• 10.1 HMQC
• 10.1.1 HMQC with X on the First Decoupler Channel
• 10.1.2 HMQC with C13 on the Second Decoupler Channel

• 10.2 HSQC EXPERIMENTS
• 10.3 HSQC WITH FLIP BACK WATER SUPPRESSION
• 10.4 HMQC-NOESY (OPTIONAL FLIP BACK WATER SUPPRESSION)
• 10.5 HETCOSY (1H-31P CORRELATIONS)

Two dimensional heteronuclear experiments are essentially identical to their homonuclear counterparts. The only minor difference between them is that the second indirectly detected dimension contains chemical shift information about the Heteronucleus. Heteronuclear chemical shift correlation experiments correlate the chemical shift of one nucleus with the chemical shift of another. In this section we will deal with three types of correlation experiment. The Heteronuclear Multiple Quantum Correlation experiment (HMQC) and the Heteronuclear Single Quantum Correlation (HSQC) produce essentially the same result. The HSQC experiment is a more sensitive experiment but for large molecules the greater number of pulses can often lead to a degradation in signal to noise. In these cases the HMQC experiment is preferred.
The Heteronuclear COSY(hetcosy) again produce a chemical shift correlation but it does so by a COSY type transfer and has a fundamentally different appearance to either of the previous experiments. The HETCOSY experiment is more widely used in 1H-31P correlation spectra of nucleic acids and this will be described below.

The HMQC experiment was previously used to calibrate the X nucleus pulse indirectly in section 7.2 and the set up is essentially the same except in this case we assume that you have already calibrated the pulse. As mentioned before we normally use the 3rd Channel for C13. THis section provides guidlines and set up files for using the first and second decoupler channels for C13. However, we strongly encourage you to use the 3rd Channel version whenever possible.

uclib: hmqc_N15_protein.fid, hmqc_C13_protein.fid. hmqc_C13_DNA.fid
1. Enter the command "hmqc"
Respond with a <RETURN> to the question about the C13 experiment
2. Set up the 1H Parameters:
- Set tn='H1' and set pw, tof, sw, and np appropriately.
- Set ss=4 and nt=16
- Set ni=1 and phase=1 for 1D test
3. Remove unwanted parameters:
- Set mbond='n', null=0,
4. Set up Presaturation Parameters for samples in H₂O:
- Set satmode='y', presat=1.5, satpwr=10, satfrq=tof
- Set sspul='n' and hs='n'
5. Set up C13 or N15 parameters accordingly:
- set dn and dof to appropriate values
- set homo='n'
- set pwxlvl=60 and pwx to calibrated value
- set j=90 for N15 and j=145 for C13
6. Set up the X Nucleus Decoupling
- Set dm='nny' and dmm='ccg'
- Set dpwr and dmf to required values (See Section 8.2)
7. Acquire a 1D spectrum
- The spectrum should contain only a subset of those resonances which are directly bonded to the heteronucleus.
- Adjust the value of alfa if necessary
8. Set up 2D Parameters
- For N15 set ni=128 and phase=1,2 and set sw=2600
- For C13 see section 10
- Set nt = a multiple of 32
- set d2=1/(2*sw1) BRACKETS!!
9. Start the experiment

uclib: hmqc_C13_Protein_3RF.fid. hmqc_C13_DNA_3RF.fid
1. From the directory /home/uclib/setup2d load the appropriate setup file
- Set tn='H1' and set pw, tof, sw, and np appropriately.
- Set ss=4 and nt=16
- Set ni=1 and phase=1 for 1D test
2. Remove unwanted parameters:
- Set mbond='n', null=0,
3. Set up Presaturation Parameters for samples in H₂O:
- Set satmode='y', presat=1.5, satpwr=10, satfrq=tof
- Set sspul='n' and hs='n'
4. Set up C13 parameters :
- Set dof2 to -4480 for Proteins and 4050 for DNA
- set homo='n'
- set pwxlvl=60 and pwx to calibrated value
- set j=145 for proteins and 175 for DNA
6. Set up the C13 Decoupling
- Set dm2='nny' and dmm2='ccg'
- Set dpwr2 and dmf2 to required values (See Section 8.2)
7. Acquire a 1D spectrum
- Adjust the value of alfa if necessary
8. Set up 2D Parameters
- Set sw to 5600 for Proteins and 4500 for DNA
- Set nt = a multiple of 32
- set d2=1/(2*sw1) BRACKETS!!
9. Start the experiment

uclib: hsqc_N15_protein.fid, hsqc_C13_protein.fid, hsqc_C13_DNA.fid, hsqc_C13__protein_3RF.fid, hsqc_C13_DNA_3RF.fid
In principle the parameters for the HSQC experiment are identical to those used for the HMQC. However it uses a different pulse sequence. Again we suggest that you use the second decoupler channel for those experiments involving C13.
1. In the directory /home/uclib/setup2d load the appropriate HSQC file:
2. Check the parameters as given for the HMQC sequences

