Chromatographic analysis and physicochemical evaluation of the essential oil of Bauhinia monandra Kurz flowers

The study of chromatographic techniques, classical and modern, describes the simplicity and, at the same time, the advances that this area has undergone in recent years in quality scientific research and also in learning in undergraduate and postgraduate courses around the world. This paper investigate a characterization by thin layer chromatography (TLC) and gas chromatography coupled with mass spectrometry (GC-MS), as a method developed by graduate students that involve a combination of a classic and modern technique, as well as results about the physicochemical properties of the essential oil of Bauhinia monandra flower. Essential oil was extracted by Clevenger, the TLC was performed in different eluents and developers, and thus the retention factors (Rfs), and the chemical profile by GC-MS were obtained. The essential oil of the flowers showed a yield of 0.06%, positive solubility in ethanol 70%, refractive index of 1.3621, optical rotation of +36.4αD and relative density of 0.941 g mL-1 at 20 °C. In the TLC analysis 18 Rfs were observed after the use of different developers, with the predominant class of oxygenates compounds. In the GC-MS analysis, 7 compounds were observed, being two majorities, characterized as panaxene with 20.51% and the α-guaiene with 33.39%. The essential oil of B. monandra flower showed a predominance of 70.22% of sesquiterpenic compounds. The allied techniques, classic and modern, demonstrated different ways of evaluating the essential oil through its chemical composition, both techniques showed high efficiency and precision, in addition was an appropriate project developed by postgraduate students.


Introduction
Techniques, such as, thin layer chromatography (TLC) and gas chromatography coupled with mass spectrometry (GC-MS) are important subjects in the development of studies on separations of compounds in various areas of chemistry. Chromatography is a widely studied technique as a tool to solve different problems in research and industry, and it is taught in undergraduate and postgraduate courses (Dawan et al., 2017;Lebot;Legendre, 2016;Silva et al., 2009).
The TLC is one of the most common techniques in the separation of compounds, presenting low cost of material and reagent, being well discussed in textbooks and various scientific articles, it is also considered the basis for separation chromatographic studies.
Another chromatographic technique of great importance is the GC, considered one of the modern techniques, has wide use in large research centers, University, pharmaceutical industries, forensic science, being a technique of separation, identification and determination of compounds with greater robustness and sensitivity, however, presents hight costs with maintenance of the equipment, in the purchase of gases and reagents of hight purity degree (Liu et al., 2018;Chiaradia et al., 2008).
However, the quality of scientific information has been demanding greater efficiency, accuracy and precision of data obtained in the area of chromatography, reducing the use of classical techniques, which are addressed in teaching and in simpler studies especially to phytocomposites. Advanced techniques such as high performance liquid chromatography (HPLC) and GC with detection by mass spectrometry (MS) are focused on high efficiency in the execution of experiments and in obtaining results with superior quality and reproducibility, which is not observed in classic techniques. (Tahtah et al., 2016;Collins, 2011;Lances, 2009).
Traditional techniques provide great assistance, as previously mentioned, in studies with biopharmaceuticals in particular of vegetable origin. It is through the TLC that the researcher can elucidate the classes of compounds present in a specific plant organ, making a survey of a certain number of natural species and their biological activity. With these species is possible to produce in the laboratory, tinctures and extracts, using steam-dragging or hydrodistillation, crushing and supercritical fluid. This information can be confirmed when combined with the empirical knowledge of the rural population who use plants as a means of treating and curing countless forms of disease (Ghosh et al., 2019;De Oliveira et al., 2018;Fadel et al., 2018;Yadav, et al., 2017;Nóbrega et al., 2017).
In this study, both TLC and GC-MS chromatographic techniques were used to characterize the essential oil extracted from Bauhinia monandra flowers, since does not have data about its characterization in the literature. The specie B. monandra is exotic in Brazil, and very important due to its medicinal use in the treatment of diabetes, and different biological activities such as antioxidant, antifungal, diuretic and hypoglycemic (Araújo-Melo et al., 2017;Fernandes et al., 2012;Menezes et al., 2007;Aderogba et al., 2006;Argolô et al., 2004). This specie is native to the tropical regions of Southeast Asia, such as China, India and Vietnam. In the Brazilian territory, it has adapted well to several regional climates, it is one of several species of the genus Bauhinia widely used in ornamentation, being a floristic option for the cities, avenues, parks and gardens, because it is an eccentric species due to the size of the numerous flowers, as well as the exhaled aroma produced from the essential oil (Anoliefo;Gill, 1992).
The present paper aims to evaluate the classes of compounds present in the essential oil of Bauhinia monandra flowers by TLC and GC-MS and also to obtain information of its physicochemical properties. Research, Society and Development, v. 9, n. 8, e454985979, 2020 (CC BY 4.

