Effect of Polarity of Column on the Separation Performance of Essential Oil?

CAO Yan-hong,FENG Shun

(Key Laboratory of Oil&Gas Fine Chemicals,Ministry of Education&Xinjiang Uyghur Autonomous Region,College of Chemistry and Chemical Engineering,Xinjiang University,Urumqi Xinjiang 830046,China)

Abstract: In this study,the Effect of polarity of column on the separation performance of three essential oils was investigated systematically by GC-MS.Compositions of three commercial essential oils of Lavandula angustifolia,Mentha piperita L.and Rosmarinus of cinalis L.from Yili region of Xinjiang,China were analyzed on three capillary columns with different polarities.The theoretical plate number of the characteristic components,the number of total peaks and the amount of identified components were combined to evaluate the separation performance of columns.Results showed the best separation of the oil can just be reached by a column with certain polarity according to the polarity distribution of components by a single run.And more,to reach a comprehensive analysis of oils,it was suggested to perform analyses on two columns with different polarities.This work also provides valuable reference for the column selection in two-dimensional gas chromatography.

Key words:polarity,Column,separation performance,essential oil,gas chromatography-mass spectrometry

0 Introduction

Essential oils are largely widespread around the world and become increasingly popular in numerous applications,they have been extensively involved in our daily life.In order to develop full understandings of essentials oils,a comprehensive study of oil composition was strongly demanded.Now,GC-MS technique played as the most important and powerful tool for its excellent qualitative and quantitative capacity[1],and had been applied as a standard technique to analyze essential oils.In GC or GC-MS analysis,whether each component can be separated from others depends mainly on two factors:the chromatographic conditions and the type of column.Between them,the polarity of column played a key role.Conventionally used columns are mainly 100%polydimethylsiloxane phase,5%diphenyl/95%dimethyl polysiloxane phase and polyethylene glycol phase,or their selectively equivalents.But it seemed the choice of column just depended on the experience of operators,and till now no systematical study was reported on the selection of type of column.

Taking into account this problem,the Effect of polarity of column on the separation performance of three com-mercial essential oils from Yili region of Xinjiang,China,including Lavandula angustifolia oil(LA),Mentha piperita L.oil(MP)and Rosmarinus of?cinalis L.oil(RO),was investigated systematically based on GC-MS technique.We hope this research can provide some suggestion for the study and the quality control of the essential oils.

1 Experimental

1.1 GC-MS system

Analyses were performed by a Shimadzu GCMS-QP2010 Ultra system(Kyoto,Japan)consisting of a GC2010 gas chromatograph and a QP2010 Ultra quadrupole mass spectrometer.All samples were introduced into the GC inlet system by a Shimadzu AOC-20i autosampler.To all of three oils,carrier gas was helium(99.999%)at a flow rate of 1.0 mL/min with a split ratio of 1:50,and injection volume was 1.0μL.Commercial oil samples were provided by Product Quality Supervision and Inspection Research Institute of Xinjiang.Prior to be analyzed,samples were diluted with n-hexane(HPLC grade,Honeywell Burdick and Jackson company)to ca.1.0 mg/mL.

In this work,the most popularly used three types of columns were chosen including Column 1,Rxi-5Sil MS(low polarity,Restek,Bellefonte,PA,USA),Column 2,Rxi-17Sil-MS(medium polarity,Restek,Bellefonte,PA,USA)and Column 3,GsBP-Inowax-MS(high polarity,General Separation Technologies,Delaware,USA).For readily comparison,all of them were 30 m×0.25 mm i.d.with afilm thickness of 0.25μm.

1.2 Analytical conditions

To reach the best separation and determine the maximum identified components from oils on different columns,the GC and MS conditions were optimized and described below:

LA analysis.The injection temperature was 230?C.Initial temperature was set at 55?C and stood for 3 min.For Column 1,the temperature program was from 55?C to 80?C at 1?C/min,then increased,finally at 5?C/min to 190?C;For Column 2,from 55?C(held for 1 min)to 90?C at 5?C/min(held for 8 min),and then ran to 150?C at 8?C/min followed by an increase to 220?C at 12?C/min;and for Column 3,temperature programmed from 40?C(held for 2 min)to 95?C at 1?C/min,finally increased to 220?C at 8?C/min.The temperatures of the transfer line and the ion source were 230?C and 200?C,respectively.Ions were generated at electron impact(EI)energy of 70 eV.All data were collected in the full scan mode(m/z 40–450).

