WO2007097486A1

WO2007097486A1 - Analysis method of flavor components

Info

Publication number: WO2007097486A1
Application number: PCT/KR2006/000688
Authority: WO
Inventors: Jae-Han Shim; Mi Ra Kim
Original assignee: Jae-Han Shim; Mi Ra Kim
Priority date: 2006-02-22
Filing date: 2006-02-28
Publication date: 2007-08-30
Also published as: KR100777060B1; KR20070084964A

Abstract

Disclosed is a method of analyzing flavor components. Featuring the use of a gas chromatography apparatus equipped with a solvent-free solid injector (SFSI), the method comprises installing an injector of SFSI into an injector port provided for a gas chromatography (GC) apparatus; inserting a glass capillary tube containing a sample into the injection port, said glass capillary tube being sealed at both ends; pre-heating the injector to vaporize flavor components in the sample; crushing the sealed capillary tube to release the vaporized flavor components into a gas chromatography column for a predetermined time period; and analyzing the flavor components in a vapor state. The method enjoys the advantage of being simple and rapid and producing no environmental pollution because no solvents are used. Also, it is very useful for distinguishing Angelica spp. from each other by analyzing the secondary metabolites from the plants.

Description

ANALYSIS METHOD OF FLAVOR COMPONENTS

Technical Field

[1] The present invention relates, in general, to a method of analyzing flavor compounds and, more particularly, to a method for the analysis of secondary metabolites using solvent-free solid injector (SFSI). Background Art

[2] The identification and quantification of secondary metabolites, such as volatile flavor compounds, volatile or non- volatile flavor compounds, and chemical constituents can be done using several extraction and concentration methods, including simultaneous steam distillation and extraction (SD), supercritical fluid extraction (SFE), accelerated solvent extraction (ASE), solid phase microextraction (SPME), and purge and trap extraction.

[3] The technique of simultaneous steam distillation and extraction is the most common and oldest method of extracting essential oils. This technique utilizes, for the most part, distilled water for extraction, but organic solvents are also frequently used. Featuring the use of a supercritical fluid under a condition exceeding the critical temperature and pressure thereof, a supercritical fluid extraction (SFE) method allows the supercritical fluid to readily penetrate into solid samples to extract analytes in a short time. A relatively new technique, known as pressurized liquid extraction or accelerated solvent extraction (ASE), which has gained acceptance in recent years as an alternative to conventional solvent extraction for the separation of secondary metabolites in many analytical and industrial processes, requires only small volumes of organic and aqueous solvents, and allows faster extractions of the volatile flavor compounds from solid or semi-solid samples. ASE operates at high pressures and temperatures above the boiling point of the organic solvent. SPME is a process in which analytes are adsorbed onto the surface of a small fused-silica fiber coated with a suitable polymeric material and placed in a syringe-like protective holder, followed by thermal desorption of the analytes into a suitable instrument to separate and identify them. A gas purge and trap (P & T) technique was used, in which volatile constituents were trapped in a cartridge, followed by automated thermal desorption of analytes into a chromatography injector. The volatile constituents are generated by bubbling a sample in a liquid or solid phase with an inert gas.

[4] In order to analyze flavor compounds, however, these above-mentioned methods require solvent-based extraction and purification processes. That is, because water and organic solvent are mixed and used in the extraction process, several solvent extraction processes are required to remove not only the water but also the impurities from the extracts.

[5] Accordingly, those methods suffer from the disadvantages of being complicated, losing volatile compounds, and being difficult to quantify. Disclosure of Invention Technical Problem

[6] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a simple and rapid method for analyzing flavor components without using solvents to extract target samples and without losing flavor components.

