Correlation spectroscopy (COSY)
The first and most popular two-dimension NMR experiment is the homonuclear correlation spectroscopy (COSY) sequence, which is used to identify spins which are coupled to each other. It consists of a single RF pulse (p1) followed by the specific evolution time (t1) followed by a second pulse (p2) followed by a measurement period (t2).[5]
The two-dimensional spectrum that results from the COSY experiment shows the frequencies for a singleisotope, most commonly hydrogen (1H) along both axes. (Techniques have also been devised for generating heteronuclear correlation spectra, in which the two axes correspond to different isotopes, such as 13C and1H.) COSY spectra show two types of peaks. Diagonal peaks have the same frequency coordinate on each axis and appear along the diagonal of the plot, while cross peaks have different values for each frequency coordinate and appear off the diagonal. Diagonal peaks correspond to the peaks in a 1D-NMR experiment, while the cross peaks indicate couplings between pairs of nuclei (much as multiplet splitting indicates couplings in 1D-NMR).[5]
Cross peaks result from a phenomenon called magnetization transfer, and their presence indicates that two nuclei are coupled which have the two different chemical shifts that make up the cross peak's coordinates. Each coupling gives two symmetrical cross peaks above and below the diagonal. That is, a cross-peak occurs when there is a correlation between the signals of the spectrum along each of the two axes at these value. One can thus determine which atoms are connected to one another (within a small number of chemical bonds) by looking for cross-peaks between various signals.[5]
An easy visual way to determine which couplings a cross peak represents is to find the diagonal peak which is directly above or below the cross peak, and the other diagonal peak which is directly to the left or right of the cross peak. The nuclei represented by those two diagonal peaks are coupled.[5]
To the right is an example of a COSY NMR spectrum of progesterone in DMSO-d6. The spectrum that appears along both the horizontal and vertical axes is a regular one dimensional 1H NMR spectrum. The bulk of the peaks appear along the diagonal, while cross-peaks appear symmetrically above and below the diagonal.
COSY-90 is the most common COSY experiment. In COSY-90, the p1 pulse tilts the nuclear spin by 90°. Another member of the COSY family is COSY-45. In COSY-45 a 45° pulse is used instead of a 90° pulse for the first pulse, p1. The advantage of a COSY-45 is that the diagonal-peaks are less pronounced, making it simpler to match cross-peaks near the diagonal in a large molecule. Additionally, the relative signs[clarification needed] of the coupling constants can be elucidated from a COSY-45 spectrum. This is not possible using COSY-90.[6] Overall, the COSY-45 offers a cleaner spectrum while the COSY-90 is more sensitive.
Another related COSY techniques is double quantum filtered (DQF COSY). DQF COSY uses a coherence selection method such as phase cycling or pulsed field gradients, which cause only signals from double-quantum coherences to give an observable signal. This has the effect of decreasing the intensity of the diagonal peaks and changing their lineshape from a broad "dispersion" lineshape to a sharper "absorption" lineshape. It also eliminates diagonal peaks from uncoupled nuclei. These all have the advantage that they give a cleaner spectrum in which the diagonal peaks are prevented from obscuring the cross peaks, which are weaker in a regular COSY spectrum.[7]
Many NMR users are very familiar with the 1H COSY experiment. It is used extensively by organic chemists. Many people do not realize that the COSY method can be used for many other abundant isotopes as well. The figure below shows the 11B COSY spectrum of ortho-carborane. The data were collected with 1H decoupling and allow the complete assignment of the 11B NMR spectrum. Note that in this case, the 11B - 11B J couplings are unresolved in the 1D spectrum.
COSY
A particularly popular 2D NMR experiment is the COSY experiment.
COSY experiments are often 1H-1H experiments but there are many other correlations in the literature.
The COSY transfer , which proceeds through J-coupling relies on quantum mechanical effects and cannot be explained with classical models. By applying a single (p/2)x rf- mixing pulse, it is possible to transfer coherence of spin a into coherence of spin b where these spins couple. Only with coupling do off-diagonal peaks occur. Consequently, COSY spectra are a terrific way to establish connectivity of networks.
The (H,H) COSY experiment establishes the connectivity of a molecule by giving cross peaks (these are the off diagnonal peaks) for pairs of protons that are in close proximity. For the example of Glutamic acid below, we obtain cross peaks for the proton pairs (2,3) and (3,4). We do not observe a crosspeak for the pair (2,4), because these protons are not directly adjacent.
500 MHz (H,H) COSY Spectrum of Glutamic acid. 1-D spectra left and top. 10 mg of compound in 0.5 mL of D2O, 5 mm sample tube, 256 spectra, digital resolution of 2.639 Hz/data point. Total measurement time ca. 3h.
A relayed COSY experiment goes one step beyond a COSY experiment by showing cross peaks not just for pairs of adjacent protons, but for triples as well. As a result, we observe additional cross peaks like the one for the pair 2,4 in Glutamic acid below. Relayed COSY experiments can give cross peaks for protons that are too distant to show coupling in the 1D NMR spectrum.