Material and Methods
The study is an academic research that consist of a field and laboratory part realized in the first semester of 2019. It was developed as a part of the Advanced Chromatography discipline to three postgraduate students of Program in Agrochemistry "stricto sensu" offered by the Goiano Federal Institute (IF Goiano), Campus Rio Verde, Goiás, Brazil. The study is of a qualitative and quantitative "quali-quanti" nature based on the studies by Pereira et al. Filho. An excicata was herborized and deposited in the Herbarium of the IF Goiano, with the following record, Voucher HRV: 1345.
The essential oil of B. monandra flowers was extracted in a Clevenger-type system (Marconi, Mod. MA553/2000) from aliquots of 500 g of inflorescence. Each aliquot was processed (Philips Walita, Mod. Viva Collection) with 500 mL of distilled water. The solution was transferred to the system, maintained under reflux for 3 hours. The hydrolat was collected and transferred to a 500 mL separation funnel (Laborglas, Borosilicate), and then washed three times with 10 mL of dichloromethane (Alphatec, P.A. -ACS, purity 99%). An organic fraction was collected and dried with anhydrous sodium sulfate (Vetec, P.A -ACS, purity 100%), and then filtered on qualitative filter paper (Unifil, C42). The supernatant was kept in a 125 mL becker bottle (Qualividros, Boron 3.3) wrapped with aluminum foil, in an environment protected from light and heat above 25 °C.
Then, the yield of essential oil mass obtained was calculated according to equation 1, described by Menezes Filho et al. (2020).
The refractive index test was performed in a refractometer (Hanna Instruments, Mod. HI96800) with refractive indexes between (1.3330 to 1.5080) and resolution of 0.0001 to 20 °C, according to Alarcón et al. (2019).
The optical rotation test was determined in polarimeter provided with a 10 mL cell at a temperature of 20 °C and line αD of the sodium lamp at 589.3 nm (Novainstruments, Mod. WXG-4) with a measurement range of -180° to +180° on the Vernier scale. The sample was prepared with 10% (w/v) of essential oil in 96% ethanol (LChemicals, P.A -ACS, purity 99.5%), as described by Alarcón et al. (2019).
For relative density, a pycnometer (RBR, Mod. 0892) of 1 mL was used. The pycnometer was clean and dry and weighed empty and the mass determined. Then 1 mL of essential oil was added, and subsequently its mass was determined and annotated. The density was expressed in g mL -1 at 20 °C and determined according to equation 2 as described by Alarcón et al. (2019).

Eq. [2]
Where Pic means Pycnometer, Picvz is an empty pycnometer and V is the volume of EO in mL.

