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ltrasonic nebulization extraction coupled with on-line gas chromatography foretermination of trans-anethole in spices
u Wang, Yue Liang, Ziming Wang, Chenling Qu, Dan Li, Yuhua Shi, Hanqi Zhang ∗
ollege of Chemistry, Jilin University, Changchun 130012, PR China
r t i c l e i n f o
rticle history:eceived 31 May 2009eceived in revised form 4 August 2009ccepted 5 August 2009vailable online 13 August 2009
eywords:
a b s t r a c t
Ultrasonic nebulization extraction (UNE) coupled with on-line gas chromatography (GC) was proposedfor the determination of trans-anethole in fruits of Illicium verum Hook. f. and Foeniculum vulgare Mill.The extraction was performed in a common self-made extraction system. In the UNE the analyte wastransferred and enriched from the solid sample to gas phase. The sample gas containing analyte obtainedby UNE was introduced into the sampling loop with the purging gas (N2). And then the sample gas inthe sampling loop was introduced into the GC column. Several experimental parameters of on-line UNE-
GC, including sampling time, flow rate of purging gas, standstill time and temperature of tubing, wereoptimized. The calibration curve ranging from 0.05 to 1.5 mg g−1 for determining the trans-anethole wasobtained. The recoveries for determining trans-anethole are between 99.2% and 111.2% and RSDs are lessthan 8.3% when the UNE was applied. The analytes can rapidly be extracted and transferred from thesolid sample to gas phase. The analytes in the gas phase are easier to be introduced into GC system thanthose in the solid and liquid phase. Compared with off-line systems, the proposed on-line system is moresuitable to detect volatile compounds.
. Introduction
Fruits of Illicium verum Hook. f. and Foeniculum vulgare Mill. areidely used as cooking spices and medicinal materials. The essen-
ial oils are the active components in fruits of the two kinds oflants. Many investigations about their antimicrobial and insec-icidal behaviors have been reported [1–4]. In the essential oils,rans-anethole is the major component. The trans-anethole is onef the common rough materials and reaction solvent in the chemi-al industry. For example, alkylanisole derivatives producing fromrans-anethole are valuable intermediates for the manufacturef pharmaceuticals, antioxidant, etc. [5]. Anisaldehyde produc-ng from trans-anethole is an important chemical and chemicalntermediate in the pharmaceutical, perfumery and agrichemicalndustry [6].
Several methods, such as hydrodistillation (HD) which ishe state standard method of China [1,7,8], ultrasonic-assistedxtraction (UAE) [9,10] and microwave-assisted extraction (MAE)
11–13], were usually applied to the extraction of the volatile com-ounds from spices. Recently, these methods coupled with somenrichment techniques, e.g. solid-phase extraction (SPE) [14,15],olid-phase microextraction (SPME) [16,17], and single-drop
microextraction (SDME) [18,19], were applied to the extractionand determination of the volatile and semivolatile compounds on-line.
The ultrasonic nebulization extraction (UNE) was first studiedby our group in the extraction of quercetin from Rhizoma AlpiniaeOfficina-rum [20] and Anthraquinones from Rheum palmatum L.[21]. These experiments confirmed that the UNE is a feasible andalternative method for extraction of effective constituents fromplant samples. But the UNE was not applied to the extraction ofvolatile compounds. It can be seen from literature [21] that thehigh frequency ultrasonic wave (1.7 MHz) can result in “ultrasonicfountain” and aerosol at the same time. In the UNE process, thevolatile compounds in the spices were transferred from the spicesto the extraction solvent and then to gas phase. Namely, the UNEmethod is different from other conventional extraction methods,analyses can rapidly be extracted, transferred and enriched fromthe solid sample to gas phase.
The purge and trap (P&T) method [22–26] is used to transferthe volatile compounds from a liquid or solid matrix to gas phaseby an inert gas flow and has been applied to the analysis of water[22], soil [23], body fluids [24], and so on [25,26]. It is a simple and
fast method to detect the volatile and semivolatile compounds inliquid. But for the solid sample, it needed pre-extraction off-line,which made operation steps increase.
