**2.1 General procedure of microscale fractional bulb-to-bulb distillation**

Commercially available, a bulb-to-bulb distillation apparatus (see Fig. 2) is usually used to purify a small amount of organic material (10-500 mg) by distillation under vacuum. Glass bulbs of about 3-3.5 cm in diameter are connected in series. The joint between bulbs, for example, between bulb A and bulb B, is made of ground glass. The general procedure for performing a distillation on this apparatus is given below.

Fig. 2. Sketch of bulb-to-bulb distillation apparatus.


Cooling all the bulbs outside the oven is a critical part of this procedure. Failure to do so will result in low recovery and poor separation.

Using this method, we can perform separations on small amounts of material (down to 10 mg) to obtain fractions with different boiling points.

#### **2.2 Separation of odor constituents of representative incense by bulb-to-bulb distillation technique**

We used fractional bulb-to-bulb distillation to evaluate the odors of oils obtained from incense materials (sandalwood, frankincense, etc.). We extracted the essential oils of the incense first with hexane and then with methanol. We compared the odors of the hexaneextracted essential oils to the original base materials. We found that the odors of the extracted oils were similar to the odors of the base materials. We performed the following fractional distillations to obtain the minimum odorant groups constituting the fragrances of

Separation of Odor Constituents by Microscale Fractional Bulb-To-Bulb Distillation 203

Fig. 4. Separation of odor constituents from hexane extract of sandalwood chips.

H NMR Spectrum of the hexane extract of sandalwood chips (200 MHz, CDCl3).

Generally, compounds with a formyl group (aldehyde and formate) are important odor constituents. These compounds, especially aldehydes, are common decomposition products of the corresponding carboxylic acids. Aldehydes, because they are prone to decomposition, are difficult to collect from an extract by chromatography or distillation. The bulb-to-bulb distillation method, however, is suitable for handling these compounds, because the heating

We performed bulb-to-bulb distillation of the hexane extract. Two fractions were obtained, and the residue was composed of -santalol and -santalol. The first fraction was a mixture of santalol hydrocarbon derivatives and the second fraction was santalyl aldehydes and

Fig. 5. <sup>1</sup>

time is shorter than that in a typical distillation.

formates, as determined by NMR spectroscopy (Fig. 6).

the scented materials (Fig. 3). We found that the fragrances of these scent materials could be expressed by combining these groups. Each group consists of several organic compounds with similar structures and thus similar odors.

Fig. 3. General procedure for separating constituents from extract by microscale fractional bulb-to-bulb distillation.

#### **2.2.1 Sandalwood**

High-quality sandalwood—from which the essential oil is collected—is a valuable and expensive material because it can only be obtained from mature sandalwood trees. Sandalwood is a medium-sized evergreen parasitic tree and is found in India, Malaysia, and Australia. The highest quality of Sandalwood trees for incense and perfume are grown in India (especially East India). Many investigations on the composition of sandalwood essential oils have been carried out, and more than 300 constituents have been identified. The main constituents are -santalol and -santalol. These compounds have distinctive woody odors. Many studies have been done on sandalwood, and the structure–odor relationships of -santalol and its related compounds have been investigated in detail.

Recently, we reported that the odor of sandalwood chips is formed by a combination of santalols and their aldehyde and formate derivatives (Hasegawa et al., 2011). Here, we will examine the interesting relationship between the structure and odor of -santalol and its derivatives having modified side chains. Recently, we identified new odor constituents of sandalwood by the method shown in Fig. 4

We applied the distillation method introduced in this chapter to the evaluation of sandalwood odor.

We collected the hexane extract and the steam-distilled oil from sandalwood chips and compared their odors with the odor of sandalwood chips. The odor of the extracted oil was found to be similar to the odor of the base material.

The 1H NMR spectroscopy revealed that the main constituents were -santalol and santalol with an extremely small amount of compounds with a formyl group (Fig. 5).

the scented materials (Fig. 3). We found that the fragrances of these scent materials could be expressed by combining these groups. Each group consists of several organic compounds

Fig. 3. General procedure for separating constituents from extract by microscale fractional

High-quality sandalwood—from which the essential oil is collected—is a valuable and expensive material because it can only be obtained from mature sandalwood trees. Sandalwood is a medium-sized evergreen parasitic tree and is found in India, Malaysia, and Australia. The highest quality of Sandalwood trees for incense and perfume are grown in India (especially East India). Many investigations on the composition of sandalwood essential oils have been carried out, and more than 300 constituents have been identified. The main constituents are -santalol and -santalol. These compounds have distinctive woody odors. Many studies have been done on sandalwood, and the structure–odor relationships of -santalol and its related compounds have been investigated in detail.

Recently, we reported that the odor of sandalwood chips is formed by a combination of santalols and their aldehyde and formate derivatives (Hasegawa et al., 2011). Here, we will examine the interesting relationship between the structure and odor of -santalol and its derivatives having modified side chains. Recently, we identified new odor constituents of

We applied the distillation method introduced in this chapter to the evaluation of

We collected the hexane extract and the steam-distilled oil from sandalwood chips and compared their odors with the odor of sandalwood chips. The odor of the extracted oil was

The 1H NMR spectroscopy revealed that the main constituents were -santalol and santalol with an extremely small amount of compounds with a formyl group (Fig. 5).

with similar structures and thus similar odors.

bulb-to-bulb distillation.

sandalwood by the method shown in Fig. 4

found to be similar to the odor of the base material.