Eq. [3]
Where Rf is the retention factor, ds is the distance that the compound has moved from the base, dm is the distance traveled by the spot in the plate.
The composition of the essential oil was also evaluated by GC-MS. In this experiment a PerkinElmer GC Clarus 580 equipment coupled with MS Clarus SQ 8S was used, the injector, interface and source temperatures were 250, 270 and 270 °C respectively, using electron impact at 70 eV. The analysis was performed using the following heating method, 60 °C for 2 min., rising to 270 °C at a ratio of 12 °C min. -1 and remaining for 1 min, helium gas flow was 1 mL min -1 , 1 µL of sample dissolved in ketone (Alphatec, P.A -ACS, purity 100%) was injected. The column used was a DB-5MS (30 m, 0.25 mm ID, 0.25 µm).
The identification of the compounds was performed from the n-alkane calibration standar series (C7 -C40) (Sigma-Aldrich), and the mass spectrum was compared to the literature of Adams (2007) and by the Nist 11 Spectroteca.
The statistical analysis consisted of triplicates from the arithmetic mean, followed by (±) standard deviation. The statistical software used was PAST 3 (free version, 2019).

Results and Discussion
The essential oil of B. monandra flower showed a yield of 0.06 ± 0.09%, the organoleptic characteristics showed as a translucent, homogeneous, crystal clear, aromatic and oil liquid to the touch.
It is remarkable the number of studies on the essential oil yield of the genus Bauhinia in the literature as shown in the Table 1. Sharma et al. (2013) in their study found an essential oil yield of 0.3% for Bauhinia variegata flowers obtained by hydrodistillation. However, Da Silva et al. (2020) extracted the essential oil of two species, Bauhinia rufa and Bauhinia dumosa, and found yield of 0.01 and 0.03%, respectively.  Experiments to evaluate the composition of the essential oil were performed and Table   2 presents the results of the analysis by TLC and respective retention factors (Rfs) for compounds detected in the analysis of essential oil of the flowers of B. monandra using different elution systems and developers.
Research, Society and Development, v. 9, n. 8, e454985979, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5979 The results obtained in the analysis by TLC shown that the eluent dichloromethane presented a greater number of compounds (7)  In the TLC analysis, mainly considering monoterpenes, it was identified only 3 compounds for the eluent hexane, for dichloromethane 7 compounds and especially for the solution of hexane/dichloromethane 8 compounds were observed in the separation. The eluted compounds in dichloromethane were revealed under UV365 nm light which suggests the presence of aromatic compounds, and the other 6 compounds were revealed using a dichloromethane/hexane solution combined with exposure to Iodine steam, which is considered one of the universal revealers. This may be related to a rapid reaction, where the Iodine atom is complex to oxygenated and/or aromatic functional groups (Collins et al., 2006). Development, v. 9, n. 8, e454985979, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5979 11 The results obtained using the developers Iodine, UV light and sulfuric vanillin, revealed the largest number of compounds, especially for Iodine, 11 compounds was observed. Ferric chloride has not been shown to be the best option evaluated separately, as only 3 compounds were observed after discoloration. When the plates was submitted to UV lights in short UV254 nm and long UV365 nm, 5 and 6 separate compounds were revealed suggesting the presence of conjugation and fluorescence, respectively.
An interesting feature was observed during the analysis by TLC, most of the compounds visible under UV light were observed due to the previous use of ferric chloride or sulphuric vanillin in the eluent dichloromethane. The application of sulphuric vanillin and ferric chloride in combination with hexane: dichloromethane eluents and in petroleum ether even after exposure to UV light did not reveal any class of compounds. However, the developers vanillin sulphur and ferric chloride combined with eluent acetone or acetone/petroleum ether and exposed to UV254 nm, showed different plots suggesting the presence of classes of phenolic compounds.
From Table 1, it is observed that the oxygenated compounds presented Rfs between 0.58 and 0.59 revealed with UV254 nm light, between 0.06 and 0.56 revealed with UV365 nm light, and with Iodine steam between 0.12 and 0.50. With this evaluation, it can be discussed that polar compounds with lower Rfs interacted more with silica, and that for the higher Rfs did not present such interaction, this is due to the interactions of polarities of the eluents used, and of the groups of compounds evaluated.