MP analysis.The injection temperature was 230?C.Initial temperature was set at 50?C and stood for 3 min.For Column 1,the temperature program was from 50?C to 70?C at 3?C/min,followed by an increase to 80?C at 2?C/min,then to 96?C at 5?C/min(held for 3 min),ramped to 110?C at 2?C/min,then to 150?C at 4?C/min,and ran to 180?C at 5?C/min,finally to 230?C at 20?C/min;for Column 2,oven temperature programmed from 50?C,increased at the rate of 5?C/min to 110?C(held for 2 min),at last to 215?C at 8?C/min;and for Column 3,from 40?C to 100?C(held for 2 min),then ran to 110?C at 1?C/min,finally ramped at 15?C/min to 180?C(held for 4 min).The temperatures of the transfer line and the ion source were 230?C and 200?C,respectively.Ions were generated at electron impact(EI)energy of 70 eV.All data were collected in the full scan mode(m/z 20–550).

RO analysis.The injection temperature was 210?C.For Column 1,initial temperature was 55?C,raised to 65?C at 1?C/min(held for 2 min),then ran to 90 at the rate of 3?C/min,finally increased to 190?C at 6?C/min;for Column 2,from 40?C to 100?C at 3?C/min,finally to 200?C at 6?C/min;and for Column 3,temperature initiated at 40?C(held for 2 min),ramped to 70?C at 4?C/min,to 105?C at 8?C/min,then increased to 120?C at 1?C/min,finally ran to 180?C at 8?C/min(held for 4 min).The temperatures of the transfer line and the ion source were 220?C and 200?C,respectively.Ions were generated at electron impact(EI)energy of 70 eV.All data were collected in the full scan mode(m/z 40–450).

The identity of the components was assigned by matching their spectral data with those detailed in the Wiley 229.L,Wiley 7.L and NIST 08.L libraries.The chromatographic and spectral data were acquired and processed using GC-MS solution software(http://www.shimadzu.com),calculated RI values were further used to validate the identified components by comparing those in literatures[2?10]

1.3 Data analysis

Baseline correction and automatic peak detection were performed with the slope of 1 600 min and full width at half-maximum of 1.6 for LA,1 500 min&1.5 for MP,and 1 400 min&1.4 for RO with Shimadzu software(GCMS Solution,Shimadzu,Japan).Then redundant peaks,such as noise,fragments and adductions,were eliminated,and all known artifact peaks were not considered in thefinal data.Each sample was represented by a GC-MS total ion chromatograms(TIC),and ion peak areas of compounds were integrated.Relative amount was expressed as mean peak area percent±SD based on the peak area normalization method.Every case was performed triplicates.To eliminate variations in quantity in different runs,Lavandulyl acetate,menthyl acetate and camphene were used as internal standards in LA,MP and RO analyses respectively

Fig 1 TIC of essential oils analyzed on their optimum columns under optimized conditions

2 Results and discussion

The oil samples were analyzed by GC-MS on three columns,respectively.Fig.1 showed the TIC under optimized conditions.Many peaks were observed in the chromatogram,and most of them were baseline separated,which indicated good separation was reached under optimized GC and MS conditions.

To compare the separation performance of columns,the column efficiency should be evaluatedfirst.In this study,the theoretical plate number was used and the result was listed in Table 1.1,8-cineole exists in three oils,and also be stated as the characteristic component of them.Here it was used as the standard component,and its theoretical plate number was used to evaluate the column efficiency.Theoretical plate numbers of 1,8-cineole represented in Column 1,Column 2 and Column 3 were 1.55×105,3.11×104and 7.79×104,respectively,which showed there was not big difference among column efficiencies of them,and can be used into further study under the same criterion.And theoretical plate numbers of 1,8-cineole in three oils were kept constant which meant the setup of GC and MS reached its optimal status under the optimized conditions.