uclib: sehsqc.fid
The HSQC experiment can be combined with the Flip Back water suppression technique to improve the sensitivity of amide resonances. This version contains a so called "Sensitivity Enhancement" and should not be used for C13 experiments. In this experiment a second set of FIDS is collected that is 180[ring] out of phase with the first set. These two sets of FIDS are later combined during the processing.
1. Calibrate a selective pulse on the water (see Section 7.3)
2. From the directory /home/uclib/setup2d/sehsqc load the setup file
3. Set up the 1H Parameters
- Set tn, tof, tpwr and pw appropriately
- Set d1, sw and np appropriately
- Set nt to a multiple of 8
4. Set the N15 Parameters
- Set dn=`N15' and dof =1700
- Set pwx and pwxlvl to calibrated values
- Set dm=`nny', dmm=`ccg'
- Set dpwr and dmf to calibrated values (see Section 5.3) Remember GARP gives decoupling over 5xB₁
- Set j=90
- Set ni=1, phase=1 and enhance=0
- Set taud=0.0028
5. Set the Selective Pulse Parameters
- Set shape =`gauss'
- Set shpwr to calibrated value (See 1)
- Set sh1pw, sh2pw, sh3pw and sh4pw to the calibrated value
- Set tau=sh4pw+g3t+.0006
6. Optimize Water suppression
- Set up arrays over ± 500µs of the calibrated value for the shaped pulses in the order sh4pw, sh3pw, sh2pw sh1pw
- In each case choose the value that gives the best water suppression. It is not as easy to judge this as in other experiments. Check the appearance of the FIDs with dfsh and dfsh(`imag')
7. Set up 2D Parameters
- Set ni=128 and phase=1,2
- Set enhance=0,1
- Make array=`phase,enhance'
- Set nt=a multiple of 16. Remember you must set nt to half its value in a normal HSQC experiment.
8. Start the experiment

uclib: hmqcnoesy.fid
This sequence combines an HMQC correlation with a NOESY. The final cross spectrum contains NOESY cross-peaks that are edited according to the chemical shift of the directly attached N or C atom. The sequence is written in such a way that you can use either presaturation (e.g. for sample in D₂O) or the Flip Back Watergate sequence for those samples with rapidly exchanging protons. If you are using the Flip back method you must calibrate a selective pulse on the water as outlined in Section 7.3 The set up file as provided is for a 1H/15N experiment with Flip-Back Water Suppression.
1. From the directory /home/uclib/setup2d load the file "hmqcnoesy.fid"
2. Set 1H Acquisition Parameters
- Set the values of tof, tpwr, pw, sw, np and d1 appropriately
- Set nt=32
3. Set the X Nucleus Parameters
- Set the dn, dof, pwx, pwxlvl and sw1 as appropriate
- For C13 set j=145 for N15 j=90
- set dm=`nny', dmm=`ccg'
- Determine the power and pulse length for GARP decoupling outlined in Section 5.6.3
- Set dpwr to the required power and dmf=1/90[ring] pulse length at chosen power level
- Set ni=1 and phase=1 (For 1D test)
4. Flip Back Solvent Suppression Method
- For Flip Back Water suppression set wg_flg=`y'
- Set mix >= 0.1 sec
- Set shpwr to power for selective 90[ring] pulse
- Set sh1pw, sh2pw and sh3pw to selective 90[ring] pulse length
- Set nt=1
- Optimize the duration of the shaped pulses in the order sh3pw, sh2pw and sh1pw
- Adjust the value of tau so that the final refocusing delay is compensated.
- Optimize the value of tof
5. Presaturation Method
- Set wg_flg=`n'
- Set satpwr=10 and presat=1.5
- For no solvent suppression set presat=0
- Optimize the value of tof
6. Setup 2D Parameters
- Set ni=128 or 256, set phase=1,2
- Set nt to a multiple of 32

uclib: hetcosy_p31.fid
The hetcosy experiment is a COSY type experiment. This means that the cross peaks appear as antiphase doublets in both dimensions as in a DQF-COSY. The experiment is very insensitive. In part due to the small 1H-31P coupling constants. Solvent suppression is achieved by a train of 180[ring] pulse, but this also helps to generate an NOE between 1H and 31P, although this effect is small.
1. From the directory /home/uclib/setup2d/hetcosy_p31 load the setup file
2. Set up the 1H Acquisition Parameters
- Set tof, sw, tpwr, pw and as appropriate
- Set cycles=30-40 This is the number of 180[ring] pulse in the saturation train
- Set loopdly=0.05 This is the delay between 180s
3. Set up P31 and 2D Parameters
- Set pwx, pwxlvl (< 55 dB) and dof
- Set sw1 appropriately
- Set ni =64 or 96q
- Set phase=1,2
- Set nt to something big e.g. 512
4. Start the experiment

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