Although the TLC does not provide specific measures, only knowledge about class of compounds and number of spots, as a preliminary analysis, it is effective when used in combination with other techniques such as GC-MS. Several chromatographic methods are developed to standardize the analysis mainly for compounds originating from medicinal plants, which include GC-MS to ensure that there is quality, efficacy and safety in the results with the aim of developing new drugs for pharmacological use (Valle Jr. et al., 2016).
According to Chiaradia et al. (2008), Andrey (2003, and Kitson et al. (1996), the combination of GC and MS is relatively simple, where the gas chromatograph is compatible with the use of the vacuum produced for MS. The ionization methods with the highest employability in GC-MS are electronic impact (EI) and chemical ionization (CI). In EI the analyte, in gas phase, is bombarded with high energy electrons, with a power of 70 eV, producing fragmented ions, which help in the identification of compounds. In this technique, molecules in the form of gas absorb this energy by triggering several processes, and the analyte is ionized by removing a single electron (M + or M -). In this process, the energy use is Development, v. 9, n. 8, e454985979, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5979 12 10 eV and the rest of the energy generates the fragmentation in the analyte. However, there is a disadvantage in the application of IE, the fragmentation of a compound occurs with rapid reaction, the molecular ion may not be observed in the GC-MS total ion chromatogram (TIC), losing important information in MS.
Besides the analysis by TCL, the essential oil of the B. monandra flowers was evaluated by GC-MS. After the chromatographic analysis, the GC-MS total ion chromatogram (TIC) and fragmentogram or mass spectrum are obtained. Figure 1 shows the TIC obtained for the essential oil of B. monandra flowers from GC-MS analysis. By the TIC it is possible to observe two peaks of maximum intensity with retention times of 11.07 and 12.50 minutes, which when compared with the equipment library and with data of alkane patterns, correspond to the panaxene and α-guaiene compounds, respectively. In Figure 2 the characteristic GC-MS mass spectra of the panaxene and α-guaiene majority compounds are presented, the comparison between the mass spectrum fragments obtained with that of the NIST 11 library showed a similarity of 84.5 and 83.6 % as shown in Table 3, both compounds present m/z = 204, characterized by similar fragment ions as m/z = 93, m/z =105 and m/z = 121. Development, v. 9, n. 8, e454985979, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5979 Table 3 presents the compounds with retention times, Kovats' literary indexes, relative percentage areas for the peaks and the similarity with the library for each compound. Only 7 compounds were observed, panaxene with 20.51% and α-guaiene with 33.39%, as the two highest peaks detected in relation to the total area of compounds analyzed. The essential oil of B. monandra flower showed about 70.22% sesquiterpene compounds and only 7.13% monoterpenes. These data are in accordance with the analyses made by TLC where 7 compounds were detected. to assemble the experiments and discuss theories and practices, and it was possible to develop a work that resulted in a publication.

Conclusions and Suggestions
This is the first study reporting the essential oil of Bauhinia monandra flowers, an exotic species in Brazil. As for the essential oil yield, it presents similar to the few data in the literature consulted for this genus. The organoleptic and physicochemical properties are similar to other essential oils of numerous plant species assessed. However, the focus of the study was to characterize the essential oil and compare the techniques by TLC and GC-MS among the study of numerous chromatographic techniques.
In this study, it is possible to conclude that the analysis by TLC presents relative ease acquisition of the commercial ready plate and the reagents. However, the technique by GC-MS presents greater robustness and greater efficiency in the separation of compounds, which were identified and characterized. The study of chromatographic separations described in this study presents two proposal, characterization of a new essential oil and elucidate the teaching of chromatography in higher education and in postgraduate studies, mainly in the area of natural products.
To future work, experiments should be carried out, evaluating the essential oil of B.
monandra toward different types of oxidizing agents to application in the pharmaceutical and Development, v. 9, n. 8, e454985979, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5979 15 chemical industry, also as an antioxidant in the food industry and, as a biological agent in the form of micro and nanocapsules, as well nanospheres incorporated with chitosan or calcium chloride in phytopathogens and pathogens also to application in the agricultural and biotechnology industries.