H

Figure 9.20

H2 and H6. But where do we go from here? The Hd/e peak connects to Hb and to Hc, but we cannot tell which is H3 and which is H5 because we lost the specific trails of the two spin systems when they "crossed" at Hd/e.

We can resolve this ambiguity with a TOCSY spectrum. This is just a homonuclear 2D (!H-!H) experiment with the TOCSY spin lock as the mixing portion of the pulse sequence. With the fragment CH!-CH2-CH3-Cq4-CH5-CH6-CH7 we expect to see H1 correlated

to H2 as it was in the COSY spectrum, but we will also see H1 correlated to H3 because the TOCSY spin lock transfers coherence in multiple /-coupling jumps. Likewise, H7 will correlate to H6 but also to H5 (Fig. 9.22). Thus there is no ambiguity and we can directly "read off" the two spin systems. Starting with Ha on the diagonal at the lower left, we can extend a line up or to the right and pass through the Hc and the Hd/e crosspeaks. Starting with Hf on the diagonal at the upper right, we can move down or to the left and pass through the Hd/e and Hb crosspeaks. We can assign peak c to H3 and peak b to H5. The "new" crosspeaks that represent multiple /-coupling jumps are shown in gray. The disadvantage is that we lose the information about the order of protons in the spin system: without the COSY data, we don't know if the order is CHa-CHd/e-CHc or CHa-CHc-CHd/e. We saw in Chapter 8 that to some extent the intensity of TOCSY transfer peaks can help us to put them in order because in long spin systems the "smearing" of coherence tends to be a diffusion-like process, giving less intensity to peaks that result from larger numbers of jumps. But to be certain of the exact order of protons in a spin system requires a COSY spectrum, which is limited to single "jumps" through / couplings.

9.4.1 Examples of 2D COSY Spectra

Figure 9.23 shows the 600 MHz DQF-COSY spectrum of 3-heptanone. As a starting point, we will use the two CH2 groups closest to the ketone carbonyl. These protons should be shifted downfield to the region of 2-2.5 ppm (compare to acetone at 2.1 ppm). There are two overlapped peaks on the diagonal (lower left) that can be identified as separate peaks because their crosspeaks do not line up: the downfield peak lines up vertically and horizontally with crosspeaks at 0.90 ppm, whereas the upfield peak lines up with crosspeaks at 1.42 ppm. Following the spin system from the downfield diagonal peak (2.30 ppm), we can move to the right and up (or up and to the right) to the diagonal peak at 0.90 ppm. This "box" (dotted lines) is a dead end: there are only two resonances in the spin system. So we can

F2 (ppm) Figure 9.23

assign it to H2 (2.30 ppm) and H1 (0.90 ppm). This spin system represents the ethyl group: CH2-CH3. By process of elimination, the upfield peak of the overlapped pair (2.27 ppm) must be H4 on the other side of the ketone carbonyl. Following the crosspeaks on either side of the diagonal (dotted lines) we move to H5 (1.42 ppm), then to H6 (1.18 ppm), and finally to the methyl group H7 (0.77 ppm). The dotted lines show how this spin system of four resonances (n-butyl group) is distinct from the spin system of two resonances (ethyl group). Note how the methyl groups are the most upfield, both because they are farthest from the carbonyl group and because there is an inherent upfield shift as we move from CH to CH2 to CH3 (¿1.6, 1.2, and 0.85 in a saturated hydrocarbon).

For comparison, the 600 MHz TOCSY spectrum of 3-heptanone is shown in Figure 9.24. The first thing you notice is that there are a lot more crosspeaks! This does lead to more clutter but now instead of having to "walk" from diagonal to crosspeak to diagonal, you can move horizontally or vertically and spell out the entire spin system along one line. The top line, starting at the right with the H7 peak on the diagonal, moves left through crosspeaks for H6, H5, and H4 (it is only coincidence that they are in order). Because the crosspeaks are in-phase, we can even read off their splitting patterns: triplet for H7, quartet for H6, quintet for H5, and triplet for H4. This fine structure is only visible in the F2 (horizontal) direction; in F1 the resolution is poor because we collect far fewer data points in the t1 FID. Along this top horizontal line the magnetization started with the H7 methyl group (F1 = 0.77 ppm), transferring in one /-coupling "jump" to give the crosspeak at F2 = H6, two "jumps" of TOCSY transfer to give the H5 crosspeak, and three "jumps" to reach H4.

There are typically two kinds of artifacts in COSY spectra: t1 noise (vertical streaks) and DQ artifacts. The t1 streaks extend up and down from the most intense and sharpest peaks along the diagonal. These result from any instability that can cause random variations

F2 (ppm) Figure 9.24

between different FIDs in the 2D acquisition (different t\ values). If after the first Fourier transform the peak heights in the interferogram (Fig. 9.2) are not exactly reproducible from spectrum to spectrum, this introduces random "noise" in the ti FID and after the second Fourier transform we have noise in the Fi spectrum which is inserted back into the data column. This "noise" only shows up in the columns which correspond to peaks in the spectrum, so it looks like vertical streaks in the 2D spectrum. Because they are truly random variations in intensity, however, there are no "tricks" of smoothing or subtraction that can remove them. We can only try to reduce any source of variation (unstable electronics, temperature variation, sample spinning, building vibration, etc.) during the 2D data acquisition. For this reason, we never spin the sample during a 2D acquisition. The DQ artifacts show up along two lines extending from the lower left and lower right corners of the 2D spectrum to a point on the top in the exact center in F2 (Fig. 9.25), at the F2 frequency of each peak on the diagonal. Again, the stronger peaks in the diagonal tend to give the most intense artifacts. These can be distinguished from crosspeaks because they are not symmetrically disposed about the diagonal. If you have any doubt about a crosspeak, always check for its partner on the other side of the diagonal.

