Measuring methods available and examples of their applications


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1H NMR (Proton nuclear magnetic resonance)

The most basic experiment is a simple one-dimensional proton NMR spectrum. 1H NMR is a suitable method for purity control, analysis of mixtures and confirmation of molecular structure. It is widely used in the pharmaceutical, medical, and food industries for quality control. The experiment is highly sensitive and measurement time is short.

Simple NMR spectra are recorded in solution, and solvent protons must not be allowed to interfere. Deuterated (symbolized as D) solvents for the specific use in NMR are preferred. The most common solvents used include deuterated water, D2O, deuterated acetone, (CD3)2CO, deuterated methanol, CD3OD, deuterated dimethyl sulfoxide, (CD3)2SO, and deuterated chloroform, CDCl3. Approximate minimal concentrations of samples are 0.1 mM for 1H (corresponding 0.015 mg, based on molecular weight 300 and sample volume 0.5 ml). Deuterated solvents permit the use of deuterium frequency-field lock (deuterium lock) to eliminate the effect of the natural drift of the NMR's magnetic field B 0 {\displaystyle B_{0}}.

Proton NMR spectra of most organic compounds are characterized by chemical shifts in the range +15 to -4 ppm and by spin-spin coupling between protons. The integration curve for each proton reflects the abundance of the individual protons.

Figure 1. 1H NMR spectrum  of Strychnine in DMSO-d6 with assignment

One example of the many applications shows detection of methanol in an alcoholic beverage below.

Maximum allowable content of methanol in alcoholic beverages is specified by the Regulation (EC) No. 110/2008; for example, 12 g of methanol per 1 l of ethanol for most fruit distillates.

Figure 2. 1H NMR spectrum of fruit distillate, time of analysis approx. 5 min.

 

13C NMR (carbon nuclear magnetic resonance)

For most organic compounds, information on the number and character of the carbon is essential for confirmation and proof of their structure. As the most common carbon isotope 12C does not possess magnetic moment, NMR spectroscopy relyes on 13C whose natural abundance is only 1.1%. This, combined with four times lower gyromagnetic ratio compared to 1H, results in much lower measurement sensitivity than in the case 1H NMR and the measurements are therefore more time-consuming. Routine 13C NMR spectra are measured usually with broadband proton decoupling and and sensitivity enhancement  (3x) by NOE effect. In our CF, a very sensitive cryoprobe (175 MHz at 16.45 T) is available for the measurement of 13C -NMR, which significantly reduces the measurement times (tens of minutes).

In analogy to proton NMR,  13C NMR allows the identification of carbon atoms in an organic molecule just as proton NMR identifies hydrogen atoms. As such, 13C NMR is an important tool in chemical structure elucidation in organic chemistry.

From a 1H -decoupled carbon (13C) spectrum useful information about 13C chemical shifts is obtained. Alternatively, carbon multiplicity and 1H-13C coupling constants can be determined from the gated decoupled methodology and quantitative measurements can be made from the inverse gated experiments.

Assignment of the 13C spectrum is usually performed with the help of additional NMR methods. As a general strategy:

  • Carbon multiplicity is determined from 1D APT or DEPT experiments.
  • Correlation with 1H nuclei via 1J(CH) is achieved from HSQC or HMQC-type experiments.
  • Correlation with 1H nuclei via nJ(CH) is achieved from HMBC-type experiments.

Examples

Figure 1. Strychnine nitrate in DMSO-d6 (approx. 50 mM)  1H -decoupled  13C NMR spectrum

with assignment. Spectrometer: AVANCE III HD 700, Probehead:  5 mm CPTXO 13C/15N-1H/D with z gradients, Experiment time: 10 min

Figure 2. Cefotaxime Acid (cephalosporin antibiotic), 1H-decoupled  13C NMR spectrum

with assignment, Spectrometer: AVANCE III HD 700, Probehead:  5 mm CPTXO 13C/15N-1H/D with z gradients, Experiment time: 10 min

For quantitative applications, it is necessary to obtain reliable integration values. In this case, the 13C spectrum is recorded with a 90º pulse and a long relaxation period (5*T1(13C)) is used to avoid partial signal saturation. It can be used for example for characterization of synthetic polymers.

