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10.1515/umcschem-2015-
ANNALES
UNIVERSITATIS MARIAE CURIE-SKŁODOWSKA
LUBLIN – POLONIA
VOL. LXX, 2 SECTIO AA 2015
Application of SPE for selective fractionation of essential
oils constituents from plant materials
Joanna Szewczyk*, Michał .P. Dybowski and Andrzej L. Dawidowicz
Maria Curie-Skłodowska University, Faculty of Chemistry,
Department of Chromatographic Methods, Maria Curie-Skłodowska Sq.3,
20-031 Lublin, Poland
Solid-phase extraction (SPE) is simple and inexpensive sample
preparation procedure which can be applied for the isolation/
fractionation of essential oil compounds from wide variety of
samples, such as foodstuffs, biological and environmental. Due to
the complex nature of the examined matrices and frequently low
concentration level of target components, analytical procedures
require the use of initial sample preparation stage. The paper shows
the possibility of essential oil components fractionation from
different plant materials using SPE method.
The results presented in this paper shows that the proposed
SPE procedure allows for easy and total fractionation of essential
oil constituents (especially low-molecular oxygen compounds)
from the sample matrix.
Keywords: SPE, essential oils from herbs, fractionation
1. INTRODUCTION
Essential oils (EO) isolated from aromatic herbs and plants are multi-
component mixtures of monoterpenes and sesquiterpenes, and their
oxygenated derivatives such as aldehydes, alcohols and esters [1-4].
As EO are commonly used by food, pharmaceutical, cosmetic and
fragrance industries, hence, it is important to evaluate the aroma-active
88 Joanna Szewczyk et al.
essential oil components, generally existing in different concentration range from high level of concentration to traces. Nevertheless, presence of traces has also significant impact as they are responsible for the specific natural flavor and odor. Therefore, it is important to applied the sample preparation stage used in order to recover EO from plant sample, enabling maintain the natural percentage of its primary compounds [5-7]. Many approaches of EO components fractionation have been reported in literature. Traditional methods applied in order to isolate of EO constituents from plant material are hydrodistillation, steam- distillation and liquid-liquid extraction [8-10]. Steam distillation is pharmacopoieal method recommended to separate EO from plant matrixes [11]. However because of time consumption and low extraction efficiency in analysis of organic components from numerous plant samples, in recent years it is frequently replaced by more effective technological approaches [12–13], such as pressurized liquid extraction (PLE) [14], supercritical fluid extraction (SFE) [15] and matrix-solid phase dispersion (MSPD) [16]. Among assisted extraction techniques, solid phase extraction (SPE) [17] is actually the most commonly studied application. Due to allows for selective and rapid preparation step, is applied to a wide variety of samples such as foodstuffs, biological and environmental [18–21]. SPE complies with the requirements of green chemistry by reduction or total elimination of solvent consumption during in analytical procedures. Its versatility allows the application of SPE for many purposes, i.e. purification, isolation, pre-concentration and class fractionation [18, 22]. This method is a good approach for the extraction/concentration of components from aromatic plants, as many solid phases are available enabling increase of selectivity of the process. In addition, enrichment of the aromatic components from extract can be accomplished by using a small amount of organic solvent during the elution step from the solid phase [23–24]. This paper reports the possibility of essential oil components fractionation using SPE method. The article shows results for following plant materials: peppermint, juniper, thyme, lemon and grapefruit_._
90 Joanna Szewczyk et al.
essential oil compound, provide determination of the accuracy of the proposed analytical SPE procedure.
