2D 1H-13C HSQC

2D Gradient Enhanced 1H-13C HSQC

The following figure is the Varian gradient selected 2D 1H-13C HSQC pulse sequence (gHSQC) with the main parameters displayed (NMR500).

Procedure (on NMR500)

• Temperature must be regulated.
• Turn spin OFF.
• Gradients amplifier should be turned on (displays RUN). Type pfgon='nny' su to allow gradient pulses.
• Lock and shim your sample as usual, except also adjust X and Y shims a bit (because spin is off now). Keep lock at ~80% after shimming.
• Find proper sw and tof to use for 1H channel
• Under exp1 (type jexp1 to go there) collect a standard 1H spectrum with nt=1 (or 4 for cleaner spectrum)
• Put the box cursor to enclose the signal region and to include ~10% flat baseline area at each end of the peak region. Type movesw. This command sets a new spectral width sw and spectrum center tof.
• Run another 1H spectrum with nt=1or 4 with the new setting. Reference the spectrum carefully.
• Decide what 13C dof (center of spectrum) to use for 13C channel.
• In the following we assume we want the 13C center to be at 70ppm with a spectral width of 140ppm to give a 13C range of 0 to 140ppm.
• Under exp1, following last step with 1H spectrum referenced, type dn='C13'
• Type setrefppm and enter the center ppm value you want. For 70ppm, enter 70.
• Write down the dof value printed out above the command line. This is the offset value for 70ppm for 13C.
• Load standard ghsqc parameter file
• Type jexp2 to goto exp2. From exp2, type mf(1,2) to move fid from exp1 to exp2. Type wft f full to display spectrum. Check reference.
• Type setexp('ghsqc') su to load standard ghsqc parameters. Solvent, and 1H spectral width and center are copied over from 1D spectrum.
• Type su. Tune 1H channel and X nucleus channel. Accuracy of pre-calibrated pw depends on probe tuning.
• Recable the X nucleus probe input cable.
• Disconnect the X nucleus cable at the filters in front of the broadband preamp (left side). Connect the X-channel probe input cable to one end of the long, 13C bandpass filter (labeled 13C on filter) on the floor. Connect the other end of the filter to the X-channel decoupler cable coming from the back of the magnet leg interface (long, tall box to the right of the magnet).
• Re-shim Z1 and Z2 a bit after this cabling change.
• Check/set proper 1H transmitter power and pulse width. If solvent is CDCL3, tpwr=57 pw=9.0. For other solvents, calibrate pw according to this procedure.
• Use default parameters values (~12 mins experiment) except the following:
• pw=XXXpw90=pw (9.0 for cdcl3 as solvent or value calibrated with your solvent at tpwr=57)
• dof=XXX (value found above)
• sw1=XXX Estimate it based on how much you need to enclose all 1H-attached 13C peaks around dof. As an example, for a 160ppm width, type sw1=160*dfrq.
• Optionally, for better quality data (may results in longer data collection time):
• ss=32 (more dummy scans before data collection)
• nt=4 (or 8, 16, 24, ... 4*n) for more sensitivity and less artifacts
• ni=200 (better resolution along indirect dimension)
• d1=2 (for less artifacts and sensitivity, but doubles data collection time)
• Type time to check experiment time.
• Type go to collect data.
• Watch for ADC or receiver overflow:
• In arrayed experiments such as 2D's, a fixed gain (not autogain) must be set. Watch the receiver overflow light and the ADC overflow error message immediately after the experiment starts. If the receiver overflows (red light blinks on VT display) or ADC overflow message is seen above the command area anytime during the experiment except during the dummy scan period, it is likely the data are ruined. Stop the experiment and reduce gain by 2-4 at a time until the overflow disappears. The default (gain=40) works most of the time unless there is an extremely strong 1H signal in your sample. With a gain above 20, it makes only small differences.
• After the experiment is done, recable the X (decoupler) channel connection to the original configuration.

Default parameters

The default setting (modified from Varian's default values) in our parameter set uses the following for a quick HSQC experiment:

• ni=128 (128 complex points along indirect dimension)
• d1=1 (recycle delay between scans is 1 sec)
• ss=16 (16 dummy scans before actual data collection)
• nt=2 (2 scans)
• C13 (dof) centered at 70ppm
• C13 sw1 ~ 140ppm
• mult=2 (1H multiplicity detection on)
• phase=1,2 (phase-sensitive, hypercomplex mode in indirect detection)
• gain=40 (reduce it by 2 at a time if receiver or ADC overflows)
• sspul='y' (solvent suppression and spin predelay randomization with homospoil gradients turned on)

Data Processing

You can use vnmrJ to process the data, but vnmrJ's graphics display for 2D spectrum leaves much to be desired. A separate program is strongly recommended for detailed processing and analysis.

One of the most widely used free processing/display software package for multi-dimensional NMR data processing is nmrPipe/nmrDraw. This program package has been installed on NMR500 and the data station. Details on how to use them will be updated.

Example spectrum collected on NMR500

Expansion of the upper right region:

H.Zhou updated Oct 2010