pu - erh tea is a special tea species in china and has become one of the most popular beverages in southwestern china and southeast asian , owing to its special flavour properties and potential healthy benefits .
it is originated from yunnan province ( china ) through a special post - fermentative process , using crude green tea prepared from the leaves of c. sinensis var .
assamica as original materials . because pu - erh tea has a malty flavour and low - stimulation taste in tea infusions   which may fit female appetite , recent years , interest in the flavor and the healthy properties and the related scientific investigations were increasing [ 3 , 4 ] .
up to now , a little information on the relationship of the chemical composition to the flavor is available .  
volatile flavor component is one of the most important factors to influence the flavor , taste , and quality of pu - erh tea , in which the contents are different from the green and black tea because of different processing procedure and variety of species and cultivar .
investigation on the components in green and black tea are reported elsewhere , yet few on those in pu - erh teas . in order to explore the influence of those components to flavor , taste , and quality , quantitative analysis of main volatile flavor components in pu - erh tea
for analysis of volatile components or essential oils , several extraction techniques , including soxhlet extraction , liquid - liquid extraction ( lle ) , simutaneous distillation - solvent extraction ( sde ) , solid phase microextraction ( spme ) , and headspace microextraction ( hsme )   and so forth , had been used to diferent matices .  
soxhlet extraction is a classical method for decades in extraction of organic compounds from solid sample , and this apparatus has been developed to several types for special use .
thorough  extraction method because the organic phase cooled from condensation tube continuously passes through the target solid sample for hours .
however , poor recovery commonly occurred for extraction of high - volatile or heat - labile compounds
. lle is a conventional method for isolation of all boiling range volatile compounds , based on the compatibility of compounds with organic phase selected .
the main disadvantage is solvent - consuming , tedious and , low - recovery for some target compounds .  
sde , proposed by godefroot and so forth , has been widely applied to analysis of volatile components in tea samples .
although low recovery has been found for extracting the most volatile or heat - labile components , this technique has achieved higher recoveries and greater repeatability of volatile or semi - volatile and heat - stable components than other isolation technique such as spme   or hsme when low water temperature in the circulating system was used .
spme and hsme are relatively new techniques that are able to address the need for concentrating the components in the headspace [ 10 , 11 ] . both of them use a small piece of fused silica , on which a liquid or solid phase , similar to a gc stationary phase , has been coated to absorb the desired components and concentrate them on the fibre . thus the two techniques are more sensitive for the isolation of high volatile components than sde , while less sensitive and lower repeatability for the low volatiles than sde .
conventionally , pu - erh tea is condensed to a pie - like tea - biscuit in the final processing procedure .
extraction of the volatile flavor components will be different from the green and black tea . in order to select the best extraction technique for studying the volatile flavor components of pu - erh tea , a modified sde was evaluated for quantitative determination of the analytes using ethyl decylate as internal standard , and the two classical techniques , steam distillation - liquid / liquid extraction and soxhlet extraction ,
pu - erh tea samples were obtained from dayi limited incorporation ( menghai , yunnan , china ) . the sample was dried at 40c in electric oven for 6  h , ground to 3060 mesh , and sealed for use . the reference volatile chemicals were purchased from sigma ( st . louis , mo , usa ) . the stock solution was prepared by dissolving single solid / liquid standard in dichloromethane to an appropriate concentration depending on the content in pu - erh tea .
ultra - pure water was obtained from a milli - q water purification system ( pall co , il , usa ) .
the other solvents used in the test were all of analytical - grade and disdillated before use .
dichloromethane and ethyl decylate were employed as solvent and internal standards , respectively . for each extraction , 15  g of tea sample ,
10  g sodium sulphate , 100  l internal standard solution and 300  ml ultra - pure water were placed in a 1  l flask , 50  ml dichloromethane was in a 100  ml flask , and temperature of the circulating water system was operated at 8c .
stream distillation was stopped after 2  h , while the solvent extraction was continued for a further 15  min .
the extract was concentrated to 1  ml at 10c by a nitrogen - purge apparatus ( shanghai anpel scientific instrument co. ltd ) .
the concentated solution was dehydrated with anhydrous sodium sulphate for at least 12  h , of which 2  l was injected to gc or gc - ms system for analysis . for sd - lle ,
15  g of tea sample , 10  g sodium sulphate , 100  l internal standard solution and 500  ml ultra - pure water were placed in a 1  l distillation flask , respectively .
the liquid was transferred to a 500  ml of separation funnel and then extracted three times ( 30  ml  3 ) using dichloromethane .
the extracted organic phase was combined and concentrated to 1  ml at 30c by a nitrogen - purge apparatus after internal standard was added . 15  g of tea sample containing 100  l internal standard solution were placed in soxhlet 's apparatus and 50  ml of dichloromethane in an 150  ml distillation flask . at both ends of the sample in soxhlet 's apparatus , there are 2  cm - height of celite to help fix the sample .