[7] Another object of the present invention is to provide a simple method of precisely distinguishing the secondary metabolites emitted from different species of Angelica roots. Technical Solution

[8] In order to accomplish the above objects, provided is a method for analyzing flavor components using a gas chromatography apparatus equipped with a solvent-free solid injector (SFSI), comprising: installing an injector of SFSI into an injector port provided for a gas chromatography (GC) apparatus; inserting a glass capillary tube, containing a sample, into the inside of the injector, said glass capillary tube being sealed at both ends; pre-heating the injector to vaporize flavor components in the sample; crushing the sealed capillary tube to release the vaporized flavor components into a gas chromatography column; and analyzing the flavor components in a vapor state.

[9] In the method, the sample may be in a solid state or a liquid state.

[10] The sample is selected from a group consisting of a plant, cement, sputum and formalin. The plant is an Angelica species selected from among Angelica gigas Nakai, Angelica acutiloba Kitagawa, and Angelica sinensis Diels.

[11] In a modification of the method, the injector of the SFSI has a plurality of holes through which the vaporized flavor components are introduced into the gas chromatography column.

[12] The pre-heating is conducted at 150 to 35O⁰C and most preferably at 25O⁰C.

[13] The pre-heating is conducted for a period ranging from 3 to 30 min, and most preferably for 7 min.

[14] In the method, the vaporized flavor components are released into a gas chromatography column for a period ranging from 1 to 7 min, and most preferably for 5 min.

[15] Preferably, the vaporized flavor components include decursin and decursinol angelate.

Advantageous Effects

[16] In the method according to the present invention, a sample, even if it is in a solid state, does not need to undergo a solvent extraction process, but can be used as it is. That is, the method of the present invention is simple and rapid. In addition, the method does not produce environmental pollution because it does not need a solvent. Further, the method can be used to analyze flavor components precisely, not only because the vaporized samples are not lost, but also because the vaporized samples are swept at once into the gas chromatography column. In addition, the method can precisely distinguish different Angelica species through the analysis of flavor components.

[17] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. Brief Description of the Drawings

[18] FIG. 1 is a perspective view showing the SFSI used in the present invention.

[19] FIGS. 2 to 4 are schematic views showing the extraction of secondary metabolites using a GC apparatus equipped with SFSI.

[20] FIG. 5 is a graph showing the effect of the pre-heating temperature of the injector on the detection of secondary metabolites of Angelica gigas Nakai.

[21] FIG. 6 is a graph showing the effect of the pre-heating time period of the injector on the detection of secondary metabolites of Angelica gigas Nakai.

[22] FIG. 7 is a graph showing the effect of the time period during which vaporized samples are introduced into the GC on the detection of secondary metabolites of Angelica gigas Nakai.

[23] *Description of the elements in the drawings

[24] 100: SFSI; 110: A bolster.

[25] 120: Injector; 125: The hole for exhausting vaporized samples.

[26] 130: glass capillary plunger.

[27] 150: Glass capillary; 160: Samples.

[28] 200: Gas chromatograph apparatus; 210: Injector port.

[29] 220: Heating device; 230: Column.

[30] 240: Analysis device.

Best Mode for Carrying Out the Invention

[31] Reference should now be made to the drawings, in which the same reference numerals are used throughout the different drawings to designate the same or similar components.

[32] With reference to FIG. 1, the solvent-free solid injector (SFSI) used in the present invention is shown in a perspective view. FIGS. 2 to 4 are the illustration of the extraction method of secondary metabolites using GC equipped with the SFSI.

[33] As shown in FIG. 1, an SFSI (100) comprises a bolster (110), an injector (120), a glass capillary plunger (130), and a control screw (140). As the SFSI (100), a Keele GC injector may be used. In this regard, it could be modified to allow the easy passage of volatiles from the vaporized samples.

[34] The injector (120) is a part which is installed into an injector port of GC. A glass capillary, in which samples are loaded and sealed, is inserted into the inside of the injector. Furthermore, the injector (120) is also modified to form a plurality of holes (125) therein, which serve to introduce vaporized samples into a GC column.

[35] The plunger of the glass capillary (130) plays a role in crushing the glass capillary, in which vaporized samples are loaded and sealed.

[36] With reference to FIGS. 1 to 4, a description will be given of a method for analyzing flavor components using gas chromatography (200) equipped with SFSI (100), below.

