When performing 2D-NOESY measurement by NMR, I was told that the mixing time should be set to about T1.
However, if we wait for the mixing time of T1, the magnetization may have changed from negative to positive due to the effect of relaxation.(I heard that the negative magnetization becomes null(0) at about 0.7×T1.)
Then, why are NOESY diagonal peaks detected with a negative sign? Is it less susceptible to mitigation?
I would be grateful if you could tell me the reason.
Diagonal peaks do not normally go negative.
How's your phasing? Off diagonal peaks can go positive or negative.
Are you sure you're doing a NOESY and not a magnetization transfer.
David Stephenson
I’m very grateful for your answer.
As you say, when I usually use NOESY, the diagonal peaks are positive. However, when I read the textbook, I saw a figure with a negative diagonal peak.
So, I understand that the spectrum I usually see is the one with the diagonal peak set positively.
Is that my understanding different? Is the diagonal peak actually positive due to the effect of relaxation?
NOESY is picking up residual (internuclear) dipole-dipole interactions.
Magnetization is exchanged between spins by a "flip-flop" process which makes the off-diagonal peaks 180 degrees out of phase with the diagonal.
In some systems there is chemical exchange this does not change the phase so gives positive of-diagonal peaks.
As for relaxation - the polarization has to make it through the mixing time or there will be no signal.
You should read Page-310 of Prof. Ray. Freeman's book SPIN Choreography (Basic Steps in High Resolution NMR). The relationship between molecular motion and observation frequency is explained. The sign of NOE is reversed for small compounds and proteins. In the case of oligosaccharides, I think the effect of water on the sugar should be considered.
Dear Hiroki
whether the diagonal is positive or negative is for the user to decide. But the physics of the molecule will decide if the cross peaks are the same phase or opposite phase to the diagonal. For small molecules nOe is positive and NOESY peaks will be opposite phase to the diagonal. For intermediate sized molecules the nOe can be zero and for larger molecules the nOe is negative and the NOESY peaks are the same phase as the diagonal peaks. The mixing time does not have to be equal to T1. nOe will build up with the same rate as T1 but making the mixing time too long will result in loss of intensity. To short and it has not built up enough. The length of mixing time will also depend on the size of the molecule. The larger the shorter and the smaller the longer.
Clemens
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