extraction was carried out at 50c for 2  hours , and extraction was concentrated to 1  ml at 30c by a nitrogen - purge apparatus after internal standard was added .  
the concentrated extracts were chromtographed by an hp 6890 series gc system ( agilent , usa ) .
a 30  m  0.25  mm db-5 quartz capillary column ( supelco , bellefonte , pa , usa ) with 0.25  m film thickness was used to resolve the volatiles .
temperature programming was as follows : initial oven temperature was set at 60c and kept for 3  min , then raised to 200c at a ramp of 4c / min and kept for 2  min ; to 210c at of 1c / min and kept for 2  min ; and finally , it was raised to 270c and kept for 7  min .
nitrogen was used as carrier gas with column head pressure at 12.26  kpa in constant pressure mode .
programming split / splitless injection temperature was set at 260c with split ratio of 10 : 1 and fid detector at 280c .
auto system shimaszu qp 2010 gc - ms was employed for qualitative analysis to confirm the target components .
the oven temperature was set at 50c and kept for 2  min , then raised to 60c at a ramp of 1c / min and kept for 2  min , to 200c at of 4c / min and kept for 2  min , and finally , to 270c at 10c / min and kept for 5  min .
the mass spectrometry was operated at 200c in the electron impact mode ( 70  ev ) , scanning from m / z 40 to 600 in 0.3  s with an 0.2  s interval time of the scan ; the temperature of the gc - ms interface was 280c ; the voltage of the photoelectric multiplier tube ( pmt ) was 200  v. the mass spectral identifications of the target components were carried out by comparing to the nist 107 ( national institute of standards and technology , gaithersburg , usa ) mass spectral library as well as to wiley 6.0 ( wiley , new york , ny , usa ) mass spectral library .
for gc separation of volatile components in tea samples , bp-20 sge column ( polar column , 30  m  0.25 mm i.d .
film thickness 0.25  m ) was commonly used for quantitative analysis . in this work ,
film thickness 0.25  m ) was employed to separate the target components with temperature programming described above , and the chromatograms of standard compounds by gc - fid and gc - ms was shown in figures 1(a ) and 2(a ) . from the chromatograms , it can be seen each peak of the target compounds was baseline separated , and separating degree between two vicinity peaks was beyond 2 , confirming weak - polar capillary column can be used to separate the target components if gc separation condition was well optimized . according to gc - fid chromatogram , we calculated the relative factor of each of the standard compounds , it was shown in table 1 , and the relative factor was used to determination of the corresponding components in real pu - erh teas .
figures 1(b ) and 2(b ) show the chromatograms of pu - erh raw tea that was obtained from gc - fid and gc - ms determination , in which the target peaks of real sample could be easily discerned and accurately quantified .
the mass spectra of each target peaks in figure 2(b ) was checked by nist or wiley mass spectral library respectively , and campared with those in figure 2(a ) , confirming that each mass spectra of the components was the same as those of the corresponding standard compound , and no interference was appeared in the target peaks . comparing figures 1(b ) and 2(b ) , we found that both gc - fid and gc - ms separation methods can be applied to quantitative analysis of volatile flavor components in pu - erh teas .
nevertheless , quantitative determination of the target components using gc - fid was more cost - saving than doing gc - ms , so the rest determinations for all of the samples were carried out in gc - fid . during sde operation , dichloromethane and diethylether
are the most suitable extraction solvents for extraction of volatile components because of their weakly polar characteristics [ 15 , 17 ] . in this experimental
, we select dichloromethane as extraction solvent for sde , due to its property of low combustion , and higher boiling point ( 40c ) than diethylether ( 35c ) thus liable to storage .
zhu et al . had applied sde to extract volatile constitutes in green tea , confirming of 2  h was adequate to extract all the target compounds .
initially , we operated sde apparatus as zhu et al . done , and found that there are about 6% of target components dwelling on the tea residue .
this is because pu - erh tea was condensed to a pie - like shape , and the components were more difficult to release from tea matrix although the teas had been ground to 3060 mesh .
then we modified this method by adding 10  g of sodium sulphate to the 1  l of steam flask containing 300  ml of water , and then operating sde device .
after 2  h of sde and a further 15  min of the solvent extraction , the tea residue was re - extracted by sde , we found the area of the target peaks were less than 2% of the total . obviously , boiling point of water in steam flask
was enhanced by adding sodium sulphate , accelerating the releasing velocity of target compounds . to confirm the repeatability , parallel
the relative standard deviations ( rsd ) were in range of 1.44%12.6% , which were shown in table 3 .