 

13C NMR structure characterization of polyolefines

Quantitative measurement of 13C NMR spectra is performed in our laboratory using a 10 mm dual 13C-{1H} NMR probehead  on the NMR Bruker AVANCE III 500 spectrometer at the field of 11.74 T (500 MHz and 125 MHz for 1H and  13C nuclei, respectively). The analysis of the NMR data enables to characterize various polyolefins such:

  • 13C NMR structure characterization of polyethylene (HDPE, LDPE, LLDPE): determination of comonomer(s), number of side chain branches
  • 13C NMR characterization of poly(ethylene-propylene) block and random copolymers: calculation of ethylene and propylene content, sequence length distribution
  • 13C NMR tacticity characterization of polypropylene on the level of pentads

The detailed analysis of these NMR data is performed by Polymer Institute Brno. www.polymer.cz

Figure 1. 13C NMR spectrum of poly(ethylene-propylene) block copolymer

 

13C NMR APT (Attached Proton Test)

The Attached Proton Test (APT) experiment is a simple method to assign C-H multiplicities in 13C NMR  spectra.  It provides information on all carbon muliplicities within a single experiment.

The APT (or J-resolved) experiment yields methine (CH) and methyl (CH3) signals negative and quaternary (C) and methylene (CH2) signals positive (In North America, oposite phasing is usually used). It is slightly less sensitive than DEPT but a single experiment shows all carbon signals at once unlike DEPT which needs three different spectra.

Even though this technique does not distinguish fully between CHn groups, it is so easy and reliable that it is frequently employed as a first attempt to assign peaks in the spectrum and elucidate the structure. It is sometimes possible that a CH and CH2 signal have coincidentally equivalent chemical shifts resulting in signal cancelation in the APT spectrum due to the opposite phases. For this reason the conventional 13C{1H} spectrum or HSQC are usually also acquired.

Examples of  APT spectra

Figure 1. Cefotaxime Acid (cephalosporin antibiotic of third-generation). APT spectrum of Cefotaxime showing CH and CH3 negative while CH2 and C are positive. Spectrometer: AVANCE III HD 700, Probehead:  5 mm CPTXO 13C/15N-1H/D with z gradients, Experiment time: 12 min

Figure 2. Comparison of the 13C NMR spectra with proton decoupling (red) and APT experiment for the molecule of cholesterol. APT spectrum shows CH and CH3 negative while CH2 and C are positive.

 

13C DEPT   (Distortionless Enhancement by Polarization Transfer)   

The DEPT experiment is a NMR method used for determining the presence of primary, secondary and tertiary carbon atoms. This experiment differentiates between CH, CH2 and CH3 groups by variation of the selection angle parameter (the tip angle of the final 1H pulse): 135° angle gives all CH and CH3 in a phase opposite to CH2; 90° angle gives only CH groups, the others being suppressed; 45° angle gives all carbons with attached protons (regardless of number) in phase.

Signals from quaternary carbons and other carbons with no attached protons are always absent (due to the lack of attached protons).

The polarization transfer from 1H to 13C has the secondary advantage of increasing the sensitivity over the normal 13C spectrum (which has a modest enhancement from the Nuclear Overhauser Effect due to the 1H decoupling).

Figure 1. Various DEPT spectra of Strychnine in DMSO-d6 compared to the conventional 13C{1H} spectrum

DEPT 13C NMR spectra phase table

 

COSY (COrrelation Spectroscopy)

The most frequently used two-dimension NMR experiment is the homonuclear correlation spectroscopy (COSY), which allows to identify spins which are coupled to each other.

The 2D spectrum that results from the COSY experiment shows the frequencies for a single isotope, most commonly hydrogen (1H) along both axes. 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).

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.

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.

The analysis of fine structure of the COSY crosspeaks allows extracting the J-couplings that carry information about dihedral angles.

The most common COSY experiment is COSY-90, in which the p1 pulse tilts the nuclear spin by 90°. Another modification 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. Overall, the COSY-45 offers a cleaner spectrum while the COSY-90 is more sensitive.

Another related COSY technique is double quantum filtered (DQF COSY). This experiment has the advantage that it gives a cleaner spectrum in which the diagonal peaks are in-phase with the crosspeaks.

Figure 1. 1H COSY spectrum of Brucine in DMF-d7, gradient enhanced (magnitude mode)

Figure 2. Cefotaxime Acide (cephalosporin antibiotic), COSY 90 in MeOD-d4

Figure 3. Strychnine nitrate in DMSO-d6,  gradient COSY (magnitude mode), Spectrometer: AVANCE III HD 700, Probehead:  5 mm Inverse Broadband with z-Gradients, Experiment time: 8 min

 

TOCSY (TOtal Correlation Spectroscopy)

The TOCSY experiment is similar to the COSY experiment, in that cross peaks of coupled protons are observed. However, cross peaks are observed not only for nuclei which are directly coupled, but also between nuclei which are connected by a chain of couplings.

