Determination of Rhodamine B Pigment in Food Samples by Ionic Liquid Coated Magnetic Core/Shell Fe3O4@SiO2 Nanoparticles Coupled with Fluorescence Spectrophotometry

The Ionic liquids (IL) 1-octyl-3-methylimidazole hexafluorophosphate ([OMIM]PF6) was selected from three ILs along with (1-butyl-3-methylimidazole hexafluorophosphate ([BMIM]PF6), 1-hexyl-3-methyl-imidazole hexafluoro-phosphate ([HMIM]PF6), were coated by Fe3O4@SiO2 nanoparticles with core-shell structure to prepare magnetic solid phase extraction agent (Fe3O4@SiO2@IL) and establish a new method of magnetic solid phase extraction (MSPE) coupled with Fluorescence spectrophotometry for separation/analysis of Rhodamine B. The results showed that Rhodamine B was adsorbed rapidly byFe3O4@SiO2@[OMIM]PF6 and eluted by ethanol. Under the optimal conditions the linear range, detection limit (DL), correlation coefficient (R) and relative standard deviation (RSD) were found to be 0.40-140.00 μg L, 0.06μg L, 0.9993 and 0.45% (n=3, c=10.00 μg L -1 ), respectively. The pre-concentration factor of the proposed method was 25-fold. The Fe3O4@SiO2 NPs can be used repeatedly for 10 times. This method has been applied to the determination of Rhodamine B in food samples.


Introduction
Rhodamine B (RhB), a red pigment, is widely used as colorant in textiles, dyeing, paper and printing [1]. RhB is harmful if swallowed by human and animals, and causes irritation to the skin, eyes and respiratory tract " [2], [3], [4]", and has been considered and used as color additive in foodstuffs. Because of the low price and bright color of rhodamine B, some traders use it as a food additive illegally [5], harming the health of consumers [6]. Thus, due to this, it was considered worthwhile to make efforts to develop a simple method for the determination of RhB in different samples. Various techniques have been developed for the determination of RhB to date, including ultraviolet spectrophotometry [7], fluorescence spectrophotometry [8], capillary chromatography [9], high-performance liquid chromatography (HPLC) [10]. However, direct determination with spectrophotometry is difficult owing to low concentration of RhB in natural samples. So that, it is important to develop a new method for determination/separation procedure of RhB.
Magnetic solid phase extraction (MSPE) is a technique that uses a magnetic material as absorbent matrix in solidphase extraction technique [11]. In MSPE procedures, the magnetic adsorbents are exposed in the sample solution to adsorb the analyte and then collect the analyte by an external magnetic field, which greatly simplifies the SPE procedure and enhances extraction efficiency [12]. To  @graphene) as sorbents for magnetic solid-phase extraction nitrobenzene compounds in environmental water samples extraction [14]. Liu et al. used magnetic coremesoporous shell microspheres with C 18 -functionalized interior pore-walls (C 18 -Fe 3 O 4 @mSiO 2 ) as adsorbent measured the residue of cephalosporins in milk [15]. However, it has not been reported for determination of RhB using Fe 3 O 4 @SiO 2 @IL.
In the present work, we have prepared magnetic solid phase extraction agents, Fe 3 O 4 @SiO 2 @ILsthrough selfassembly. This extraction agent combines the advantages of the ILs and the magnetic nanoparticles (MNPs). Compared with the previously reported works " [16], [17], [18]", this adsorbent based MSPE providing a rapid, and efficient sample preparation process, which enables the treatment of large volume samples in a short period of time. A novel MSPE method coupled with Fluorescence spectrophotometry was therefore established for separation/analysis of RhB from food samples.

Equipment and Reagents
FTIR spectra were measured with a Bruker Tensor 27 spectrometer (Bruker Company, Germany). Samples were pressed into KBr pellets and recorded at the frequencies from 4000 to 400 cm -1 with resolution of 4 cm -1 . A Tecnai 12 TEM (Philips, Netherlands) was used to obtain micrographs of the MNPs. Chromatographic separation of analytes was achieved with Fluorescence spectrophotometry.

