Synthesis and Characterization of a Magnetic Nanomaterial and Its Use in the Spectroscopic Determination of the Organic Dye Carmine Using Cloud Point Extraction

INTRODUCTION
Carmine (CAR) dye, also known as carminic acid, is a natural red dye that is obtained from the female cochineal insect, which is found on certain species of cactus in Central and South America. The dye is used for centuries as a coloring agent in food, cosmetics, and textiles. In food, it is listed as “E120” and is commonly found in processed meats and desserts.
The dye is considered safe for consumption by most people, but it can cause allergic reactions in some individuals. Vegetarians and vegans may also choose to avoid carmine dye as it is derived from an animal source [1,2].
Acceptable daily of carmine intake (ADI) is average 5 mg based on body weight. Their use may exceed the tolerable limit. Therefore, it is very important to observe the carmine dye levels in high-consumption food products. And the amounts of carmine added to foods and drinks should be controlled [3].
 Until now, various methods like high performance liquid chromatography (HPLC) [4,5] differential pulse polarography (DPP) [6], stripping voltammetry (SV) [7], , and spectrophotometry [8] have been recommended for the determination of carmine in food samples. UV-visible spectrometry is an important tool in this area, , it is often used in many areas such as environment, food, and chemistry. There are two chief limitations to the spectrometric determinations of food dyes. The major is the lower analytical quantity than the quantitative limits of the UV-visible spectrometry, and the other is the possible interference effect of other chemical species existing in the samples. Preconcentration methods such as ion exchange (IE) [9,10], solid phase extraction (SPE) [11,12], solvent extraction (SE) [13], and cloud point extraction (CPE) [14-17], were widely used to resolve these problems .CPE concederd environmentally friendly less reactive consumptionThis method is an alternative to conventional liquid liquid extraction (LLE) due to its high enrichment factor, lower toxic reagent use, less desirable sample size, removal of large amounts of organic solvents, usage of non-toxic surfactants, simpler, safer and more economical [18].
The combined extraction methods are very favorable in overcoming or minimizing definite limitations of each individual technique, accomplishing enhanced selectivity and alleviating time [19].
 This study aims to prepare the Fe3O4 nanocomposite, encapsulate it with tetraethyl orthosilicate (TEOS) and use it as an adsorbent to increase the extraction efficiency of CAR Dye at the optimum conditions of acidity function, temperature and time required for extraction using the cloud point technique.

MATERIALS AND METHODS
Apparatus
Jasco 7850 UV Visible Spectrophotometer with 1 cm (0.5 mL) quartz cell was used to record the absorption spectrum and absorption measurements. A 3D device (4000 rpm, UromAzma Corporation) was applied to accelerate the phase separation process. Metrohm 632 pH meter was used with integrated glass pH electrode Measurements All measurements were made at the University of Al-Qadisiyah.
 
Chemicals 
In this study, no similarly purification was once required in all chemical compounds and reagents. Carmine dye (C44H43AlCa2O30), MR 1158.936 g·mol−1, dye purity ≥ 90%, accredited with the aid of the Biological Spots Committee) used to be got from Sigma-Aldrich. 0.1g of dye was once weighed and dissolved with 10ml of ethanol in a 100ml volumetric vial and the volume was completed with distilled water to dissolve the amount of dye needed to prepare a stock solution of 1000mg/L CAR. Distilled water was used throughout this study. For absorbance of CAR dye solutions, a Shimadzu UV-1601PC spectrophotometer set at wavelength 603 nm was used for measurement.

Synthesis of TEOS functionalized magnetic nanoparticles:
The magnetic nanoparticles (MNPs) were prepared by using increased chemical co-precipitation method. FeCl3·6H2O (11.68 g) and FeCl2·4H2O (4.30 g) had been dissolved in 200 mL deionized water beneath nitrogen atmosphere with vigorous stirring at 85 °C. Then, 20 mL of 25% aqueous ammonia solution used to be brought to the solution. The shade of the bulk answer modified from orange to black immediately. The magnetic precipitate was once washed twice with deionized water and as soon as with 0.02 mol L−1 sodium chloride solution. The above organized magnetic suspension used to be placed in a 250 mL round-bottom flask and allowed to settle.An exterior first-rate magnet used to be utilized to isolate the Fe3O4 nanoparticles from the solution and the supernatant was once discarded . Then coating of MNPs with TEOS was once carried with the addition of an aqueous solution of TEOS (10 %, v/v, 80 mL), accompanied by using glycerol (60 mL). The mixture was once then stirred and heated at ninety °C for two h under a nitrogen atmosphere. After that, the resulting modified nanoparticles (TEOS– Fe3O4) had been washed with deionized water (3×250 mL), methanol (2×150 mL), deionized water (3×250 mL) and dried as black powders in a vacuum oven at 45 °C for 2 h [20].

Recommended extraction procedure
In 10 ml test tube, 2 ml of dye (carmine) reply with a attention of 10 mg/L used to be taken and 2 ml of the surfactant Triton X-114 used to be delivered to it and 1 ml of buffer solution with pH 2 used to be as soon as delivered and the extent used to be completed with distilled water free of ions at the mark limit. It used to be positioned in a water bathtub at a temperature of 70° C for 15 minutes till it fashioned a turbidity and then the take a appear at tube used to be positioned in the centrifuge for 3 minutes till the organic layer was once separated from the aqueous layer. Then the natural layer used to be taken and the nanomaterial 0.05 g used to be once added to it. It used to be as soon as placed in sonication for 5 minutes, then the nanomaterial used to be withdrawn with the resource of the utilization of a magnet and two ml of ethanol used to be added to it and placed in the sonication for 15 minutes, and the absorbance was observe for it at the wavelength of 603 nm. In the equal way, the plank used to be prepared, alternatively except the presence of dye.

