DEPT

DEPT (Distortionless Enhancement by Polazization Transfer) is a 13C detected, multiplicity analysis experiment that differentiates CH, CH2 and CH3 carbons from each other. Quaternary carbons are suppressed in the experiment. Varian supplies a single DEPT pulse sequence (Dept). The editing is done in the pulse sequence via a 1H pulse set to a 45, 90, or 135 degree rotation. The actual setting is through a multiplier mult to a 90 degree pulse as shown in the table below:

In the following, as commonly found in literature, we will refer to the individual elements of dept as dept45, dept90 and dept135.

Varian Dept Pulse Sequence

Note:

• DEPT requires accurate 90 degree 1H and to a lesser degree 13C 90 degree pulse width to give a clean editing results.
• Varian's processing macro adept requires data with an array of 4 experiments with mult=0.5, 1, 1, 1.5 with 1 run twice for even sensitivity. The macro processes the raw data (wft adept) to produce four individual spectra: All (CH+CH2+CH3), CH only, CH2 only, and CH3 only.
• If you already have a 13C spectrum, it is often sufficient to run dept90 and dept135 separately in order to distinguish CH, CH2, and CH3 from each other. In this case, judge from chemical shifts or other info to phase the peaks up or down according to the table above. If desired, optionally run dept45 to get the phase correct before phasing the dept90 and dept135 spectra.
• Optionally, you can run a customized 5-element array experiment deptnoe (DEPT + 13C direct detection with NOE).

Procedure (on NMR500 with SWP probe)

Notes for all DEPT experiments:

• Turn ON temperature regulation (done by FTS on nmr500)
• Turn OFF spinning
• Cable connection remains the same as for 13C direct detection
• Tune both 1H channel and X-channel for 13C. This is critical for clean editing.
• If some imperfection is to be tolerated in the final spectra, use the default pulse widths. Otherwise, for best quality or if you observe excessive poor editing results, calibrate 1H 90 degree pulse width with a precision of 0.5 usec at tpwr=57. The default setting assumes cdcl3 as solvent at 22C, but should also apply to solvents with similar dielectric properties.

Option 1 (recommended)

• Lock and shim with spin off. Keep lock level at ~80%.
• Type setexp('dept') to run dept45, dept90, and dept135 together as an array in a single experiment. In this experiment, the four elements with mult=0.5, 1, 1, 1.5 are set to run in interleaved mode with bs=16. In the this mode, each element of the array is run bs scans and then the cycle starts from the 1st element again for another bs scans. Each cycle takes ~ 2.2 mins.
• Change solvent to yours
• Tune 1st channel to 13C and 2nd channel to 1H
• Optional: Change pw at tpwr=57 to calibrated value if available
• Change nt to a large number of multiple of 16 (nt=16000). For long unattended experiment, set nt according to time available.
• Type au to start data acquisition. au allows certain macros to be executed every bs or other interval.
• Default gain=34. Reduce it by 2 at atime if receiver overflow light blinks on VT display panel, or ADC overflow message displays above command line.
• Stop the experiments by typing stop whenever you feel the sensitivity is sufficient. If you use aa or click the Abort Acquisition button, make sure you wait till a whole array cycle has completed (when the FID number in the bottom status panel is #1). Terminating the experiment in the middle of the array makes the intensity uneven among array elements.

• Processing:

  You can always click Process->Autoprocess to process DEPT data after the 1st array cycle is done. But manual processing is often needed for best result.

• During Data Collection:
• Type procarray to process all elements. Auto-phasing and scaling is applied. Spectra are displayed in stacked plot mode.
• Alternatively, type wft ds(1) f full to process all and display the 1st spectrum after the 1st bs cycle is done. Type aph to phase it and use manual phasing if needed. Once the 1st spectrum is phased up, the phases of other spectra are set properly. Type wft dssh dscale to display all four in horizontal view, or wft dssa dscale in stacked plot or vertical view. To shrink the peak heights to proper scaling, typing vs=vs*2 or vs=vs*0.5 etc. followed by dssa dscale.
• To separate the spectra CH, CH2 and CH3, type adept after wft. Try wft adept dssa.
• After Data Collection:
• To look at raw (unedited) data from each dept element, follow the processing procedure above.
• If total spectral separation of CH, CH2 and CH3 is desired, click the autoprocess button . The macro applies an automatic phasing, add/subtract the raw data into All, CH, CH2 and CH3 spectra. Note some add/subtract artifacts may be visible. Alternatively, for manual processing, type wft adept to do editing. After this command, type ds(1) f full aph (manually correct phasing if necessary), followed by dssa. Note adept should always act on data processed after wft command. Always proceed adept with wft.
• To print the either edited or unedited spectra, type pldept.
• Or: type autodept to auto-process and print both edited and edited DEPT.

Helpful Processing and Printing Commands for DEPT

• procarray (auto-process, scale, and display array)
• plarray (plot array)
• autodept (auto-process and print both unedited and edited DEPT spectra)
• pldept (print either unedited or edited DEPT spectra)
• deptproc (performs DEPT editing, applies auto-phasing and scaling, and display stacked plot)
• adept (performing DEPT editing only. No auto-phasing/scaling or display is done. Always call wft before adept)

Option 2 (For individual dept45, dept90, or dept135 runs)

• Lock and shim with spin off. Keep lock level at ~80%.
• Type setexp('dept') to load customized parameters for DEPT experiment.
• Change solvent to yours
• Tune 1st channel to 13C and 2nd channel to 1H
• Optional: Change pw at tpwr=57 to calibrated value if available
• To run dept90, type mult=1
• Or: to run dept135, type mult=1.5
• Or: to run dept45, type mult=0.5
• Or: to run dept90 and dept135 in one experiment in an array mode, type: mult=1.0, 1.5
• Set nt to a large number multiple of 16 (nt=16000). For long unattended experiment, set nt according to time available.
• Type go to start data collection.
• Default gain=34. Reduce it by 2 at a time if receiver overflow light blinks on VT display panel, or ADC overflow message displays above command line.
• Stop the experiment anytime the normal way by typing aa or click the Abort Acquisition button.
• Processing:
• For single element data, type wft. Apply manual phasing for dept90 and dept135 according to Table 1 (DO NOT use autophasing for these two). For dept45, type aph for auto-phasing (with manual adjustment if necessary).
• For arrayed data, type procarray for auto-processing and display. Or, type wft f full dssa dscale to display arrayed spectra. Type ds(1) f full to select the first one to phase.

Default parameters

• ss=8 nt=64 bs=16
• d1=1 at=1 gain=34 lb=1
• tpwr=56 pw=12.5 (for 13C)
• pplvl=57 pp=9.0 (for 1H, in cdcl3)
• mult=0.5, 1, 1, 1.5
• 13C center tof: ~ 75ppm, sw ~ 180ppm covering -15ppm to 165ppm.
• 1H center dof: ~ 4.2ppm with 1H decoupling during 13C aquisition

Example

Sample: Strychnine at ~ 25mg/mL (~ 100mM) in cdcl3 Data collected October 2010 (Option 1, ~9 mins with nt=64)
The 4 elements of Dept with mult=0.5, 1, 1, 1.5 (from bottom to top) correspond to dept45, dept90, dept90, and dept135. These raw dept elements are sufficient for multiplicity analysis. Further processing with adept is optional.
Spectra further processed with adept command. From top to bottom: 1: CH3 2: CH2 3: CH 4: All (CH+CH2+CH3)

H. Zhou updated Oct 2010