2.4. Chromatographic analysis
Qualitative analyses of components in the prepared samples of essential oils were carried out using GC/MS QP2010 (Schimadzu, Kyoto. Japan) equipped with 30 m × 0.25 mm id. 0.25 μm film thickness ZB5-MS fused silica capillary column (Phenomenex). The column was operated with helium as carrier gas (1cm^3 /min). The temperature of injection was 310°C and the volume of injected sample was 1 μL. During injection the split mode was applied (purge time- 0.7 min) The following temperature program was administered: 1 min at 50°C and it was then linearly raised at the rate of 6°C/min to 310°C. EI mode at 70 eV was used in the mass spectrometer and 220°C was the temperature of ion source. The mass range was from 35 to 360 amu. Qualitative analysis was performed by comparing the retention indexes and MS spectra for the obtained peaks with the analogous data from mass spectrometry library (NIST’05). Quantitative analyses were carried out using gas chromatograph with a flame ionization detector (GC/FID, Schimadzu 2010). Hydrogen was used as carrier gas (1mL min-1). Experimental conditions were the same as for GC/MS. Peaks identification was performed basing on the experimentally determined indexes of retention.
- RESULTS AND DISCUSSION
Table 1 shows the percent amounts of mint essential oil components retained in the SPE column after its washing out with methanol-water mixtures of different composition. The values were obtained by relating the peak area of essential oils components eluted from the SPE column (fraction II) to the peak area in an appropriate reference solution. It should be remembered that essential oil reference solution contained 50 mL of the oil in 5mL hexanic solution and that the SPE column was loaded with 50 mL of the oil and then, in the second elution step, was washed out with n-hexane to reach 5 mL of hexanoic eluate. The application of C-18 sorbent and 70% methanol causes total or almost total elution of certain components from the sorbent (cis-4-thujanol, p-menthon, isomentol, menthol, piperitone) with
Ion-imprinted polymers: synthesis, characterization and applications… 91
simultaneous retention of other essentials oil components. The average recovery of components eluted from the SPE column by 70% methanol equals almost 100%, whereas an average content of compounds retained in SPE column after its elution with 70% methanol exceeds 85% (without taking into account the above ingredients, for which the recovery was below 5%) – see the last line of Table 1, which is a satisfactory result of preliminary separation for analytical and preparative purposes.
Table 1. The percentage of mint essentials oil components retained by SPE cartridge after its washing out using methanol/water mixture (i.e. composition of fraction II); n = 5, RSD < 3.5%.
No. Compound RI*
Methanol concentration (%) 30 40 50 65 70 75 80
- 3-Thujone (^) 962 (^100 100 100 100) 100 94.13 90.
- Pinene 977 100 100 100 100 100 97.48 91.
- β-Phellandrene 1046 100 100 100 100 100 97.21 92.
- cis-Sabinene hydrate 1068 100 100 100 100 100 97.29 91.
- 3-Octanol (^) 1088 (^100 100 100 100) 100 98.64 93.
- 3.5-Dimethyloctane 1094 100 100 100 100 100 91.64 80.
- α-Terpinene 1141 100 100 100 100 100 97.45 91.
- o-Cymene 1161 100 100 100 100 100 94.48 91.
- D-Limonene (^) 1173 (^100 100 100 100) 100 97.65 91.
- Eucaliptol 1184 100 96.14 91.42 88.49 20.82 9.53 4.
- γ-Terpinene 1251 100 100 100 100 100 97.78 92.
- cis-4-Thujanol 1291 100 92.65 75.14 0.81 T T T
- Terpinolene (^) 1338 (^100 100 100 100) 100 92.79 86.
- Linalool 1384 100 95.32 75.19 12.18 10.48 9.50 9.
- 2-(methylbuthyl)-iso-valerat 1414 100 100 100 100 93.83 87.93 39.
- p-Menthanon 1634 100 100 100 100 30.81 12.58 1.
- Menthofurane (^) 1662 (^100 100 100 100) 100 95.58 91.
- p-Mentone 1671 100 91.97 76.50 23.71 6.03 2.5 2.
- Isomenthol 1689 100 98.00 83.68 30.01 7.27 4.8 0.
- Menthol 1730 100 95.20 81.94 4.24 0.99 0.84 T
- γ-Terpineol (^) 1782 100 75.32 66.92 35.98 (^) 33.59 31.63 27.
- Pulegone 1911 100 96.98 82.74 21.37 21.03 14.74 6.
- Piperitone 1955 100 91.67 77.57 0.79 0.77 0.62 0.
- Menthyl acetate 1995 100 100 100 100 92.83 91.16 75.
- o-Menth-8-en (^) 2136 (^100 100 100 100) 100 100 100
Ion-imprinted polymers: synthesis, characterization and applications… 93
As results from the comparison, peaks relating to: cis-4-thujanol, p-menthon, isomentol, menthol, piperitone in the Table 1 were decreased on the chromatogram B, indicating removal these compounds during fractionation process. Data from Table 1 and Figure 1 show that the application of column packed with octadecyl silica sorbent can be used for the isolation of mint essential oil components. To check validity of this statement, it was decided to carry out the same experiments using by SPE C-18 procedure for various plant materials. In the case of juniper essential oil (Table 2) washing out the SPE column with 75% methanol causes complete removal of some components: borneol, terpinen-4-ol, α-terpineol (low-molecular oxygen compounds). The average recovery of the remaining components of juniper essential oil exceeds 94%.
Table 2. The percentage of juniper essential oil components retained by SPE cartridge after its washing out using methanol/water mixture (i.e. composition of fraction II); n = 5, RSD < 3.5%.
No. Compound RI Methanol concentration (%) 10 15 25 50 65 75 100*
- α-Pinene 935 100 100 100 100 100 100 1.
- Camphene 952 100 100 100 100 100 100 1.
- Sabinene 972 100 100 100 100 100 100 1.
- β-Pinene 980 100 100 100 100 100 100 2.
- β-Myrcene 990 100 100 100 100 100 100 1.
- Pseudolimonene 1003 100 100 100 100 100 100 2.
- m-Cymene 1021 100 100 100 100 100 100 2.
- Limonene 1031 100 99.90 95.11 91.64 90.41 88.65 1.
- Eucaliptol 1035 100 100 100 100 100 100 2.
- γ-Terpinene 1060 100 100 100 100 100 29.96 0.
- Terpinolen 1089 100 100 100 100 100 100 2.
- Camphor 1152 100 100 100 100 100 100 2.
- Borneol 1176 100 100 100 93.66 T* T T
- Terpinen-4-ol 1178 100 100 100 94.61 6.78 T T
- α-Terpineol 1189 100 99.35 93.27 91.49 1.80 T T
- Bornyl acetate 1287 100 100 100 100 100 95.69 0.
- α-Cubebene 1345 100 100 100 100 100 89.61 3.
- α-Copaene 1377 100 100 100 100 100 100 8.
- β-Elemene 1393 100 100 100 100 100 94.97 3.
94 Joanna Szewczyk et al.
cont. Table 2.
No. Compound RI*
Methanol concentration (%) 10 15 25 50 65 75 100
- (E)-Caryophyllene 1424 100 100 100 100 100 100 2.
- α-Caryophyllene 1453 100 100 100 100 100 100 2.
- Cadina-1(10),4-dien 1519 100 100 100 100 100 100 10. Average recovery (%) 100 99.97 99.47 98.70 90.43 94.68 2.
- – Kovats Retention Index; T – less than 0.05%.
The total elution of six components from thyme essential oil: linalool, camphor, borneol, α-terpineol, terpinen-4-ol, thymol was achieved using 75% methanol. Average recovery of remaining EO components after SPE procedure of thyme herb essentail oil was more exceed 86% (see Table 3).
Table 3. The percentage of thyme essential oil components retained by SPE cartridge after its washing out using methanol/water mixture (i.e. composition of fraction II); n = 5, RSD < 3.5%.
No. Compound RI Methanol concentration (%) 15 25 50 65 75 100*
- Tricyclene 927 100 100 100 100 94.73 5.
- α-Pinene (^) 935 100 100 100 100 95.98 5.
- Camphene 952 100 100 100 100 94.79 4.
- β-Pinene (^) 980 100 100 100 100 90.44 4.
- β-Myrcene 990 100 100 100 100 92.60 4.
- Pseudolimonen (^) 1003 100 100 100 100 100.0 99.
- α-Phellandren 1006 100 100 100 100 93.77 78.
- α-Terpinene 1012 100 100 100 100 93.59 41.
- p-Cymene 1023 100 100 100 100 89.05 T
- Eucaliptol 1035 100 100 100 100 96.91 0.
- γ-Terpinene 1060 100 100 100 100 94.15 T
- Terpinolene 1088 98.91 96.31 95.46 90.88 81.23 16.
- Linalool (^) 1090 98.89 97.77 97.37 26.50 T* T
- Camphor 1139 100 96.98 95.33 64.42 T T
- Borneol (^) 1176 100 98.56 97.20 19.72 T T
- Terpinen-4-ol 1178 97.95 98.28 95.52 53.58 T T
96 Joanna Szewczyk et al.
cont. Table 4.
No. Compound RI*
Methanol concentration (%) 10 15 25 50 65 75 100
- trans-Limonene oxide 1147 100 100 100 100 100 48.48 T
- α-Terpineol 1189 100 100 100 81.39 23.87 2.85 T
- cis-Carveol 1230 100 100 100 100 20.08 11.21 T
- Neral 1247 100 100 100 75.31 44.72 3.19 T
- Linalool acetate 1256 100 100 100 100 88.83 82.41 T
- Geranial 1276 100 100 100 100 78.15 4.03 T
- Geraniol acetate 1382 100 100 100 100 100 100 1.
- (E)-Caryophyllene 1424 100 100 100 100 100 100 5.
- α-Caryophyllene 1453 100 100 100 100 100 100 7.
- Caryophyllene oxide 1589 100 100 100 100 100 100 3.
Average recovery (%) 100 100 100 97.57 86.58 78.03 4.
- – Kovats Retention Index; T – less than 0.05%.
In the case of grapefruit essential oil washing out the SPE column with 75% methanol causes total elution of α -terpineol, ostaol and almost total elution of 1-octanol and linalool. Average recovery of other components is more than 87% (see Table 5).
Table 5. The percentage of grapefruit essential oil components retained by SPE cartridge after its washing out using methanol/water mixture (i.e. composition of fraction II); n = 5, RSD < 3.5%.
No. Compound
Methanol concentration (%) RI 10 25 50 65 75 100*
- α-Pinene 935 100 100 100 100 100 2.
- Sabinene 972 100 100 100 100 100 2.
- β-Myrcene 990 100 100 100 100 100 1.
- Octanal 1001 100 100 98.58 61.29 55.09 2.
- Limonene 1031 100 100 100 100 100 2.
- trans-Ocimene 1038 100 100 100 100 100 T
- 1-Octanol 1078 100 98.50 94.04 3.70 1.58 T
- Linalool 1090 100 100 96.74 8.36 3.90 6.
- Nonanal 1102 100 100 98.32 87.61 66.08 2.
- Limonene oxide 1134 100 100 88.65 91.29 60.50 7.
- (R)-(+)-Citonellal 1159 100 100 96.82 69.74 49.49 T
Ion-imprinted polymers: synthesis, characterization and applications… 97
cont. Table 5.
- – Kovats Retention Index; T – less than 0.05%.
The comparison of the chromatographic data for juniper, thyme, lemon, and grapefruit EO leads to the analogous conclusion as in case of SPE application to fractionation of mint essential oil components, i.e. oxygen compounds of low molecular mass occurring in all the used oils can be eluted from the SPE column using 75% methanol. The average recovery of these compounds in methanolic fraction is almost 100%, whereas the average recovery of the remaining essential oil compounds (fraction II) is above 94% for juniper, above 86% for thyme, above 89% for lemon and above 87% for grapefruit – see Table 2-5. Results proved that using of methanolic solution in the initial step of SPE causes the selective fractionation of essential oil components. Hence, it was decided to check what situation will be observed, when we replace character of solvent by e.g. dioxane or acetonitrile. The percentages of essentials oil components from mint retained in the SPE column after its washing out using ACN/water and 1,4-dioxane/water mixtures of different composition are presented in Table 6. As results from the data, the concentration increase of the organic modifier in the SPE eluents causes a decrease of the amount of almost all mint essential oil components deposited on the SPE column. After the total removal of some components in the first fraction, the average recovery of the rest in second fraction is lower than 60%. Only one component – α-terpinene – still remain in the SPE column after washing it
No. Compound
Methanol concentration (%) RI 10 25 50 65 75 100*
- α-Terpineol 1189 100 100 100 5.47 T* T
- Decanal 1209 100 100 100 100 84.28 T
- α–Cubebene 1345 100 100 100 100 100 3.
- α-Copaene 1378 100 100 100 100 100 9.
- (E)-Caryophyllene 1424 100 100 100 100 100 T
- α-Caryophyllene 1453 100 100 100 100 100 T
- Germacrene D 1487 100 100 100 100 100 T
- Cadina-1(10),4-dien 1519 100 100 100 100 100 4.
- Elemol 1546 100 100 100 100 91.68 6.
- Nootkaton 1814 100 100 100 100 75.05 T
- Ostaol 2144 100 100 98.69 81.20 T T Average recovery (%) (^100) 99.93 98.72 82.21 79.38 4.
Ion-imprinted polymers: synthesis, characterization and applications… 99
cont. Table 6.
No. Compound
Concentration (%) of ACN 1,4-Dioxane 50 60 70 50 60 70
- Muurolene 100 100 99.32 97.9 93.41 89.
- α-Cubenene 98.67 95.32 84.1 90.17 85.72 83.
- Eriksene 98.96 92.94 83.33 82.53 78.03 76.
- δ-Cadinene 100 97.71 87.07 100 91.06 81.
- Wiridofloren 93.76 80.69 42.89 88.02 62.81 8. Average recovery (%) 74.94 77.37 59.04 84.52 62.56 52.
- – Kovats Retention Index; T – less than 0.05%.
Results from Table 6 indicate that applied 70% acetonitrile allows for the total elution of following EO components from SPE sorbent: cis-4- thujanol, γ-terpineol, piperitone and almost total elution of pulgeone, menthol, isomenthol, p-menthol, and p-menthanon. Average recovery of remaining EO components was 70.57%. In case of 70% dioxane application, the total elution of EO constituents was only in the case of γ-terpineol. For components (numbered 12, 18-20, 22 and 23) the elution was more than 95%. Recovery of other components was only 64.64 % (without taking into account the above constituents, for which the recovery was below 5%).
- CONCLUSIONS
The presented SPE method allows to carry out fractionation/isolation of essential oil components from plant materials. It is especially effective for isolation of low-molecular oxygen compounds, for which is easy and almost complete from the remaining compounds. Total removal of some components from essential oil can be achieved by application of methanol/water mixture as the eluent. The SPE method for fractionation of essential oils may be adapted to isolation of valuable components (e.g. menthol from mint essential oil) for foodstuffs, cosmetics or pharmaceutical applications.
100 Joanna Szewczyk et al.
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Andrzej L. Dawidowicz. Born in Lublin (Poland) in 1950. Graduated from Maria Curie-Skłodowska University in Lublin (1973). Received his Ph.D. and D.Sc. degree (1978 and 1986, respectively) in physical and analytical chemistry from University of Maria Curie-Skłodowska in Lublin. Since 1991 titular professor. Since 2000 Head of Chroma- tographic Methods Department. Since 1973 member of Polish Chemical Society. Member of numerous University teaching and research committees. Since 1996 Head of Doctoral Studies at the Faculty of Chemistry. His main field of interest is clinical, pharmaceutical, food, forensic and environmental analysis, development of sample preparation methods for chromatographic analysis, and studies of porous materials. He is an author over 200 papers.