MSPE Procedure
The MSPE procedure was done as follows. First, Fe 3 O 4 @SiO 2 @[OMIM]PF 6 MNPs were added to a 40 mL water sample containing RhB (pH=3.0), and the mixture was placed on a slow-moving platform shaker and allowed to equilibrate for 10 min at 30°C. Then, a strong magnet was applied to the bottom of the beaker, isolating the Fe 3 O 4 @SiO 2 @[OMIM]PF 6 MNPs from the suspension. After 5 min, the suspension was decanted and the residual solution of Fe 3 O 4 @SiO 2 @[OMIM]PF 6 MNPs was transferred to centrifuge tube. The Fe 3 O 4 @SiO 2 @[OMIM]PF 6 MNPs were aggregated again by positioning a magnet to the outside of the tube wall so that the residual solution could be completely removed by pipette. Finally, the isolated MNPs were mixed with 3 mL of ethanol and sonicated for 5 min to elute the pre-concentrated target analytes. After wards, a magnet was positioned on the outside of the centrifuge tube, and the supernatant solution was collected and F-7000 FL spectrophotometer (Hitachi, Japan) was used for all the fluorescence measurement.

Extraction
At room temperature, the sample solution after treatment and the pH buffer were added into a tube, and then distilled water was added to 40.0 mL. 0.1 g IL MNPs was added into the tube. The mixture was shaken on the timing multifunctional oscillator for 20.0 min and then centrifuged. ethanol of 3.0 mL as elution was added into the used IL MNPs. The mixture was ultrasonically vibrated at room temperature and then centrifuged to obtain supernatant solution. Supernatant solution was determined with fluorescence spectrophotometer.

Fluorescence Measurements
In a test tube (10.0 mL), 0.05 g 0.01mol/L IL-MNPs, 0.3mL of ethanol, 2.0 mL of buffer solution (pH=3.0) and adequate Rhodamine B standard solution or sample solution were added; the solution was diluted to the mark with distilled water. Then fluorescence spectra were recorded in the range of 300-700 nm upon excitation at 570 nm.

Sample Preparation
An amount of 5 g of chili powder/hot pepper/ Chinese prickly ash were accurately weighed into the beakers respectively, dissolved in 80.0 mL of ethanol. The resultant clear solution was evaporated to dryness at 60°C The residue was diluted with distilled water into a 100.0 mL flask and stored in darkness for detection.

Characterization of the MNPs
The synthesized Fe 3 O 4 @SiO 2 @ILsMNPs were characterized by FT-IR spectroscopy, thermo-gravimetrical analysis and magnetic characterization. In the curve c, d and e, the peaks at 2920 cm -1 and 2850 cm -1 corresponded to the C-H stretching vibration; the peaks at 1575 cm −1 corresponded to the characteristic absorption of imidazole groups in the Fe 3 O 4 @SiO 2 @ILs; in addition, the peak of 840 cm -1 was attributed to the P-F stretching vibration in the Fe 3 O 4 @SiO 2 @ILs [28], which indicated that ILs had been successfully immobilized on the surface of Fe 3 O 4 @SiO 2 .
Thermo-gravimetric analysis (TGA) was conducted in a nitrogen atmosphere, and the heating rate employed was 5°C min -1 all cases from 25 to 1000°C. Figure 2 presents the TGA of Fe 3 O 4 @SiO 2 and Fe 3 O 4 @SiO 2 @[OMIM]PF 6 . The figure, illustrates a weight loss of 2.7% at 240°C corresponding to the water content. Compared curve a and b, an additional weight loss of 6.6% is observed from 200 to 650°Cwhich is due to the ILs coating.

Optimization of Adsorption
The factors affecting the adsorption process of RhB such as pH, temperature and solution volume were studied and the adsorption behavior of Fe 3 O 4 @SiO 2 @PIL on RhB was compared with that of MNPs.

Effect of pH
As shown in Figure 3, the adsorption efficiency of RhB was varied with the pH, which was between 3.0 to 12. It could be concluded that the adsorption (retention) efficiency of RhB on IL MNPs was above 90.0%. It reached the highest value 94.44% when pH was 3.0. As the dissociation constant (pKa) of RhB is 3.2, the major existence form of RhB was molecule form at pH 3.0, the hydrophobicity of molecule form was stronger than its ionic form, Therefore, pH 3.0 was selected for the subsequent assays. Liquid Coated Magnetic Core/Shell Fe 3 O 4 @SiO 2 Nanoparticles Coupled with Fluorescence Spectrophotometry

Effect of Adsorption Temperature and Time
The adsorption efficiency of RhB on IL MNPs and MNPs at different temperatures (5.0-60°C) were studied (Figure 4). The adsorption efficiency of RhB on Fe 3 O 4 @SiO 2 @IL was higher than that on MNPs and was always above 70% from 0.5°C to 15.°C and was above 85% from 0.20°C to 50.°C. The experiment was carried out at room temperature.  Effect of adsorption time on extraction efficiency shows the extraction process was completed within 15.0 min, and the adsorption efficiency remained almost stable (86.0%). 15.0 min as the adsorption time for RhB was adopted ( Figure 5).