A.LA on Column 1;B.MP on Column 3;C.RO on Column 3

Determination of all characteristic components could be used as the judgment whether the column was suitable for the analysis of a certain essential oil.So three columns were used to analyze the three essential oils atfirst,and the results were listed in Table 1.It can be seen that to certain oil,some of columns can determine all of characteristic components,and some not.The experimental result meant the polarity of column has some Effects on the oil separation.

The number of peaks and the amount of identified components were used to further explore the Effect of column polarity on the separation performance of column(Fig.2).It was showed that both of them varied a lot.To LA,totally 48 compounds were identified by three columns with 28 compounds co-identified,The best separation was obtained by Column 1,which is universally considered to be low polar column category,as many as 44 compounds with an average of 51 peaks were determined.All characteristic components specified in ISO standard[11]could be detected by Column 1,Camphor on Column 2 and ?-caryophyllene on Column 3 was not detected(Table 1).The LA(linalyl acetate-linalool chemotype)mainly consisted of linalyl acetate,linalool,lavandulyl acetate,cis-?-ocimene,trans-caryophyllene and 4-terpineol,belonging to non-and low-polar compounds,which meant that the separation column with non-or low-polarity would be suitable for the analysis of LA.In previous studies,the most widely used columns were also non-or low-polar[12?14],and our study also testified the view of point.The sequence of separation performance was Column 1>Column 3>Column 2.

To MP,Menthol-menthone chemotype,45 compounds were determined through three columns with 28 coidentified compounds.Column 3 with the highest polarity was the best one resulted in an average of 55 peaks and 40 identified compounds.Each column can identified all characteristic components stated in ISO standard[15]except iso-menthone was unidentified on Column 1(Table 1).The main components of MP are menthol,menthone,men-thyl acetate and 1,8-cineole.[9,16],which are relative polar compounds belonging to alcohol,ketone and their ester.It suggested the column with high polarity would be suitable for the analysis of MP.However,the previous studies were most used low-or medium polar columns,for example DB-5 and CP-Sil8CB column,but the amount of identified compounds was all less than that in this work.The result showed the column with high polarity should be chosen for MP analysis.The sequence of separation performance was Column 3>Column 1>Column 2.

Table 1 The characteristic components of LA,MP and RO and their theoretical plate number.(n=3)

To RO,even if the theoretical plate number of characteristic components was very high,but only 38 compounds were identified by combining results of three columns,and 24 was co-identified.Since no ISO standard for RO of the cineoliferum chemotype,the Industry Standard of Xinjiang[17]was used as the reference and also listed in Table 1.All characteristic components of RO can be identified by each column.Column 3,with the highest polarity,showed best results with an average of 49 peaks and 35 identified components.The results was a little con flicted with previous reports,in which the low-polarity columns were used[18,19].The main components of RO are 1,8-cineole,α-pinene,camphor,camphene and limonene,whose polarities are relatively high.The polarity distribution of them meant that the best separation would be reached on a relative high polar column.Just as that of MP,The sequence of separation performance was Column 3>Column 1>Column 2.

Fig 2 Comparison of analytic results of three essential oils through three columns

Column 2,with medium polarity,showed the worst separation ability to all the three oils because the polarity distribution of studied three oils was dominated by either non-,low-or high-polar components.More importantly,it can be clearly seen that amounts of identified components by combining qualitative results of the low and high polar column together were as many as those identified by all of three columns(Fig 2B),which suggested two columns with different polarity were needed for oil analysis to get a comprehensive understanding of‘sample.

3 Conclusions

Here,the Effect of polarity of column on the separation performance of three kinds of commercial essential oils,LA,MP and RO,was investigated systematically based on GC-MS technique.It was found the theoretical plate number of the characteristic components,the number of total peaks and the amount of identified components were varied significantly for certain oil on different columns.The result suggested that the separation column should be selected by the polarity distribution of components in oil,not just depending on the experience of operator,and the medium polar column generally cannot reach good separation for analysis of the essential oil.It should be pointed out that a comprehensive analysis of oils can be reached by combining two columns,one with low or non-polarity and the other with high polarity.The results also give some guideness for the establishment of column coupling system for two-dimensional gas chromatography.