Determining Chemical Structures with NMR Spectroscopy the ADEQUATEAD Experiment Application Note Author Paul A. Keifer, Ph.D. NMR Applications Scientist Agilent Technologies, Inc. Santa Clara, CA USA Abstract VnmrJ 3 software provides easy-to-use, interactive tools for setting up advanced experiments. This allows even novice users to obtain critical information about their research samples using the most advanced NMR experiments available. This application note is one of a series designed to provide step-by-step guidance for using sophisticated experiments to collect the exact data needed for your analyses. Introduction A key use of NMR spectroscopy is to determine chemical structures. The most important step in doing this for organic molecules is to determine their carbon framework. This application note describes a valuable tool that can be used in this process: the 1 H-detected adequate sensitivity double-quantum spectroscopy (ADEQUATE) experiment. In 1996, Reif and coworkers developed several versions of the 1 H-detected ADEQUATE experiment. 1,2 Their 1,1-ADEQUATE version was further optimized in 2003 to use adiabatic carbon pulses, 3 and is now a standard experiment in VnmrJ 3. It is available as a push-button protocol labeled ADEQUATEAD, where the additional AD reflects the inclusion of adiabatic pulses. This experiment correlates two directly-connected carbons to protons attached by one bond to one of the carbons.
An Example of ADEQUATEAD Data: Acquisition and Interpretation Figure 1 shows an ADEQUATEAD spectrum acquired using the single-quantum (SQ) mode, and the corresponding 1 H and 13 C spectra for a sample of menthol in deuterated benzene (C 6 D 6 ). Menthol has a molecular formula of C 10 H 20 O, and because it is a fully aliphatic compound, all of the carbon resonances lie between 15 and 75 ppm ( 13 C). F1 (ppm) 20 30 40 50 B C The SQ ADEQUATEAD data correlates two directly connected carbons to the protons attached over one bond to one of the carbons. One way to interpret the data from this experiment is to start with the carbon axis. To do this, select a single carbon signal (along the vertical F1 axis in Figure 1) and note which proton resonances are correlated to it. For example, as outlined by the horizontal blue boxes near the bottom of Figure 1, the carbon signal at 70 ppm (Box A) shows correlations (along F2) to proton signals at 1.9, 1.0, and 0.9 ppm. Likewise, moving up the carbon scale you can see that the carbon at 50 ppm (Box B) shows correlations to protons at 3.2, 2.2, 1.45, and 0.8 ppm. 60 70 80 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 F2 (ppm) Figure 1. A two-dimensional (2D) ADEQUATEAD spectrum of menthol, acquired using SQ mode and an adiabatic sequence, along with the corresponding one-dimensional (1D) 1 H and 13 C spectra. A You can also begin the analysis from the proton spectrum. For example, as outlined by the vertical blue box near the top of Figure 1, the proton at 2.2 ppm (Box C) shows correlations (along F1) to carbons at 16, 21, and 50 ppm. 2
The SQ ADEQUATEAD data is interpreted with a thought process much like the one used to interpret heteronuclear multiple bond correlation (HMBC) data. The difference is that SQ ADEQUATEAD data show only two-bond H-C correlations, whereas HMBC data can arise from two, three, or four-bond H-C correlations. The advantage of SQ ADEQUATEAD data (over HMBC) is that the structural information it provides is unambiguous because SQ ADEQUATEAD data traces out only direct carbon-carbon bonds. HMBC experiments provide a larger quantity of information (more correlations), but the quality of that data is lower. This leads to ambiguity for the structural information provided by HMBC data and makes the interpretation harder than for SQ ADEQUATEAD data, especially if the chemical structure is not yet known. The disadvantage of the ADEQUATEAD experiment is that it is less sensitive than HMBC, and therefore takes longer to run. F1 (ppm) 20 30 40 50 60 70 80 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 F2 (ppm) Figure 2. A 2D HSQC spectrum of menthol, acquired using the ghsqc sequence, and the corresponding 1D 1 H and 13 C spectra. Methine and methyl correlations are shown in orange, and methylene correlations are shown in blue. When interpreting SQ ADEQUATEAD data, it is also very helpful to have the one-bond heteronuclear single quantum correlation (HSQC) data for the molecule in question. This is because these two datasets are complementary, each helping you interpret the other. HSQC data indicates which protons are directly attached to which carbons, and this provides a good place to start a structure elucidation. The HSQC experiment in VnmrJ 3 also has a very handy multiplicity-edited option that identifies the number of protons bound to each carbon, as represented by the phase of the correlations in the 2D spectrum (indicated by different colors in the 2D plot). 3