The gradient enhanced 2D TOCSY experiment allows to obtain a 2D TOCSY spectrum with a single scan per t1 increment provided that the S/N ratio is adequate. The main advantage of such approach is the large reduction in the total acquisition time compared with a conventional 2D TOCSY experiment. The TOCSY experiment permits to correlate all protons resonances belonging to the same spin system via the homonuclear JHH coupling constants.

Figure 1. Strychnine nitrate in DMSO-d6,  2D – TOCSY , Spectrometer: AVANCE III HD 700, Probehead:  5 mm Inverse Broadband with z-gradients, Experiment time: 76 min

 

2D NOESY  (Nuclear Overhauser Effect Spectroscopy)

NOESY experiments are an important tool to identify stereochemistry of a molecule in solvent.

Two-dimensional NOE option allows to measure in a single experiment NOEs between all the hydrogen atoms in molecule. Instead of scalar interactions (through bonds), dipolar interactions through space are indicated. NOESY is particularly suitable for large molecules (biopolymers) to solve their 3D structure. One application of NOESY is in the study of large biomolecules such as in protein NMR, which can often be assigned using a sequential walk. For smaller molecules, the method is used to assign spatially near protons and to solve stereochemistry. The disadvantage is that for molecules with the molecular weight of approximately 700-800, the NOE effect is close to zero resulting in weak ar absent signals. Care must be taken, however, as many other processes lead to reduced NOEs including spin-lattice relaxation, temperature, increased solvent viscosity, increased molecular weight, and dissolved paramagnetic impurities including oxygen. Also important is the value used for the mixing time. The intensity of the NOE is in first approximation propotional to 1/r6, with r being the distance between the protons: The correlation between two protons depends on the distance between them, but normally a signal is only observed if their distance is smaller than 5 Å.
The spectrum obtained is similar to COSY, with diagonal peaks and cross peaks. For small molecules, diagonal peaks (black) have opposite phase than cross peaks (red) in the NOESY spectrum.

Figure 1. Brucine in DMF-d7, 2D – NOESY, mixing time 800 ms, Spectrometer: AVANCE III 500, Probehead:  5 mm CPPBBO (Prodigy) with z gradients, Experiment time: 70 min.

 

2D ROESY (Rotating frame nuclear Overhauser Effect Spectroscopy)

ROESY method is useful for certain molecules whose rotational correlation time falls in a range where the Nuclear Overhauser effect is too weak to be detectable, usually molecules with a molecular weight around 1000. ROESY has a different dependence between the correlation time and the cross-relaxation rate constant. In NOESY the cross-relaxation rate constant goes from positive to negative as the correlation time increases, giving a range where it is near zero, whereas in ROESY the cross-relaxation rate constant is always positive.

Measurements are carried out under spinlock conditions. Experiment provides essentially the same information as NOESY. Red peaks in the figure are negative and black signals are positive. Spectrum is symmetrical about the diagonal and cross peaks means spatial proximity (within 5 Å). For large molecules, ROESY allows distinguishing between true NOE signals and exchange peaks as they have oposite signs. In NOESY, both types of peaks have the same sign.

Figure 1. NOE dependence on molecular weight

Figure 2. Strychnine in DMSO-d6,  2D–ROESY, mixing time?, Spectrometer: AVANCE III HD 700, Probehead:  5 mm CPTXO 13C/15N-1H/D with z gradients, Experiment time: 72 min

Figure 3. Strychnine in DMSO-d6,  2D–ROESY, the aliphatic area expanded

 

2D HSQC (Heteronuclear Single-Quantum Correlation spectroscopy)

The 2D HSQC experiment permits to obtain a 2D heteronuclear chemical shift correlation map between directly-bonded 1H and X-heteronuclei (usually 13C or 15N). It is widely used because it is based on proton-detection, offering high sensitivity.

HSQC detects correlations between nuclei of two different types which are separated by one bond. This method gives one peak per pair of coupled nuclei, whose two coordinates are the chemical shifts of the two coupled atoms. HSQC works by transferring magnetization from the proton to the other nucleus (the heteroatom) using the INEPT pulse sequence.