**2.2.1 Sandalwood** 

sandalwood odor.

Fig. 4. Separation of odor constituents from hexane extract of sandalwood chips.

Fig. 5. <sup>1</sup> H NMR Spectrum of the hexane extract of sandalwood chips (200 MHz, CDCl3).

Generally, compounds with a formyl group (aldehyde and formate) are important odor constituents. These compounds, especially aldehydes, are common decomposition products of the corresponding carboxylic acids. Aldehydes, because they are prone to decomposition, are difficult to collect from an extract by chromatography or distillation. The bulb-to-bulb distillation method, however, is suitable for handling these compounds, because the heating time is shorter than that in a typical distillation.

We performed bulb-to-bulb distillation of the hexane extract. Two fractions were obtained, and the residue was composed of -santalol and -santalol. The first fraction was a mixture of santalol hydrocarbon derivatives and the second fraction was santalyl aldehydes and formates, as determined by NMR spectroscopy (Fig. 6).

Separation of Odor Constituents by Microscale Fractional Bulb-To-Bulb Distillation 205

extract contained a large amount of odorless constituents and thus attempted to collect only odor constituents from the hexane extract. Bulb-to-bulb distillation of the hexane extract produced two groups (group A and B) with characteristic odors; the residue did

Group A consisted of several sesquiterpenes and anethole (Fig. 7). Group B consisted of almost entirely patchoulol. These two groups were found to contain the key compounds

Fig. 7. Separation of scent components from hexane extract from patchouli leaves.

The resin of frankincense is obtained from many species of trees in the genus Boswellia. Frankincense has been used as a valuable fragrance source since ancient times, and has been reported to possess a wide range of bioactivity. Many compounds have been identified in frankincense (Hamm et al., 2005; Mertens et al., 2009). To our knowledge, however, the effects of particular odor components have not been clarified in detail. There are two representative species of frankincense. The main components of frankincense are markedly different between these two species. One has many monoterpenes (e.g., -pinene) as key compounds that contribute to frankincense odor. The other contains diterpenes as the main constituents, along with octyl acetate and octanol. This latter species is used in traditional

The hexane extract of frankincense is a highly viscous oil, suggesting that it contains a large amount of compounds that contribute relatively little to the characteristic odor of frankincense. The NMR spectrum of the extract (Fig. 8) supports this assessment. We did a bulb-to-bulb distillation to evaluate the key compounds of frankincense odor. First, fraction 1 was obtained from distillation below 124 °C at 0.09 Torr. The constituents were octanol and octyl acetate (Fig. 9). Then, the temperature and pressure were maintained at 124 °C (0.09 Torr), and highly similar constituents were collected in the three different bulbs

according to the slightly different boiling point of each constituent (Fig. 9).

not have a significant odor.

**2.2.3 Frankincense** 

Japanese incense.

that contribute to patchouli odor.

Fig. 6. <sup>1</sup> H NMR spectrum of group B obtained from hexane extract by bulb-to-bulb distillation (200 MHz, CDCl3).

The obtained fractions were analyzed, and santalol derivatives with a formyl group (Group B) were found to play an important role in the odor of sandalwood chips. The diagram (Fig. 4) indicates the scent profile of sandalwood obtained by this method. - Santaol has been reported to be the principal constituent of sandalwood odor. In contrast, -santalol has been reported to be only a supporting component of sandalwood odor because -santalol has a weaker odor than does -santalol. However, we found that both -santalol and -santalol derivatives with a formyl group were important constituents of sandalwood odor.

This result demonstrates that bulb-to-bulb distillation is useful for collecting very small fractions from a mixture.

#### **2.2.2 Patchouli**

The unique woody aroma of patchouli is one of the four major woody notes derived from essential oils, and serves as an indispensable scent in modern fragrance. Although many studies have been performed, the key components that constitute this odor have not been successfully identified (Nabeta et al., 1993; Singh et al., 2002). Suitably appraising patchouli fragrance is crucial in order to produce potentially useful synthetic compounds.

Despite being a topic of investigation for many years, the complete odor profile of patchouli remains elusive for three reasons: first, the scents of individual compounds are weak; second, the compounds are structurally diverse and complex; and third, the aroma changes over time. To overcome these obstacles, we performed bulb-to-bulb distillation of the patchouli hexane extract, which had a similar odor as the base material.

The composition of the hexane extract of patchouli leaves was analyzed by 1H and 13C NMR spectroscopy. One constituent of the extract was found to be patchoulol, but its content was low and the other constituents were unidentified. We presumed that the extract contained a large amount of odorless constituents and thus attempted to collect only odor constituents from the hexane extract. Bulb-to-bulb distillation of the hexane extract produced two groups (group A and B) with characteristic odors; the residue did not have a significant odor.

Group A consisted of several sesquiterpenes and anethole (Fig. 7). Group B consisted of almost entirely patchoulol. These two groups were found to contain the key compounds that contribute to patchouli odor.

Fig. 7. Separation of scent components from hexane extract from patchouli leaves.