In this paper, a novel on-line analytical method combined theUNE and purge technique was used to detect trans-anethole in
ruits of I. verum Hook. f. and F. vulgare Mill. It has both advan-ages of UNE and on-line analysis. Compared with off-line systems,he on-line UNE coupled with GC technique has several advan-ages: (1) minimal sample contamination, (2) ideal method forhe labile samples, (3) reduction of manual operation, (4) morenexpensive settings-up, (5) reduction of analyzing time, (6) theossibility for a totally automated analytical system. In addition,ompared with P&T, the proposed method is suitable to ana-yze solid sample. And the UNE coupled with many enrichmentechniques (SPE, SPME, SDME and so on) can improve the sensitiv-ty.
. Experimental
.1. Chemicals and sample preparation
Three kinds of fruits of I. verum Hook. f. (named as Sample, 2 and 3) and three kinds of fruits of F. vulgare Mill. (nameds Sample 4, 5 and 6) were purchased from Changchun, Jilinrovince, China. The fruits of I. verum Hook. f. and F. vulgare Mill.ere crushed by high-speed disintegrator (FW-100 Test Instru-ent Co., Ltd., Tianjin, China) at 24,000 rpm, and the powders
btained were passed through 80 mesh sieve and stored in theesiccators.
The standard of trans-anethole was purchased from Acros Com-any (NJ, USA). The naphthalene was used as internal standard andurchased from Beijing Chemical Factory (Beijing, China). Both ana-
yte and internal standard were dissolved in n-hexane to obtaintock solution. These solutions were stored in the dark glass bottlest 4 ◦C. Pure water was obtained by using Milli-Q water purificationystem (Millipore Co., USA). The other reagents purchased fromeijing Chemical Factory (Beijing, China) are all of analytical grade.
Fig. 1. On-line UNE system. (a) Extracti
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2.2. Instruments and apparatus
The on-line UNE-GC system was assembled in our laboratory. Aschematic diagram of the system is shown in Fig. 1. An ultrasonichumidifier (Beijing Branson Ultrasound Co. Ltd., China) working at1.7 MHz with maximum output power of 35 W was employed asthe ultrasonic source. A self-made glass flask (100 mL, ID 10 cm)was used as extraction vessel. There were three ports on this ves-sel. One of the ports on the bottom of the vessel has the same sizeas the piezocrystal and was sealed by PVC film. Other accessories,such as an injection valve (Nanda Analysis Instrument GraduateSchool, Nanjing, China), a sampling loop (1 mL) and a few stainlesssteel tubes, were used to fabricate the on-line system. A filter-ing column consisting of a stainless steel cylinder (2 cm × 3 mmi.d.) packed with glass wool was closed by screws at either end.A thermocouple sensor (XMTD-2001 Xinghua AOTE temperatureInstrument Co., Xinghua, China) connected with a heating tape wasused to measure the temperature. In addition, an ultrasonic gener-ator (KQ2200E Kunshan Ultrasonic Instrument Co. Ltd., Kunshan,China) with 150 W of output power and 40 kHz of frequency wasused in UAE.
The extract was analyzed by GC (Hewlett Packard computerizedsystem comprising a 4890 gas chromatograph) with a HP-1 column(30.0 m × 250 �m × 0.32 �m film thickness). Nitrogen was used asthe carrier gas at a flow rate of 4 mL min−1.
2.3. Extraction procedures
2.3.1. On-line UNE-GCIn sequence 1 (Fig. 1a), the mixture of 0.1 g of sample and 15 mL
of n-hexane, which was referred to as the extract, was put intoextraction vessel and the nebulizer was switched in. During this
on, (b) sampling and (c) injection.
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exist in the aerosol. Under the gravitational effect those powdersfall into the extract after a period of time. This circulating processis beneficial to the renewal of the solvent on the surface of samplepowders and the extraction efficiency can be enhanced. If the nebu-lization stopped, the sample powders would gradually drop into the
66 L. Wang et al. / Tal
equence, the extraction vessel was full of aerosol. The valve 1 wasn extraction position and valve 2 in sampling position. 5 min later,he nebulizer was switched off. The nebulizer was at a standstill forcertain standstill time. The aerosol in the headspace of extractionessel will drop into the extract located in the bottom of extractionessel. In sequence 2 (Fig. 1b), the valve 1 was in injection posi-ion, and valve 2 was also in sampling position. The purging gasN2) (270 mL min−1) was passed through the extraction vessel forcertain purging time and the sampling loop was fill with the sam-le gas containing analyte. In sequence 3 (Fig. 1c), the valve 1 was
n extraction position and valve 2 was in injection position. Theample gas in sampling loop was introduced into GC column andnalyzed. The tubing in the UNE system was wrapped by a heatingape except the extraction vessel, and the temperature of heatingape was controlled at 80 ◦C.
