*4.3.4.4.3 HMBC spectrum*

HMBC spectrum is short for <sup>1</sup> H detected heteronuclear multiple bond correlation, which associates the 1 H nucleus with 13C nucleus of long-range coupling. HMBC could detect the long-range coupling of <sup>1</sup> H-13C sensitively (n *J*CH, n≧2). Moreover, the correlation signal peaks between protons and quaternary carbons that are two or three bonds apart could also be shown in HMBC spectra, as shown in **Figure 7**. From the HBMC spectrum, we can get the connection information of the carbon chain skeletons, the structure information of the quaternary carbons, and the structural information of the coupling systems that are cut off by heteroatoms.

#### *4.3.4.4.4 NOESY spectrum*

When two groups of protons are located at rather close spatial distances, irradiation of one group will enhance the signal strength of another, which is known as nuclear Overhauser enhancement (NOE). The NOE spectrum can determine the spatial relative position, stereoscopic configuration, and dominant conformation of some groups in the molecule, which is very important for the study of the stereostructures of organic compounds.

**65**

*Analytical Methods of Isolation and Identification DOI: http://dx.doi.org/10.5772/intechopen.88122*

*Schematic diagram of correlations between <sup>1</sup>*

*Schematic diagram of correlations between <sup>1</sup>*

2D-NOE (NOESY) spectra could show the NOE correlations of protons. The greatest advantage of NOESY is that all the NOE information between protons of a compound could be shown in one spectrum. However, not all the cross peaks are NOE correlation signals, the residual correlation signals of COSY are often shown in NOESY

*H and 13C in the TOCSY spectrum.*

*H and 13C in the HSQC-TOCSY spectrum.*

spectrum as well, which should be paid attention during spectroscopic analysis.

The TOCSY spectrum shows the correlation of the entire spin system, which is

HSQC-TOCSY is a kind of combined 2D-NMR spectrum. Comprehensive results of HSQC and HMBC are obtained by using a long pulse sequence. The correlation is shown in **Figure 9**. It is very useful for the assignment of carbon and proton signals in complex chemical structures. For example, for saponins with a series of glycosyl groups, the signals generated by glycosyl groups are often overlapped seriously in common NMR spectra, which causes difficulty to assign signals of glycosyls. HSQC-TOCSY spectrum will play an important role in this case. The spectrum includes the

H COSY.

H-1

Polarimetry is an optical method used widely in the studies of asymmetric structures, which appeared very early. The progress of the sensitive method such as ORD and CD made it possible to study stereostructures of chiral compounds more deeply. Both of them are spectra related to the optical activity of compounds, and

*4.3.5 Optical rotary dispersion (ORD) and circular dichroism (CD)*

generated the correlation peaks are shown in **Figure 8**. Not only the correlation signals of a proton with protons connected to the adjacent carbons, but also its correlation signals with other protons in a whole spin system could be shown in the TOCSY spectrum, which provides important basis for the connection of structural fragments.

H COSY. The relationships between the nuclei that

*4.3.4.4.5 Total correlation spectroscopy (TOCSY) spectrum*

H-1

different from the ordinary 1

**Figure 8.**

**Figure 9.**

*4.3.4.4.6 HSQC-TOCSY spectrum*

information of HSQC, HMBC, and <sup>1</sup>

*Analytical Methods of Isolation and Identification DOI: http://dx.doi.org/10.5772/intechopen.88122*

*Phytochemicals in Human Health*

**64**

**Figure 7.**

**Figure 6.**

1

between 1

*Schematic diagram of correlations between <sup>1</sup>*

*Schematic diagram of correlations between <sup>1</sup>*

some particular configuration systems, 1

elucidation of an unknown structure.

*4.3.4.4.2 HSQC (HMQC) spectrum*

HMBC spectrum is short for <sup>1</sup>

HMBC could detect the long-range coupling of <sup>1</sup>

relation, which associates the 1

*4.3.4.4.3 HMBC spectrum*

*4.3.4.4.4 NOESY spectrum*

structures of organic compounds.