A portion of the 600 MHz DQF-COSY spectrum of 15-^-hydroxytestosterone (Fig. 9.26) is shown in Figure 9.27. As before, Fi assignments are written to the left side or right side of a crosspeak and F2 assignments are written above or below the crosspeak. The amount of information packed into this single experiment is amazing! The assignments were obtained by using this data along with other 2D experiments that connect 1H to 13C (Chapter 11). There are several overlapped groups on the diagonal; for example, at the lower left side we see two overlapped peaks on the diagonal. The downfield one is H6^ alone and the larger upfield one is H2^ and H16a. We can see that H2^ is slightly downfield of H16a by looking

at the H2^ - H1a crosspeak (center left), which is slightly to the left of the H16a - H16^ crosspeak just above it. You can also see that the H2^ resonance is wider and appears as a triplet in F2, whereas the H16a resonance is narrower. In this way, we can see resolved crosspeaks even when they are badly overlapped or even precisely the same chemical shift on the diagonal. This is one of the huge advantages of 2D NMR.

For resolved peaks we can easily move along a horizontal or vertical line to identify all of its coupling partners. Starting at the right with the H8 resonance (F1 = 2.03 ppm), we can see COSY magnetization transfer (moving left) to H14, H9, H7a and then, passing through the diagonal, to H7^. All of these are vicinal relationships involving a single J coupling to H8. Starting at the center left side we can follow the F1 = H1a line to the right, passing the H2^, H2a, and H1^ crosspeaks, then going through the diagonal peak to a very narrow crosspeak with H19, the angular methyl group singlet. How can we have a COSY crosspeak to a singlet? There is a small "W" coupling between the axial H1a proton and the H19 methyl group due to the anti relationship of H-1a and C19 (Fig. 9.26, bottom). At least one of the C19 methyl protons is in a "W" relationship with H1a, and this leads to a COSY crosspeak even though the coupling is not resolved in the 1D 1H spectrum.

Figure 9.28 shows the 300 MHz DQF-COSY spectrum of sucrose in D2O. For convenience, the 1H spectrum is shown at the top and on the left side. To analyze the COSY spectrum you always need a starting point: a 1H resonance that is resolved and can be

unambiguously assigned on the basis of its coupling pattern and/or chemical shift. The sucrose structure (Chapter 8, Fig. 8.44) predicts only two doublet resonances: H-g1 because it is coupled only to H-g2, and H-f3 because it is coupled only to H-f4. H-g1 is bonded to an anomeric carbon, with two bonds to oxygen, so it should be downfield of H-f3. So we can assign H-g1 to the doublet at 5.36 ppm (J = 3.8 Hz) and H-f3 to the doublet at 4.2 ppm (J = 8.8). Starting at 5.36 ppm (d, J = 3.8) with H-g1 on the diagonal (Fig. 9.28, lower left) we can move to the right to a prominent crosspeak (lower right) and then straight up passing through a crosspeak (rectangular box) and returning to the diagonal at 3.5 ppm (dd, J = 10.0, 3.8). This diagonal peak can be assigned to H-g2. Moving either left or back down from the H-g2 diagonal peak we encounter a crosspeak (rectangular boxes) that leads us back to the diagonal at 3.7 ppm (t, 10 Hz). This diagonal peak corresponds to H-g3. Before continuing our walk, let's take a closer look at the H-g1 to H-g2 crosspeak.

9.4.2 Fine Structure of COSY Crosspeaks

So far we have looked at the COSY crosspeaks as "blobs" of intensity which correlate one resonance (Ha in Fi) with another (Hb in F2) at the intersection of their chemical-shift

Figure 9.28

positions (F\ = Qa and F2 = Qb). But we know that the resonances Ha and Hb are not single lines: at the very least we would have doublets for the Ha and Hb resonances if they are coupled only to each other. If we extend the four individual lines of the two doublets into the 2D spectrum, we see that there will be four peaks in each crosspeak and four peaks in each diagonal peak (Fig. 9.29). Positive intensities are shown as closed circles and negative intensities are shown as open circles. The crosspeak arises from a transfer of antiphase coherence to antiphase coherence by the mixing sequence (a single 90o pulse), so the peak intensities are antiphase (+/-) in each dimension. The upper left crosspeak (F2 = Qa, F\ = £2b) gives an antiphase doublet if we display a horizontal slice or a vertical slice through the peak. It often happens that the crosspeaks are "out in the open"—not overlapped with other crosspeaks—whereas the corresponding peaks in the 1D spectrum are overlapped and cannot be analyzed to extract J couplings. By making a slice through the crosspeak, we can measure the J coupling and assign it clearly to the coupling between the two resonances that give rise to the crosspeak (in this case, Jab between Ha with frequency Qa and Hb with frequency £2b). When analyzing a 1D spectrum one can determine J couplings accurately in resolved multiplets, but it is not always clear which of the other peaks in the spectrum is the coupling partner for each of the couplings. The 2D COSY clearly shows us which resonances are coupled, and the fine structure of the crosspeak can give us the exact coupling constant value.

In the inset of the sucrose COSY spectrum (Fig. 9.28, upper left), the H-g2 (F1) to H-g1 (F2) crosspeak is enlarged to show its fine structure. In the 1D spectrum the H-g1 peak is a doublet (J = 3.8 Hz) because it is the anomeric proton (at the "end of the line") and nil cqsy n

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