In the method, the volatile compounds in the spices were trans-erred from solid phase (spices) to liquid phase (extraction solvent)ecause of the UNE extraction, and then transferred from liquidhase to gas phase due to the gas–liquid equilibrium. The focusednergy of UNE makes the extraction solvent and sample powderspurt from the bottom of the extraction vessel, which looks likefountain. Thank to the “ultrasonic fountain”, the sample pow-
ers can be spurted from the bottom of the extraction vessel anduspended in the headspace in the aerosol and then dropped intohe bottom of the extraction vessel. In this process, because theollision between extraction solvent and sample powder occurredrequently and the extraction solvent around the sample powderas renewed rapidly, the extraction efficiency of the analytes was
nhanced. In the other hand, a lot of aerosols, which are small inize, were produced in UNE. The transfer the analytes from theerosols into the gas phase is easy because the surface area oferosols is large [27] and the interface between gas and liquids frequently renewed. The gas–liquid distribution equilibrium ofnalytes was rapidly reached. So the total amount of the volatileompounds in gas phase increased at a certain time.
.3.2. Off-line UNE0.10 g of sample powder and 15 mL of n-hexane were added into
he extraction vessel. The optimized UNE conditions were as fol-ows: extraction solvent was 15 mL of n-hexane; ultrasonic power
as 35 W; extraction time was 5 min. The extract was filtered with.45 �m micropore filter membrane and then analyzed by GC.
.3.3. UAEThe comparison between conventional UAE with on-line UNE
as carried out to evaluate the extraction efficiencies of differentethods. 0.10 g of sample powders and 15 mL of n-hexane were
dded into the extraction vessel. After UAE for 5 min, the result-ng suspension was filtered through a 0.45 �m micropore filter
embrane. And 0.3 �L extract was injected into the GC system fornalysis.
.3.4. HDAccording to Chinese Pharmacopoeia [1], 20 g of sample and
00 mL of water were put into a 500 mL flask and heated by heatingacket at 100 ◦C for 4 h. The essential oil was collected, dried withnhydrous sodium sulphate and then stored at 4 ◦C until analyzed.
.3.5. Gas chromatographyThe operating conditions were as follows: nitrogen carrier gas
ow rate 4.0 mL min−1; split ratio 1:10; injection and detectionemperature 260 ◦C; oven temperature progress from 60 ◦C (hold-ng for 3 min) to 240 ◦C at the rate of 5 ◦C min−1. The injectionolume is 0.3 �L in off-line methods. The flame ionization detectoras used and its temperature was 260 ◦C.
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3. Results and discussion
3.1. Optimization of on-line UNE-GC
Some parameters can affect the performances of on-line UNE-GC, such as purging time, flow rate of purging gas, standstill timeand temperature of tubing. When one parameter was changed,other parameters were fixed at their optimal values.
3.1.1. Effect of purging timeThe effect of purging time on peak area of trans-anethole is
shown in Fig. 2. It can be seen that the peak area increases withthe increase of purging time from 10 s to 20 s, does not significantlychanges from 20 s to 60 s and slightly decreases from 60 s to 120 s.When purging time was shorter than 20 s, the analyte concentra-tion in the sample gas in the extraction vessel and the samplingloop was not identical, and concentration of analyte in samplingloop was lower than that in the extraction vessel. On the contrary,the concentration of analyte in extraction vessel was diluted whenpurging time was longer than 60 s. Remarkably, the precision wasnot satisfactory when the purge time was 10 s. So 20 s was chosenfinally to be purging time in this work.
