE, will begin the
run. At the conclusion of the run, Spectra Analysis will automatically open.
For BASELINE Correction – Press the green “B” in the toolbar. Press MEASURE to collect the
BLANK spectrum. Note that this data is not saved as a file or displayed in Spectra Analysis. It is
saved to memory and auto-subtracted from each sample. If you need to analyze of collect the
background spectrum select NONE correction in the Control tab of Parameters Settings. Once
the background sample is collected, press the cyan “S” in the toolbar, then press MEASURE.
Spectra Analysis will automatically open and display data at the conclusion of the run.