Therefore, if the correlations for methyl and methine carbons (CH 3 and CH, respectively) are shown in orange, the correlations for methylene carbons (CH 2 ) are shown in blue. The HSQC data for this menthol sample (Figure 2) indicates that the carbon at 70 ppm is directly bonded to the proton at 3.2 ppm. The phase of this correlation, plus the values of the chemical shifts, indicates that this is a methine (CH ) carbon. As discussed previously, the SQ ADEQUATEAD data (Figure 1) shows that this methine carbon ( 13 C = 70 ppm; 1 H = 3.2 ppm) is correlated to protons at 1.9 and 0.9 (which HSQC shows are directly bonded to a methylene [blue] carbon at 45 ppm) and to a proton at 1.0 ppm (which HSQC shows is directly bonded to a methine [orange] carbon at 50 ppm). These assignments belong to the CH CH(OH) CH 2 group in menthol that is outlined in blue in Figure 3. This is because there are only two places in the molecule that have sequential CH CH CH 2 atoms, and the 70 ppm carbon chemical shift for the middle methine carbon strongly suggests that an oxygen is directly attached. This assignment can be confirmed by further examination of the SQ ADEQUATEAD data: the C-2 carbon of the CH CH(OH) CH 2 group ( 13 C = 50 ppm; 1 H = 1.0 ppm) is correlated to protons at 1.45 and 0.8 ppm (which HSQC shows are directly bonded to a methylene [blue] carbon at 23 ppm; C-3) and to a proton at 2.2 ppm (which HSQC shows is directly bonded to a methine [orange] carbon at 25 ppm; C-7). This creates the pattern CH 2 CH( CH ) CH(OH) CH 2 which fits into the chemical structure of menthol (Figure 3) in only one way. In a similar fashion, the assignments can be completed through the rest of the ring. The C-3 methylene carbon ( 13 C = 23 ppm; 1 H = 1.45 and 0.8 ppm) is correlated to new protons at 1.5 and 0.7 ppm (which HSQC shows are directly bonded to a methylene [blue] carbon at 35 ppm; C-4). This C-4 carbon is then correlated to a new proton at 1.25 ppm (which HSQC shows is directly bonded to a methine [orange] carbon at 32 ppm; C-5). This C-5 carbon is then correlated to the protons at 1.85 and 0.85 ppm that were previously assigned as belonging to the methylene carbon at 45 ppm (C-6). This means that we have completely and unambiguously assigned all of the 1 H and 13 C chemical shifts of the atoms within the six-membered ring of the menthol structure. A simple extension of this process can assign the rest of the molecule. 10 CH 3 H 2 C 4 CH 5 CH 6 2 3 H 2 C 2 CH 1 CH OH CH H 3 C 7 CH 3 8 9 Figure 3. The chemical structure of menthol. 4
Experimental All data shown here were acquired on a 500-MHz VNMRS DD2 spectrometer equipped with the OneNMR Probe. 1. To run this experiment on your own sample, simply click the ADEQUATEAD button in the (CC)corr tab of the Experiment Selector (Figure 4). The default parameters should work well, but you can also use the two parameter panels shown in the next step to optimize the acquisition. 2. The two most useful parameter panels for optimizing the ADEQUATEAD experiment are shown in Figures 5 and 6. The pull-down menu that controls the C-C evolution option was set to SQ (chemical shifts) for all data shown here; in VnmrJ 3.2, this is the default option. Figure 4. The Experiment Selector, showing the ADEQUATEAD experiment. Figure 5. The Acquire/Defaults parameter panel for the SQ ADEQUATEAD experiment. 5
To obtain useful 2D data, you need to have enough signal-to-noise to see signals in the first increment. This requires the use of moderately concentrated samples, typically in the range of 15 to 50 mg of sample for solutes of molecular weight 400 when using a 500-MHz, room-temperature probe. If there is enough signal-to-noise in the first increment, the user can increase the sensitivity of the experiment by arraying the value of gzlvl3 on the first increment. This was done for the parameters shown in Figure 6, and the manually optimized value of -7,653 yielded approximately 35 % higher signals than did the default value of -7,694. This parameter and its optimization could also theoretically influence the size of any artifacts that might be present in the 2D spectrum. Figure 6. The Acquire/Pulse Sequence parameter panel for the SQ ADEQUATEAD experiment. 6
Conclusions The ADEQUATEAD experiment is a powerful and useful method within the structure elucidation toolbox. It can suffer from low sensitivity as compared to some of the other heteronuclear 2D experiments, but it is a good way of assigning or determining the carbon framework of a molecule. In this application note, we demonstrate the assignment of the carbons found in menthol, showing the strength of this tool for assigning direct carbon-carbon bonds. ADEQUATEAD data combined with HSQC and HMBC data will normally contain all of the information needed to assign the full carbon backbone of typical organic molecules. References 1. Reif, B., et al. ADEQUATE, a new set of experiments to determine the constitution of small molecules at natural abundance. J. Magn. Reson., 1996, 118:282-285. 2. Reif, B., et al. Determination of 1 J, 2 J, and 3 J carbon-carbon coupling constants at natural abundance. J. Magn. Reson., 1996, 112:295-301. 3. Kock, M., Kerssebaum, R., and Bermel, W. A broadband ADEQUATE pulse sequence using chirp pulses. Magn. Reson. Chem., 2003, 41:65-69. 7
www.agilent.com/chem/nmr This information is subject to change without notice. Agilent Technologies, Inc., 2012 Published in the USA, February 2, 2012 5990-9685EN