3.1.2. Effect of flow rate of purging gasThe effect of flow rate of purging gas ranging from 70 to
630 mL min−1 on the peak areas was examined, and the result isshown in Fig. 3. It can be seen that the peak area of trans-anetholeincreases at first, but then decreases when the flow rate of purginggas is higher than 270 mL min−1. The flow rate of purging gas wasmeasured by soap film flowmeter in waste vent. At low flow rate,small amount of analyte was introduced into the sampling loop. Onthe contrary, the overhigh flow rate will dilute the sample gas inextraction vessel. So, the flow rate of purging gas was chosen finallyto be 270 mL min−1.
3.1.3. Effect of standstill timeIn the UNE extraction process, the sample powders of small
diameter will spurt from extract, suspend in the headspace and
Fig. 2. Influence of the purging time. Flow rate of purging gas, 270 mL min−1; stand-still time, 5 min; temperature of tubing, 80 ◦C.
L. Wang et al. / Talanta 80 (2009) 864–869 867
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ig. 3. Influence of the flow rate of purging gas. Purging time, 20 s; standstill time,min; temperature of tubing, 80 ◦C.
xtract along with the aerosol. The concentration of volatile com-ounds was highest in the headspace of extraction vessel when theebulization stopped. If the sampling was performed immediately,large quantity of aerosol was introduced into the sampling loop
n which the solution droplet can form and block the tubing. So, theampling was performed after a period of standstill. The effect oftandstill time of 0, 1, 3, 5, 8 and 10 min was tested, respectively.
hen the standstill time was longer than 5 min, a large quantityf aerosol containing sample powders would drop into the extract,hich was observed by the naked eye. The results are shown in
ig. 4. It can be seen that the extraction yield of trans-anetholeecreases with the increase of the standstill time. If the stand-till time is too long, the trans-anethole in gas phase would loss,hich results in the decrease of concentration of trans-anethole
n sample gas. So the standstill time of 5 min was selected in theork.
.1.4. Effect of temperature of tubingThe effect of tubing temperature from 20 to 120 ◦C on the peak
rea was evaluated, and the result is shown in Fig. 5. It can beeen that the peak area increases obviously along with the increase
ig. 4. Influence of the standstill time. Purging time, 20 s; flow rate of purging gas,70 mL min−1; temperature of tubing, 80 ◦C.
Fig. 5. Influence of the temperature of tubing. Purging time, 20 s; flow rate of purginggas, 270 mL min−1; standstill time, 5 min.
of temperature from 20 to 80 ◦C, whereas it decreases when thetemperature is higher than 80 ◦C. Before the sample gas was intro-duced into GC column the main component in the gas phase andthe aerosol was n-hexane whose boiling point was 68.7 ◦C. At the270 mL min−1 of flow rate, small amount of aerosol in sampling gaswas introduced into the sampling loop. When the temperature islower than 68.7 ◦C, n-hexane can precipitate on the wall of tub-ing. Therefore, the analyte transferred to the sampling loop lost. Ifthe temperature was too high the small amount of aerosol whichwas transferred to the sampling loop will vaporize and then thevolume of sample gas was expanded and the concentration of ana-lyte in sample gas decreases. Finally, the temperature of tubing waschosen to be 80 ◦C in this work.
3.2. Evaluation of the method
3.2.1. Standard curve and limit of quantificationIn the work, trans-anethole was determined by on-line and off-
line UNE-GC. The working standard solutions of trans-anethole atthe concentrations ranging from 0.05 to 1.5 mg mL−1 were pre-pared. The linear equations for on-line and off-line UNE-GC areA = 0.01574 + 4.05186C and A = −0.3107 + 2.50664C and limits ofquantification (LOQs) are 0.020 and 0.017 mg g−1, respectively. TheLOQs were determined as the lowest concentration that yielded asignal-to-noise (S/N) ratio of 10.