[37] FIG. 2 shows a GC apparatus (200) for the identification of secondary metabolites emitted from plants, which is provided with an injector port (210), a heating means (220), a column (230), and an analysis means (240).

[38] The injector port (210) is provided for installing the injector (120) of SFSI (100) into the gas chromatography apparatus therethrough. The heating means (220) is responsible for heating the installed injector (120) to vaporize the samples. Connected with the analysis means (240), the column (230) guides vaporized samples to the analysis means (240). The structures and operating principles of GC (200) are omitted, as they are well-known in the art.

[39] First, a sample (160) of interest is prepared and sealed in a glass capillary (150).

[40] At that time, the sample (160) is used without any solvent extraction. For example, if the sample (160) is a solid type, like a raw plant sample, it could be used in its original form after being cut to a proper size. On the other hand, a liquid type is directly used in proper amounts.

[41] The glass capillary (150) is initially sealed at one end and the prepared sample is introduced into the glass capillary, and then the other side is sealed.

[42] After the injector (120) of SFSI (100) is loaded in the injector port (210), the plunger of the glass capillary (130) is loosened to insert the glass capillary, containing the sample (160), into the inside of injector (120).

[43] Referring to FIG. 3, the glass capillary containing the sample (160) is inserted into the inside of the injector (120), followed by inserting the plunger of the glass capillary (130) into the injector (120) to a proper position therein using the control screw (140).

[44] Subsequently, the injector (120) is pre-heated with the heating means (220) such that the sample (160), sealed within the glass capillary (150) inserted into the injector 12, vaporizes.

[45] Afterwards, as shown in FIG. 4, the glass capillary (150) is crushed such that the vaporized sample (161) within the glass capillary (150) is released into the analysis means (240) through the column (230). At that time, the plunger of the glass capillary (130) is vertically pushed down to crush the glass capillary (150).

[46] The vaporized sample (161) is introduced into the column (230) through holes

(125) formed in the injector (120). A plurality of the holes (125) allows the vaporized sample (161) to be introduced into the column (230) quickly (simultaneously). Moreover, the holes (125) make it possible to control the time period during which the vaporized sample (161) is introduced into the GC column (230).

[47] After entering the analysis means (240), the secondary metabolites of the vaporized sample (161) are analyzed.

[48] After completion of the analysis, the SFSI (100) is removed from the GC such that non- vaporized compounds are not introduced into the column (230), thereby preventing the contamination of the column (230).

[49] [Experimental example 1]

[50] An experiment with SFSI was conducted so as to establish optimal conditions for the analysis of the secondary metabolites emitted from Angelica roots. Analysis efficiency was examined with a few flavor components of Angelica roots, that is, buty- rolactone (Retention time of GC, 7.3 min), 2-acetylpyrrole (12.5 min), and decursin (48.2 min), under various conditions. In this analysis, three species of Angelica, including Angelica gigas Nakai, Angelica acutiloba Kitagawa, and Angelica sinensis Diels, were used. The samples from Angelica gigas Nakai and Angelica sinensis Diels were cultured in Korea, while the samples from Angelica acutiloba Kitagawa were cultured in China. The analysis instrument used was a GC-FID, specifically, a Hewlett Packard series 4890 equipped with Ultra- 1 column (dimethylpolysiloxane, 50 m x 0.2 mm i.d., 0.11 um thickness), and the GC oven temperature was programmed to be kept at 5O⁰C for 5 min, increased up to 28O⁰C at a rate of 5°C/min, and then be held at 28O⁰C for 10 min. The carrier gas was nitrogen, and the temperature of the detector was at 300⁰C.