*H and 13C in the HMBC spectrum.*

*H and 13C in the HSQC or HMQC spectrum.*

H COSY spectra can show 4

H detected heteronuclear multiple bond cor-

H nucleus with 13C nucleus of long-range coupling.

H-13C sensitively (n

*J* coupling

H detected

*J*CH, n≧2).

In addition, for compounds of aromatic systems, double bond systems, and

or longer coupling relationships of hydrogen groups. It is very important for the

heteronuclear multiple quantum coherence (HMQC) can display the correlations

HMQC. In the HMQC or HSQC spectrum, the signals occurred at the crosses of chemical shifts generated by corresponding carbons and protons (**Figure 6**).

Moreover, the correlation signal peaks between protons and quaternary carbons that are two or three bonds apart could also be shown in HMBC spectra, as shown in **Figure 7**. From the HBMC spectrum, we can get the connection information of the carbon chain skeletons, the structure information of the quaternary carbons, and the structural information of the coupling systems that are cut off by heteroatoms.

When two groups of protons are located at rather close spatial distances, irradiation of one group will enhance the signal strength of another, which is known as nuclear Overhauser enhancement (NOE). The NOE spectrum can determine the spatial relative position, stereoscopic configuration, and dominant conformation of some groups in the molecule, which is very important for the study of the stereo-

H and 13C. HSQC possesses higher sensitivity and wider application than

H detected heteronuclear single quantum coherence (HSQC) and <sup>1</sup>

H-1

**Figure 8.** *Schematic diagram of correlations between <sup>1</sup> H and 13C in the TOCSY spectrum.*

2D-NOE (NOESY) spectra could show the NOE correlations of protons. The greatest advantage of NOESY is that all the NOE information between protons of a compound could be shown in one spectrum. However, not all the cross peaks are NOE correlation signals, the residual correlation signals of COSY are often shown in NOESY spectrum as well, which should be paid attention during spectroscopic analysis.

## *4.3.4.4.5 Total correlation spectroscopy (TOCSY) spectrum*

The TOCSY spectrum shows the correlation of the entire spin system, which is different from the ordinary 1 H-1 H COSY. The relationships between the nuclei that generated the correlation peaks are shown in **Figure 8**. Not only the correlation signals of a proton with protons connected to the adjacent carbons, but also its correlation signals with other protons in a whole spin system could be shown in the TOCSY spectrum, which provides important basis for the connection of structural fragments.

#### *4.3.4.4.6 HSQC-TOCSY spectrum*

HSQC-TOCSY is a kind of combined 2D-NMR spectrum. Comprehensive results of HSQC and HMBC are obtained by using a long pulse sequence. The correlation is shown in **Figure 9**. It is very useful for the assignment of carbon and proton signals in complex chemical structures. For example, for saponins with a series of glycosyl groups, the signals generated by glycosyl groups are often overlapped seriously in common NMR spectra, which causes difficulty to assign signals of glycosyls. HSQC-TOCSY spectrum will play an important role in this case. The spectrum includes the information of HSQC, HMBC, and <sup>1</sup> H-1 H COSY.

#### *4.3.5 Optical rotary dispersion (ORD) and circular dichroism (CD)*

Polarimetry is an optical method used widely in the studies of asymmetric structures, which appeared very early. The progress of the sensitive method such as ORD and CD made it possible to study stereostructures of chiral compounds more deeply. Both of them are spectra related to the optical activity of compounds, and

#### *Phytochemicals in Human Health*

could provide information of absolute configurations, dominant conformations, and reaction mechanisms of chiral compounds, that cannot be replaced by any other spectroscopic methods [28].

### *4.3.5.1 Optical rotary dispersion (ORD) spectrum*

The specific rotation [α] of a chiral compound depends upon the wavelength of the monochromatic light wave. The measurement of specific rotation as a function of wavelength is called optical rotator dispersion (ORD). The common types of ORD curves are as follows.

### *4.3.5.1.1 Plain curves*

The ORD spectrum of an optically active compound with no chromophores is plain without peaks and troughs. An ORD curve of specific rotation increases with decrease of wavelength which is called positive plain curve, while in the case of negative plain curve, negative rotation increases with decrease of wavelength (see **Figure 10**).