Figure 1. Strychnine nitrate in DMSO-D6, 1H-13C-HSQC phase-sensitive experiment with assignment. Spectrometer: AVANCE III HD 700, Probehead:  5 mm Inverse Broadband with z-Gradients, Experiment time: 14 min

Figure 2. Vinorelbin tartrate , 20 mM in DMSO-d6 gradient HSQC, Spectrometer: AVANCE III 500, Probehead:  5 mm CPPBBO (Prodigy) with z gradients, Experiment time: 50 min

 

2D HSQC multiplicity edited

The 2D multiplicity-edited HSQC experiment is a simple and popular modification of the gradient enhanced 2D HSQC experiment in which carbon multiplicity can be directly extracted from the resulting spectra in addition to the conventional heteronuclear correlations by simple analysis of the cross-peaks sign.

Figure 3. Strychnine nitrate in DMSO-D6, phase-sensitive 2D 1H-13C-HSQC multiplicity- edited experiment with assignment. Spectrometer: AVANCE III HD 700, Probehead:  5 mm Inverse Broadband with z-Gradients, Experiment time: 14 min

 

2D HMQC (Heteronuclear Multiple-Quantum Correlation)

Heteronuclear multiple-quantum correlation spectroscopy (HMQC) gives a spectrum similar to HSQC, but using a different method. The two methods give results of similar quality for small to medium-sized molecules, but HSQC is considered to be superior for larger molecules.

Figure 1. Strychnine in DMSO-D6, 1H,13C-HMQC, magnitude mode. Spectrometer: AVANCE III HD 700, Probehead:  5 mm Inverse Broadband with z-Gradients, Experiment time: 12 min.

 

2D HMBC (Heteronuclear Multiple-Bond Correlation spectroscopy)

HMBC detects heteronuclear correlations over longer ranges of about 2–4 bonds. The 2D HMBC experiment permits to obtain a 2D heteronuclear Chemical Shift correlation map between long-range coupled 1H and heteronuclei (usually 13C). It is widely used because it is based on proton-detection, offering high sensitivity in magnitude mode. In addition, long-range proton-carbon coupling constants can be measured from the resulting spectra.

The HMBC spectrum shows the typical 2D long-range correlation map. A cross-peaks means that the corresponding 1H and heteronucleus are two- or three-bonds away. Residual direct connectivites are usually present as large doublets due to 1J(CH). A modification of the HMBC method is available, which allows suppression one-bond signals, leaving only the multiple-bond signals.

Figure 1. Strychnine nitrate in DMSO-D6, 1H,13C-HMBC optimized for J(CH) long range = 10 Hz in

magnitude mode with assignment. Spectrometer: AVANCE III HD 700, Probehead:  5 mm PABBO BB-1H/D, Experiment time: 50 min

 

15N HMBC

1H-15N-HMBC is basically the same as the proton-carbon correlation HMBC experiment, but as a result you obtain the 1H-15N-correlation. In contrast to a 15N-detected NMR spectrum it is much more sensitive. Thus you need less time for a measurement. In one example (molecular weight 300 g/mol, concentration 30 mg/ml) it was possible to obtain a good quality 1H-15N-HMBC-spectrum within 60 min. In order to obtain a 15N-NMR spectrum under the same conditions you would have to measure overnight. Moreover, the 1H-15N-HMBC-spectrum provides additional information for structure elucidation. Therefore, a 1H-15N-HMBC-spectrum is preferable over a 15N-NMR spectrum in most cases.

Figure 2. Strychnine nitrate in DMSO-D6, 1H,15N-HMBC with assignment, Spectrometer: AVANCE III HD 700B, Probehead:  5 mm CPTXO 13C/15N-1H/D with z gradients, Experiment time: 70 min

 

DOSY (Diffusion Ordered SpectroscopY)

Characterization of molecular weight: Diffusion Ordered SpectroscopY - DOSY

The DOSY method measures the translational self-diffusion of molecules in solution and allows a precise analysis of a complex mixture without any prior separation of the different components.

Binding of substituents to the substrate during the synthesis of drugs can be also verified by this method.

NMR diffusion experiments provide a way to separate the different compounds in a mixture based on the differing translation diffusion coefficients (and therefore differences in the size and shape of the molecule, as well as physical properties of the surrounding environment such as viscosity, temperature, etc.) of each chemical species in solution.

A series of spin echo spectra is measured with different pulsed field gradient strengths in a pseudo-2D manner, and the resulting signal decays provide the diffusion axis after the data processing.

Figure 1. DOSY spectrum of a mixture of three compounds with different molecular weights.

 

 


Measuring methods available and examples of their applications