500 MHz H-relayed (H,H) COSY Spectrum of Glutamic acid. 1-D spectra left and top. 10 mg of compound in 0.5 mL of D2O, 5 mm sample tube, 256 spectra, digital resolution of 2.639 Hz/data point. Total measurement time ca. 3h.
With present hardware and pulse sequences, it is possible to repeat the relay step up to three times. This allows the correlation of of protons that are separated by up to six bonds (d-protons). The relaying nucleus is typically 1H, but high abundance I =1/2 hetero-elements like 31P or 19F can be used as well.
COSY pulse sequences
There are many different COSY pulse sequences, it is also between heteronuclear (eg, C, H-COSY) and homonuclear COSY spectra (eg, H, H-COSY) differ.
The most common method is the H, H-COSY experiment , which will be explained in more detail below.
In its simplest form, the COSY of two 90 °-pulses only in the evolution time t is 1 are separated:
H, H-COSY spectrum
In H, H-COSY spectrum are on both axes, the 1 H chemical shifts plotted; In principle, both the axes 1 to see H-NMR spectra. Thus, there is a symmetric to the diagonal diagram.
1 H-NMR spectrum of acetylsalicylic acid
H, H-COSY spectrum of acetylsalicylic acid
In the spectrum, only the range from 7.0 to 8.2 ppm is applied, because only here HH scalar couplings can be expected.
There are two types of signals:
- Diagonal signals: join the coordinates δ a δ a (in core A), δ b δ b (in core B) ... on, but play no role in the evaluation of the couplings between different cores, since it is only the signal of a nucleus is. The diagonal with all its signals corresponding to the 1D H-NMR spectrum.
- Cross signals: These signals are based on the scalar spin-spin coupling and are suitable for the evaluation of spectra of enormous importance.
In general it can be seen in the COSY spectrum each scalar coupling between two nuclei at four signals (two cross and two diagonal peaks) resulting connected a square; in the following example, the vicinal coupling between the H atoms is highlighted 3 and 4.
With good resolution of the COSY spectrum, the coupling constants can be determined from the fine structure of the cross and diagonal signals, but this is rarely done because of the 1-D H-NMR spectra is easily possible.
1H-1H COSY is used for clearly indicate correlation with coupled protons. A point of entry into a COSY spectrum is one of the keys to predict information from it successfully. Relation of Coupling protons is determined by cross peaks(correlation peaks) and in the COSY spectrum. In other words, Diagonal peaks by lines are coupled to each other. Figure 12 indicates that there are correlation peaks between proton H1 and H2 as well as between H2 and H4. This means the H2 coupled to H1 and H4.
Fig 12. 1H-1H COSY spectrum
1H-13C COSY (HETCOR)
1H-13C COSY is the heteronuclear correlation spectroscopy. The HETCOR spectrum is correlated 13C nuclei with directly attached protons. 1H-13C coupling is one bond. The cross peaks mean correlation between a proton and a carbon (Fig 13). If a line does not have cross peak, this means that this carbon atoms has no attached proton (e.g. a quaternary carbon atom)
Historic Development
2D Methods have greatly contributed to the explosive growth of NMR in the investigation of chemical and biological problems.
The first 2D-NMR experiment was reported by the Belgian Physicist Jeener. The most active developers of 2D methods were R. R. Ernst (Varian Research Center at Palo Alto and ETH Zürich) and R. Freemen (Oxford).
Types of 2D Experiments
2D methods can be broadly classified in shift-shift correlations (d,d methods) and those that display coupling constants and shifts (J,d methods). A 2D NMR spectrum displays signal intensity as the function of two frequency variables. An obvious advantage of the approach is the removal of signal crowding.
As an example, the resolving power of a 1H-13C HetCor experiment improves the resolving power by a factor of 1000 !
More important is the detection of spatial relationships between individual nuclei.
Measurement Time for Typical 2D NMR Experiments
2D-NMR: The Pulse Sequence
The appearance of 1D-spectra is determined by the three blocks of preparation, mixing and detection (=acquisition).
Preparation Mixing Detection (t2)
Preparation Evolution (t1) Mixing Detection (t2)
The detection period corresponds exactly to that of 1D experiments and the time t2 provides, after FT, the w2 axis of the spectrum.
The crucial difference that distinguishes the 2D methods is the introduction of a second, incrementally increased time, the so called evolution time t1. Separate FID's are collected for each incremental variation of t1. 2D-NMR uses a second Fourier Transformation that generates a second frequency axis w1 from t1.
While t2 is a "natural" time determined by the FID of the molecule, t1 can be manipulated to a wide degree.
The total number of increments is varied in exponents of 2 and is usually 128 or 256 although much lower numbers n are quite acceptable for some experiments.
2D-NMR: Description of Pulse Sequences
While It is possible to understand many 2D techniques as arrayed 1D pulse sequences 2D techniques involving phase correlation or multi quantum transition cannot be reduced to the 1D formalism.
A reasonably simple formalism to explain the results of 2D experiments in a qualitatively and quantitatively correct fashion is the so calledproduct operator formalism. An excellent introduction to 2D-NMR with the product operator formalism is the famous Review by Kessler, Gehrke and Griesinger [H. Kessler, M. Gehrke and C. Griesinger, "Two-Dimensional NMR Spectroscopy: Background and Overview of Experiments" Angew. Chem. Int. Ed. Engl. 1988, 27, 490-536.]