Effect of the Sample Volume
The adsorption efficiency of RhB varied with the increase of sample volume. The amount of RhB was fixed at 50.0 µg and the volume of the sample solution increased from 10.0 mL to 90.0 mL. The adsorption efficiency of RhB was above 89% from 10.0 to 80.0 mL and remain steady. and 77.85% when sample volume was 90.0 mL. So the largest sample volume allowed was 80.0 mL ( Figure 6).

Adsorption Capacity
The adsorption capacity is defined as the maximum amount of RhB adsorbed per gram of IL MNPs. The adsorption capacity of RhB on IL MNPs was studied ( Figure  7). When the concentration of RhB was 50.0 µg/mL (volume: 50 mL), the adsorption of RhB for 0.0225g IL MNPs reached the maximum. The adsorption capacity for IL MNPs was calculated as 105.80 mg/g.

Eluent Volume
The effect of ethanol volume was evaluated on the elution of RhB. The results in Figure 9 described that quantitative elution (above 90%) was observed as the volume of ethanol was higher than 3.0 mL. The pre-concentration factor is 25fold. The optimum volume of ethanol was chosen at 3.0 mL.

Elution Time
The elution process was completed within 10.0 min, and the elution efficiency did not change with a stable elution efficiency of 89.29% thereafter. The elution time of 10.0min for RhB was adopted (Figure 10)

The Reusability of Fe 3 O 4 @SiO 2 @IL
In order to investigate the recycling of the Fe 3 O 4 @SiO 2 NPs, they were washed with 2mL ethanol for twice after each MSPE run, and subsequently assembled with IL. Each re-prepared adsorbent was used for MSPE. The experimental results are shown in Figure 11. It was clear that no obvious loss of the adsorption capacity occurred after ten times of recycling.

Evaluation of Interferents
With a relative error of less than ±5%, the influence of some interferents that food samples contain on the determination of RhB in the presence of foreign substances was investigated. the tolerance limit for various foreign substances is in table 1. The results indicated that the majority of these substances in samples had no remarkable interference on the (RhB) determination.

Analytical Performance of the Method
Under the optimum conditions, the linear calibration curve was obtained in the concentration range of 0.40-140.00 µg L -1 . The equations of calibration graph is I (peak area) = Liquid Coated Magnetic Core/Shell Fe 3 O 4 @SiO 2 Nanoparticles Coupled with Fluorescence Spectrophotometry 11.97c+12.11 (µg L -1 ), with a correlation coefficient of 0.9993. The limit of detection (DL) was 0.06 µg L -1 . The relative standard deviation was 0.45% (n=3, c=10.00 µg L -1 ). The preconcentration factor, defined as the quotient of volume before absorption and after elution, is 25-fold.

Sample Analysis
The proposed method was applied to determine RhB in chili powder, hot pepper and Chinese prickly ash. RhB is 5.46 µg kg -1 in chili powder, with a recovery rate of RhB of 97.0% to 103.5%. and it was 4.53 µg kg -1 in hot powder with a recovery rate of RhB of 98.0%.
No RhB is detected in Chinese prickly ash, the recovery rate of RhB was from 100.7% to 100.1%.

Comparison of the Proposed Method with Relevant Literature
The comparison of current work with some other methods on the determination of RhB (Table 2) reveals that this method is either comparable or has rather pronounced advantages over them. Moreover, it is obvious that the present work has high sensitivity, wide linear range, and easy operation. Table 3 listed the linear range and the limit of detection for the analysis of RhB in real samples obtained by the reported methods, such as solid phase extraction coupled with UV spectrophotometry, solid phase extraction coupled with high performance liquid chromatography, and HPLC-CL method. Compared with other reported methods, the method adopted in the present work obviously had a satisfactory linear range and limit of detection.

Conclusion
In this work, IL MNPs was synthesized as magnetic solid phase extraction adsorbent to pre-concentrate/separate RhB from real samples. In summary, we have prepared Fe 3 O 4 @SiO 2 @IL as adsorbent, for MSPE of. The magnetic nano-adsorbent is prepared by a self-assembly technique, which is very simple and mild. Compared with traditional SPE, this MSPE based on the Fe 3 O 4 @SiO 2 @IL MNPs as an SPE adsorbent is fast, and the adsorbent can be easily separated from the sample solution. Moreover, this adsorbent has a high extraction capacity and high enrichment factors and is able to treat large-volume samples in a short period of time. The hydrocarbon chains of ILs on the surface of the adsorbent can provide adsorption sites for Rhodamine B other organic pollutants through π-π and hydrophobic interactions. The proposed method for the analysis of Rhodamine B is satisfactory. Thus, this adsorbent may also find potential application in the extraction and analysis of other analytes.