[51] Angelica gigas Nakai was analyzed as follows. 1 mg of the sample was placed into a soft glass capillary with one end sealed. Then, the other end was sealed briefly using a flame. The glass capillary tube was then introduced into CG using an SFSI. The preheating temperatures of the injector to vaporize secondary metabolites from the sample were set at 200⁰C, 25O⁰C, and 300⁰C. At these temperatures, the pre-heating was conducted for 3, 5, 7, 10, and 30 min. It was designed to take 0, 3, 5, and 7 min to sweep the secondary metabolites onto the GC column using the carrier gas.

[52] With reference to FIG. 5, the pre-heating temperatures of the injector have significant effects on the detection of secondary metabolites in Angelica gigas Nakai.

[53] As seen in FIG. 5, the analysis efficiency of butyrolactone and decursin peaks at

25O⁰C, while 2-acetylpyrrole was analyzed at the highest efficiency at 300⁰C. Accordingly, the optimal pre-heating temperature of the injection was determined to be 25O⁰C, because decursin is more abundant than the other flavor components of Angelica gigas Nakai.

[54] With reference to FIG 6, the detector responses are plotted against the pre-heating time period of the injector according to the secondary metabolites of Angelica gigas Nakai.

[55] As seen in FIG. 6, the extraction efficiency of both butyrolactone and

2-acetylpyrrole increased with an increase in the pre-heating time period, while decursin showed the highest yield at a pre-heating time period of 7 min. Thus, the optimum pre-heating time period was determined to be 7min.

[56] With reference to FIG. 7, the detector responses are plotted against the time period for which the vaporized sample was present in the column according to the secondary metabolites of Angelica gigas Nakai.

[57] As seen in FIG. 7, a maximum extraction efficiency was observed at 5 min for all the secondary metabolites, butyrolactone, decursin, and 2-acetylpyrrole.

[58] Therefore, the optimum conditions obtained for decursin or decursinol angelate can be used to distinguish Angelica gigas Nakai from other Angelica species because Angelica gigas Nakai is more abundant in the secondary metabolites than are the other species.

[59] Although described only for Angelica species, the method of the present invention can be applied for the analysis of not only secondary metabolites of other plants but also smell components of various samples, including cement, sputum, formalin, etc. Industrial Applicability

[60] Featuring the use of samples in their original states without solvent extraction, as described hitherto, the method of analyzing flavor components in accordance with the present invention is applicable to the analysis of flavor components from various samples including plants, cement, sputum, formalin, etc. In particular, the method of the present invention is very useful for distinguishing Angelical spp. from each other by analyzing the secondary metabolites from the plants.

Claims

[I] A method of analyzing flavor components using a gas chromatography apparatus equipped with a solvent-free solid injector (SFSI), comprising: installing an injector of SFSI into an injector port provided for a gas chromatography (GC) apparatus; inserting a glass capillary tube, containing a sample, into the inside of the injector, said glass capillary tube being sealed at both ends; pre-heating the injector to vaporize flavor components from the sample; crushing the sealed capillary tube to release the vaporized flavor components into a gas chromatography column; and analyzing the flavor components in a vapor state. [2] The method according to claim 1, wherein the sample is in a solid state or a liquid state. [3] The method according to claim 1, wherein the sample is selected from a group consisting of a plant, cement, sputum and formalin.

[4] The method according to claim 3, wherein the plant is an Angelica species.

[5] The method according to claim 1, wherein the injector of the SFSI has a plurality of holes through which the vaporized flavor components are introduced into the gas chromatography column. [6] The method according to claim 1, wherein the pre-heating is conducted at a temperature ranging from 150 to 35O⁰C.

[7] The method according to claim 1, wherein the pre-heating is conducted at 25O⁰C.

[8] The method according to claim 1, wherein the pre-heating is conducted for a period ranging from 3 to 30 min. [9] The method according to claim 1, wherein the pre-heating is conducted for 7 min. [10] The method according to claim 1, wherein the vaporized flavor components are released into a gas chromatography column for a period ranging from 1 to 7 min.

[I I] The method according to claim 1, wherein the vaporized flavor components are released into a gas chromatography column for 5 min.

[12] The method according to claim 1, wherein the vaporized flavor components include decursin and decursinol angelate.