2D-NMR: d,J vs. d,d Experiments
The coordinate axis of a 2D NMR spectrum are invariably frequencies, but depending on whether these frequencies represent chemical shifts or coupling constants it is convenient to classify 2D NMR in those that correlate shifts with shifts (d,d- experiments) and those that correlate shifts with coupling (d,J- and d,D-experiments).
A third mechanism of information exchange is the chemical exchange of atoms. Exchange processes can be intermolecular or intramolecular.
2D-NMR: d,J vs. d,D Experiments
Both types of coupling, the scalar coupling (through bonds) and the dipolar coupling (through space) can be used for 2D methods. They are complementary to each other:
- 1J-coupling is used to establish the connectivity of covalent frameworks.
- 3J-coupling is sensitive to the dihedral angle and is used to establish conformations.
- D-coupling establishes the proximity of non-connected atoms through NOE transfer experiments
2D-NMR: Common Pulse Sequences
To make a selection which 2D pulse sequences are the most important to an inorganic chemist is a difficult task. Kessler's review states that in 1988 (!) more than 500 pulse sequences were known.
Most pulse sequences that were developed for organic molecules apply to organometallic molecules but many more are specific to fluxional problems not often encountered in organic chemistry and of course to the correlation of hetero-atoms like 1H-11B.
The following sections will discuss the most important 2D pulses:
- COSY
- NOESY
- EXSY
- HMQC
- INADEQUATE
- Inverse Detection Techniques
Below is a structural list for 1H to C to 1H connectivities. Absent from the list, heteroatoms such as O, N and S should also be considered. The 2J coupling, the 3J coupling, outlined with a blue box, the 4J coupling, outlined in a green box, and the 5J coupling, outlined with a purple box, offers 1, 3, 6 and 14 possible combinations, respectively.
Interpreting a 1H-1H COSY spectrum
A component of structure elucidation involves the capability to interpret spectral data of an unknown compound. The interpretation of the data generally leads into a wide range of structural possibilities for the unknown. The goal of the elucidator is to narrow down the structural possibilities to a minimum set of fragments. Taking these fragments, the elucidator can piece together the parts that make structural sense.
Below is a portion of a 1H-1H COSY-45 spectrum for an unknown compound. The COSY data exhibits 5 correlations: 3 diagonal and 2 off-diagonal. The 2 off-diagonal correlations at (1.45, 2.36) and (2.36, 1.45) ppm indicate 4 possible 1H-C-1H structural configurations between the protons at 1.45 and 2.36 ppm. The issue is compounded by the fact that for each off-diagonal correlation, there are 4 possibilities to consider when trying to build a fragment(s). Keep in mind that COSY data by itself may not constitute adequate information to narrow down the possibilities. However, noting the multiple possibilities will ensure that nothing is overlooked.
Note: the intensity of the off-diagonal correlations, judged by the number of contours in respect to the diagonal correlations, may provide a clue in eliminating some of the possibilities. However, this is dependent on how the data is collected and/or on the dihedral angles of the coupled protons.
ref1-7
- Aue, W. P., Bartholdi, E., and Ernst, R. R. (1976) "Two-dimensional spectroscopy. Application to nuclear magnetic resonance," Journal of Chemical Physics, 64 : 2229-46.
- ^ Martin, G. E; Zekter, A. S. (1988). Two-Dimensional NMR Methods for Establishing Molecular Connectivity. New York: VCH Publishers, Inc. p. 59.
- ^ Akitt, J. W.; Mann, B. E. (2000). NMR and Chemistry. Cheltenham, UK: Stanley Thornes. p. 273.
- ^ a b Keeler, James (2010). Understanding NMR Spectroscopy (2nd ed.). Wiley. pp. 184–187. ISBN 978-0-470-74608-0.
- ^ a b c d Keeler, pp. 190–191.
- ^ Akitt & Mann, p. 287.
- ^ Keeler, pp. 199–203.
examples
The COSY-DQF (Cosy Double Quantum Filter) provides two advantages over the magnitude COSY. Higher resolution is possible and multiplet fine structure can be seen. This may allow proton-proton couplings to be measured. When there are several couplings to a given proton and the multiplet is complex, however, often the cross peak is not interpretable because of cancellation of multiplet components. The E.COSY experiment is usually necessary for coupling measurement.
The DQF version reduces the diagonal dispersive peaks of the COSY experiment but sensitivity is reduced by one half. In addition, uncoupled spins such as water are removed. This experiment is seldom used for small molecules.
Below is a 500 MHz spectrum of sucrose
The DQF version reduces the diagonal dispersive peaks of the COSY experiment but sensitivity is reduced by one half. In addition, uncoupled spins such as water are removed. This experiment is seldom used for small molecules.
Below is a 500 MHz spectrum of sucrose
H, H-COSY spectrum of acetylsalicylic acid
In the spectrum, only the range from 7.0 to 8.2 ppm is applied, because only here HH scalar couplings can be expected