3.2.2. Enrichment effect of UNEIn the UNE process, the frequency of acoustic vibration is about
1.7 MHz. When the vibration was transmitted through the solvent,“ultrasonic fountain” and aerosol would occur. If the analyte hasgood volatility, its concentration in gas phase obtained by UNE ishigher than that obtained by stirring extraction. The reasons maybe that compared with the stirring extraction, the transfer rate ofvolatile compounds from liquid to gas phase is more rapid andgas–liquid equilibrium is more rapidly reached when the UNE isapplied. Compared with the conventional stirring extraction, theUNE process also can be viewed as an enrichment step. In order toevaluate the enrichment effect of UNE, the stirring method with-out nebulization was applied. In the two experiments, the standardsolution at concentration of 0.2 mg mL−1 was added into the 15 mL
n-hexane in extraction vessel. In the UNE, both the nebulizationand the standstill time was 5 min; while in the stirring method, thestirring time was 10 min. The resulting sample gas in extractionvessel was introduced into GC for analysis. The results indicted thatthe concentration of the analyte obtained by the UNE was 5 times
868 L. Wang et al. / Talanta 80 (2009) 864–869
Table 1Contents of trans-anethole in fruits of Illicium verum Hook. f. and Foeniculum vulgare Mill. obtained from different cultivated areas.
ig. 6. Chromatograms of standard solution (A) and extracts obtained by off-lineNE-GC (B), and on-line UNE-GC (C). a, naphthalene; b, trans-anethole.
igher than that obtained by stirring method. It was obvious thatnrichment effect of UNE was significant.
.2.3. Sample analysisThe trans-anethole in fruits of I. verum Hook. f. and F. vulgare Mill.
btained from different cultivated areas was determined by on-linend off-line UNE-GC, respectively. The chromatograms of standardolution and extracts obtained by on-line UNE-GC and off-line UNE-C are shown in Fig. 6. The contents of trans-anethole obtained byn-line and off-line UNE-GC are shown in Table 1. From Table 1, itan be seen that the contents of trans-anethole in fruits of I. verumook. f. and F. vulgare Mill. obtained by on-line are very similar. The
esults demonstrate proposed method can be applied to the realample analysis with good accuracy. But, it was worth to notice thathere were significant differences in the contents of trans-anetholeelong to different samples, which was perhaps due to the differentultivated areas and picking periods. The contents of trans-anetholen the samples collected from different areas were compared apply-
ng of variance. For this purpose, the Student’s t test was applied.he statistical analysis indicated that there are significant differ-nces among contents of trans-anethole in the samples collectedrom different area. The difference between sample 5 and 6 wasignificant (P < 0.01) and the differences between the other samples
Fig. 7. Comparison of different extraction methods.
were very significant (P < 0.001). In addition, the RSDs and recov-eries obtained by on-line UNE-GC are 3.3–7.0% and 99.2–111.2%,respectively. The results also indicate the on-line UNE-GC can beapplied to the determination of trans-anethole in fruits of I. verumHook. f. and F. vulgare Mill. with good precision and accuracy.
3.3. Comparison of HD, UAE, off-line UNE and on-line UNE
In order to evaluate the extraction efficiency of proposedmethod, HD and UAE were applied to extract trans-anethole fromsample 1. The extraction yields obtained by different methods areshown in Fig. 7. Correspondingly, the extraction yield obtainedby HD is the lowest. So, compared with HD, the extraction yieldobtained by on-line UNE-GC is higher, but shorter extraction timewas required and lower energy was consumed. Furthermore, theextraction yield obtained by proposed method is in agreement withthose obtained by UAE and off-line UNE-GC, which indicates thaton-line UNE-GC is a feasible method for extracting volatile com-pounds from spices. In addition, it is worth to notice that on-lineUNE-GC is also a gentle and enjoyable method without any noise.
4. Conclusion
In this study, UNE coupled with on-line GC detection wasapplied to determining trans-anethole in fruits of I. verum Hook. f.
and F. vulgare Mill. The experimental conditions for on-line UNE-GCwere examined and optimized. On-line UNE-GC was then com-pared with HD, UNE and off-line UNE-GC. The extraction yieldobtained by on-line UNE-GC was obviously higher than thoseobtained by conventional HD, and RSD and recovery obtained by
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he proposed method are also satisfactory. Therefore, it can be con-luded that on-line UNE-GC is a simple, labor saving, sensitive,ovel and feasible method for extracting and determining the con-tituents in essential oil from spices. If the UNE coupled with manynrichment techniques (SPE, SPME, SDME and so on), the sensitivityf the proposed method would be improved.
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