to check accuracy of sde for volatile flavor components , a known amount of standard solutions were added to aliquot of pu - erh tea . the adding level and corresponding recoveries were listed in table 3 .
for quantitative extraction of volatile flavor components from complex matrices , common technique used is sde . because both liquid / liquid extraction and soxhlet extraction are classical techniques [ 8 , 10 ] , in this test , the modified classical techniques were compared with sde in extraction of the volatile flavor components from pu - erh tea in order to select the best one for sample preparation .
the results were shown in table 2 . generally , sde was the best one among them , because the amounts of the components extracted were greater than those by employing sd - llc or soxhlet for the high - volatile components such as benzyl alcohol , linalool oxide , and linalool .
this may attributes to the fact that sde was a closed and continuous extraction system , in which the target components can be 
furthermore , the temperature of the circulating water in sde was set at 8c , reducing the lose of the high - volatile components . as for extraction of the low - volatile components in pu - erh tea , such as 1,2,4-trimethoxylbenzene
, sde is less poor compared with soxhlet extraction , probably due to the characteristics of high - boiling point and incompatibility with water steam .  
although sd - lle shares a same extraction principle with sde , the limitation of the technique is clear in table 2 .
this is due to its open steam distillation system and following step - liquid - liquid extraction . during this procedure ,
not only high - volatile components such as benzyl alcohol , linalool oxide , and linalool and so forth had a chance to escape from the distillation system , but the analytes hardly transferred to organic phase .
we could not confirm whether isomerization reaction was happened between the two compounds during extraction , and further investigation was beyond the work .
table 2 shows that it can almost  exhaustively  extract all the 1,2,3- and 1,2,4-trimethoxylbenzene from pu - erh tea compared with sde , nevertheless it is very poor for benzyl alcohol , linalool oxide , linalool , phenethyl alcohol , and geraniol .
these findings indicate soxhlet was suitable for high - boiling point ( low - volatile ) compounds such as essential oil . as for the trace high - volatile component ,
, we analyzed several tea samples , including four pu - erh ripe tea , four pu - erh raw tea , one green tea ( huangshan , anhui ) and one black tea ( qimen , anhui ) , and the contents of the target volatile components were shown in table 4 .
it can be seen that the content of high volatile components , such as benzyl alcohol , linalool oxide , linalool in raw teas are higher than those in ripe teas , these findings are mainly due to the high temperature ( 4555c ) during the pile - fermentation process of ripe pu - erh teas , leading to the lose of these components .
however , in raw teas , the content of 1,2,3- and 1,2,4-trimethoxylbenzene are much lower than those in ripe teas , this is because microbes play an important role in the synthesis of the two compounds during the pile - fermentation process of ripe pu - erh teas , which promote reaction procedure of methylation . generally , the contents of the volatile components in the green and the black teas are higher than those in pu - erh teas , except for 1,2,3- and 1,2,4-trimethoxylbenzene , which are not found in both of teas , probably due to lack of post - pile - fermentation process in manufacture , and linalool , lower than those in pu - erh raw teas due to different cultivar , variety of species and processing procedure .
sde combined gc method was constructed for determination of volatile flavor components in pu - erh tea samples .
10 of volatile flavor components were quantitatively determined , and the recoveries and rsds were in the range of 66.4%109% and 1.44%12.6% , respectively .
the method was compared with sd - lle and soxhlet extraction , comfirming sde was suitable for pu - erh teas among them .
pu - erh raw tea and ripe tea samples were analyzed by the method , indicating the high - volatile components , such as benzyl alcohol , linalool oxide , and linalool , were rich in pu - erh raw teas , while the contents of 1,2,3- and 1,2,4-trimethoxylbenzene were much high in pu - erh ripe teas .
 a simutaneous distillation extraction ( sde ) combined gc method was constructed for determination of volatile flavor components in pu - erh tea samples . 
 dichloromethane and ethyl decylate was employed as organic phase in sde and internal standard in determination , respectively . 
 weakly polar db-5 column was used to separate the volatile flavor components in gc , 10 of the components were quantitatively analyzed , and further confirmed by gc - ms . the recovery covered from 66.4%109% , and repeatability expressed as rsd was in range of 1.44%12.6% . 
 sde was most suitable for the extraction of the anlytes by comparing with steam distillation - liquid / liquid extraction and soxhlet extraction . 
 commercially available pu - erh tea samples , including pu - erh raw tea and ripe tea , were analyzed by the constructed method . 
 the high - volatile components , such as benzyl alcohol , linalool oxide , and linalool , were greatly rich in pu - erh raw teas , while the contents of 1,2,3-trimethoxylbenzene and 1,2,4-trimethoxylbenzene were much high in pu - erh ripe teas . 
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusion