1
SUPPLEMENTARY MATERIAL
2
Fast determination of Ziziphora tenuior L essential oil by inorganic–organic hybrid
3
material based on ZnO nanoparticles anchored to a composite made from
4
polythiophene and hexagonally ordered silica
5
6
Marzieh piryaeia,b, Mir Mehdi Abolghasemic , Hossein Nazemiyeha,b*
7
a,
8
Sciences, Tabriz, Iran
9
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical
b
Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
10
c
11

12
Hossein Nazemiyeh
13
Tel: +98 41 3336 7014; Fax: +98 41 3336 7929
14
E-mail address: hosseinNazemiyeh@yahoo.com
15
Abstract
16
In this paper, for the first time, an inorganic–organic hybrid material based on ZnO
17
nanoparticles anchored to a composite made from polythiophene and hexagonally
18
ordered silica (ZnO/PT/SBA-15) for use in solid-phase fiber microextraction (SPME) of
19
medicinal plants. A homemade SPME apparatus was used for the extraction of volatile
20
components of Ziziphora tenuior L. A simplex method was used for optimization of five
21
different parameters affecting the efficiency of the extraction. The main constituents
22
extracted by ZnO/PT/SBA-15 and PDMS fibers and hydrodistillation (HD) methods
23
respectively, included Pulegone (51.25%, 53.64% and 56.68%), Limonene (6.73%,
24
6.58% and 8.3%), Caryophyllene oxide (5.33%, 4.31% and 4.53%) and 1, 8-cineole
25
(4.21%, 3.31% and 3.18%). In comparison to the HD method, the proposed technique
26
could equally monitor almost all the components of the sample, in an easier way, shorter
27
time and requiring a much lower amount of the sample.
Department of Chemistry, Faculty of Science, University of Maragheh, Maragheh, Iran
Corresponding author:
28
Keywords: ZnO nanoparticles anchored to a composite made from polythiophene and
29
hexagonally ordered silica (ZnO/PT/SBA-15) solid-phase fiber microextraction,
30
Ziziphora tenuior L
31
Experimental
32
Plant materials
33
The aerial parts of Ziziphora tenuir were gathered during the flowering period in summer
34
2013 from pave in the west of Iran. The aerial parts were dried in the shade (at room
35
temperature). A voucher specimen was deposited at the chemistry herbarium of this
36
laboratory under the code 1812 ZT.
37
Chemicals and reagents
38
Poly (ethylene glycol)-block-poly (propylene glycol)-blockpoly (ethylene glycol)
39
(EO20–PO70–EO20 or Pluronic P123) as surfactant were purchased from Sigma (Buchs,
40
Switzerland, www.sigmaaldrich.com). Zn(Ac)2, Li(OH)· H2O, Methylene chloride,
41
thiophene monomers, and all chemical solvents were obtained from the Fluka (Buchs,
42
Switzerland, www. sigmaaldrich.com) or Merck (Darmstadt, Germany, www. merck.de)
43
companies. The stock solution 1 mg mL−1 of PAHs was prepared in mixed methanol
44
solvents. The working solutions of above compounds were prepared by diluting the stock
45
solution with methanol and more diluted working solutions were prepared daily by
46
diluting these solutions with deionized water. All solvents used in this study were of
47
analytical reagent grade. Blank analyses were performed regularly to ensure that no
48
PAHs were present in laboratory reagents, atmosphere, or fibers.
49
Essential oils isolation
50
One hundred grams of air-dried aerial parts (leaves) of Ziziphora tenuir were ground to a
51
fine powder, and then put into a 1000 ml distillation flask. Five hundred milliliters of
52
distilled water was added and subjected to hydrodistillation for 4 h, using a Clevenger-
53
type apparatus as recommended by British Pharmacopeia. Oil was collected from the
54
condenser, dried over anhydrous sodium sulfate, and the yield of the sample was about
55
0.21% based on dry weight of the sample. The obtained essential oil was stored at 4 ˚C
56
until analysis by GC–MS.
57
GC–MS analysis
58
A Hewlett-Packard Agilent 7890A series GC equipped with a split/splitless injector and
59
an Agilent 5975C mass-selective (Agilent Technologies, Palo Alto, CA, USA,
60
http://www. agilent.com/chem) detector system were used for determination. The MS
61
was operated in the EI mode (70 eV). Helium (99.999 %) was employed as a carrier gas,
62
and its flow-rate was adjusted to 1 mL min−1. The separation of PAHs was performed on
63
a 30 m×0.25 mm HP-5 MS column (Agilent Technologies, Palo Alto, CA, USA) with
64
0.25 μm film thickness. The column was held at 50 °C and increased to 180 °C at a rate
65
of 15 °C min−1 and then raised to 260 °C at 20 °C min−1 and kept at this temperature for
66
5 min. The injector temperature was set at 260 °C, and all injections were carried out on
67
the splitless mode for 2 min. The GC–MS interface, ion source and quadrupole
68
temperatures were set at 280, 230 and 150 °C, respectively. Compounds were identified
69
using the Wiley 7 N (Wiley, New York, NY, USA) Mass Spectral Library. A homemade
70
SPME device was used for holding and the injection of the fabricated fiber into the GC–
71
MS injection port. The commercial SPME device and PDMS fiber (100-μm film
72
thickness) were purchased from Supelco (Bellefonte, PA, USA). The fiber was
73
conditioned in the injection port of a GC for 1 h.
74
Preparation of ZnO/PT/SBA-15 nanocomposites
75
Preparation the ZnO/PT/SBA-15 nanocomposite was synthesized following the
76
procedures described elsewhere (Zhang, Chen, Ma, Chen, Yang, & Zhang, 2010). Highly
77
ordered mesoporous SBA-15 was synthesized using a procedure reported by Zhao and
78
co-workers (Zhao et al., 1998). SBA-15 was thermally treated at 120 °C in a vacuum
79
oven to remove the physically adsorbed water. Then, 0.50 g SBA- 15 was immersed in a
80
mixture of 10 ml methylene chloride and 5 ml thiophene monomers. The mixture was
81
sonicated at ambient temperature for 1 h. When the methylene chloride and unadsorbed
82
thiophene had been slowly evaporated at 30 °C for 24 h under a vacuum oven, the
83
mixture was added to a solution of H2O/ethanol (volume ratio: 5/1), which contained 5
84
ml 30 % hydrogen peroxide aqueous solution and 4 mg FeCl3. The polymerization
85
proceeded under nitrogen atmosphere and pH=2 for over 12 h at 50 °C. The remained
86
product was directly precipitated into vigorously stirred methanol (six volumes), then
87
filtered off and washed with methanol several times, and eventually dried under vacuum
88
at 50 °C for 12 h to remove the physically adsorbed water molecules (Lee, Lee, Cheong,
89
Lee, & Kim, 2007). The PT/SBA-15 nanocomposite was directly immersed in
90
Zn(Ac)2/ethanol solution through sonication in order to adsorb Zn2+ adequately, and then
91
Li(OH) aqueous solution was added to form ZnO nanoparticles and the ZnO/PT/SBA-15
92
composite was successfully prepared (Vietmeyer , Seger, & Kamat, 2007) . Preparation
93
of the SPME fiber a piece of stainless steel wire with a 200-μm diameter was twice
94
cleaned with methanol in an ultrasonic bath for 20 min and dried at 70 °C. One
95
centimeter of the wire was limed with epoxy glue and the ZnO/PT/SBA-15
96
nanocomposite was immobilized onto the wire. The coated wire was heated to 50 °C for
97
48 h in an oven, gently scrubbed to remove non-bonded particles and assembled to the
98
SPME holder device. Finally, prepared SPME fiber was inserted into the GC injection
99
port to be cleaned and conditioned at 260 °C for 1 h in a helium environment. The
100
thickness of the uniform coating layer was calculated from the difference between the
101
coated and uncoated stainless steel wire and come out to be about 20 μm. The headspace
102
solid phase microextraction (HS-SPME) procedure SPME was performed with the
103
prepared nanocomposite fiber, mounted in its SPME device. The extraction temperature
104
was controlled using a thermostated oil bath (Abolghasemi et al., 2014).
105
106
107
108
109
110
111
112
113
114
115
Figure S1. The response (sum area of four main peaks of Ziziphora tenuir samples) for
116
the designed experiments mentioned in Table 1.
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
Figure S2. Effect of water addition on the extraction efficiency of the solid-phase
135
microextraction method.
136
137