2D NMR Spectroscopy Lecture 3
hemical shifts The chemical environment affects the magnetic field of nuclei. B eff = B o - B loc B eff = B o ( - σ ) σ is the magnetic shielding of the nucleus. Factors that affect σ include neighboring atoms and groups. The polarization of the bonds are also important. O- 2-3 low field high field ω o
The NMR scale (δ, ppm) You can use the frequency scale, but is it feasible? B loc is a lot smaller than B o, thus the range is very small (hundreds of z) relative to the absolute frequency (Mz). ν sample - ν ref δ = ppm (parts per million) ν ref Question: Is the chemical shift in ppm of an atom different at two different field strengths?
The chemical shift range for is relatively small (0 to 5 ppm) in most cases, although peaks above 20 and below -3 ppm have been observed in rare cases Imino groups DNA/RNA Aromatics Amides Protein side chain (α, β, γ, δ) Aliphatic 5 0 7 5 2 ppm
oupling Electrons have a magnetic moment and have a spin of ½. Scalar coupling is facilitated by the electrons in the bond connecting the two nuclei. This through-bond interaction results in splitting of the nuclei into (2I + ) states. Thus, for a spin /2 nucleus the NMR lines are split into 2(/2) + = 2 states. Mul$plet = 2nI + n - number of iden$cal adjacent nuclei I - spin quantum number 2 2 Quiz: ow does the spectrum of ethanol Looks like?
oupling onstants (J) The energy levels of a nucleus are affected by the spin state of neighboring nuclei. The two nuclei are said to be coupled to each other. 3 one-bond three-bond I β I β S S J (z) J (z) α I β S β I α S S I I S α I α S - The magnitude of the separation is called coupling constant (J) measured in z. - oupling patterns are crucial to identify spin systems in a molecule and to the determination of the chemical structure.
omonuclear coupling The magnitude of the J coupling is dictated by the torsion angle between the two coupling nuclei according to the Karplus equation. J (2-5 bond) = -20 z Geminal oupling ( 2 J) Dependent upon the bond angle between the nuclei. Generally, the smaller the angle the bigger the coupling constant.
Vicinal coupling ( 3 J) Karplus curve
eteronuclear coupling ( J X ) Estimate the value of the coupling constant of: 2 and 4 N 3 For 3 nuclei coupled to an, J = 50 to 250 z 5 N For 5 N nuclei coupled to an, J N = 70 to 00 z
an you identify all couplings in this chemical structure? o
D Pulse sequences z z M o y 90 y pulse M y acquisition 90 90 n
2D NMR Who is talking to who? o
2D NMR Preparation Evolution Miing Acquisition/ Detection t t 2 90 t 90 t 2 P E M D The first perturbation of the system (pulse) is called the preparation of the spin system. The variable t D is evolution time, t. Miing: information from one part of the spin system is relayed to other spins. Acquisition period (t 2 ): as for D eperiments. t is the variable delay time, and t 2 is the normal acquisition time. FT gives f and f2
The basic 2D spectrum involves repeating a multiple pulse D sequence with a systematic variation of the delay time t D, and then plotting everything stacked. We get two time domains, one that appears during the acquisition as usual, and one that originates from the variable delay.
90 90 t t 2 t=0 z z y 90 y ω o t>0 z z y 90 y ω o
z z y 90 y ω o z z y 90 y ωo z z y 90 y ωo z y 90 y ω o
Plot all the spectra in a stacking mode: A(t ) n t ω o f 2 (t 2 ) t 2 Why the intensity is decreasing with time?
A second Fourier transformation in the t domain (the first one was in the t2 domain) gives a two-dimensional spectrum: ωo ωo f f2 A spectrum with a lot of signals would be a mess. We look it from above, and draw it as a contour plot. ωo Each slice is color-coded depending on the height of the peak. ωo f f2
time - time 0.270 0.260 0.250 0.240 0.230 0 200 400 600 800 000 t2 pts t 0.220 t sec t 2 0.060 time - frequency 0.050 0.040 f2 500 600 700 800 900 pts t 0.030 t sec f 2 frequency - frequency f2 400 500 pts f f 2
o omonuclear correlation OSY OSY stands for Orrelation SpectroscopY, and for this particular case in which we are dealing with homonuclear couplings, homonuclear correlation spectroscopy.
eteronuclear correlation In a similar fashion, we can perform a 2D eperiment in which we analyze heteronuclear connectivity, that is, which is connected to which 3 or 5 N. The pulse sequence in this case involves both 3 and. 90 3 : 90 90 : t
The 2D spectrum is not symmetrical, because one ais has 3 frequencies and the other has frequencies.
Long Range couplings 3 MB (eteronuclear Multiple Bond oherence) Peaks have two- or three-bond coupling Sees through heteroatoms and quaternary carbons