RESULT AND DISCUSSION
Fourier Transform Infrared Spectroscopy (FTIR) for Fe3O4 magnetic nanoparticles and (TEOS) modified Fe3O4 magnetic nanoparticles 
When measuring the FTIR of the nanomaterial (Fe3O4) before encapsulation, the spectrum was obtained as shown in Fig. 1 by taking a sample of the uncoated nanomaterial and using the KBr potassium bromide disc method within the range of positive numbers Cm-1 (400-4000). Fig. 1 shows the FTIR data for the nanomaterial (Fe3O4) as shown. 
The infrared spectrum of Fe3O4 typically shows several bands between 400 and 4000 cm-1, corresponding to various vibrational modes of the iron oxide lattice. The exact wave numbers of these bands may vary depending on the sample preparation and measurement conditions. Some typical wave numbers for Fe3O4 in the infrared region include: - 579 cm-1: stretching vibration of Fe-O bonds - 670 cm-1: bending vibration of Fe-O bonds - 1100-1200 cm-1: stretching vibrations of the O-Fe-O bridges in the spinel structure - 1623 cm-1: bending vibration of the O-Fe-O bridges in the spinel structure and 3398 cm-1 of O-H stretching (associated with water molecules on the surface of Fe3O4) .
The FTIR spectrum of TEOS- Fe3O4 will contain absorption peaks corresponding to both TEOS and Fe3O4, as well as any functional groups that may be present on the surface of the nanoparticles. Here are some typical FTIR wavenumbers for TEOS and Fe3O4:
TEOS: 1058 cm-1: Si-O-Si stretching, 875 cm-1: Si-O-C stretching, 850 cm-1: Si-O-CH3 bending, 2923 cm-1: C-H stretching of ethyl groups.
Fe3O4: 588-465 cm-1: Fe-O stretching, 1539 cm-1: O-H bending (associated with water molecules on the surface of Fe3O4), 3399 cm-1: O-H stretching (associated with water molecules on the surface of Fe3O4).

Scanning electron microscope (SEM)
An analytical approach used to decide the surface appearance and particle measurement of organized samples. Fig. 3 shows the SEM evaluation of Fe3O4 iron oxide nanoparticles if irregular spherical shapes are located with a clear enlarge in the partial accumulation rate. While Fig. 4 complex (Fe3O4, TEOS) indicates the presence of heterogeneous nanoshells with irregular distribution resulting from the synthesis of iron oxide by means of TEOS. The enlarge in the rate of aggregation of particles shows the success of the physical bond due to iron oxide with TEOS. The average particle dimension of the superimposed iron oxide is 19 nm and 40 nm, respectively. The interpreted results are comparable to a set of preceding studies.

X-ray diffraction
X-ray diffraction is a technique used in chemistry to determine the atomic and molecular structure of a crystal. It involves shining a beam of X-rays onto a crystal and measuring the resulting diffraction pattern. This pattern is produced when the X-rays interact with the crystal lattice, causing them to scatter in different directions. By analyzing the angles and intensities of the diffracted X-rays, scientists can obtain information about the arrangement of atoms within the crystal. This technique is widely used in various areas of chemistry, including solid-state chemistry, materials science, and structural biology.
Fig. 5 shows the X-ray diffraction spectrum of Fe3O4 nanoparticles, which indicates the appearance of a wide range of peaks and the diffraction values are both peaks and peaks Ɵ=(30.1630 ,32.4432, 35.4084 ,46.5421,52.6132, 57.0007 , 62.6569 (The apparent diffraction indicates that Fe3O4 nanoparticles are spherical, and the appearance of sharp peaks indicates that Fe3O4 is highly crystalline and free of impurities.
Fig. 6 shows the X-ray diffraction spectrum of Fe3O4 nanoparticles covered with TEOS and glycerine, showing the appearance of twelve peaks for the prepared compound and the locations of these peaks are at 
 2Ɵ=(22.9822 , 32.6892 , 35.4747 , 46.9463, 52. 8191 , 52.9539 , 57. 3182 , 58.3026 , 62. 9104 (The apparent diffraction indicates that Fe3O4 nanoparticles are spherical, and the appearance of sharp peaks indicates that Fe3O4 is highly crystalline and free of impurities. The similar diffraction peaks for the coated and uncoated nanoparticles indicate that the coating agent has no significant effect on the crystal structure of the nanoparticles. 
 
Optimization of CPE Procedure
Effect of the (pH)
The effect of “pH in the range” 2 to 9 “was tested using distinct pH acetate buffer solutions. The effects are explained in Fig. 7. As can be seen from Fig. 5, at the pH= 2 the absorption reached a maximum subsequently, the absorption decreased due to partial dissociation of higher pH. Therefore, pH 2 used to be chosen as the top of the line pH for the adsorption of the natural layer of the dye for it at the wavelength of the best Absorption max = 603 nm. 
Effect of time
The perfect time that affords the perfect condition for measuring the absorbance of the dye. Effect of the “time in the range” from 5 to 25 was studied the use of Triton X-114 solution and the results are illustrated in Fig. 6. As can be viewed from Fig. 8, the absorption first accelerated with growing time, accomplishing a most of 15 minutes. Subsequently, the absorption decreased due to the “upper time partial dissociation”. Therefore, the time 15 min was once chosen as the most suitable time to measure the absorbance of the natural layer extracted from the dye at the wavelength of maximum absorbance = 603 nm (Fig. 8). 

Effect of temperature 
The impact of temperature in the vary from 30 to 90 °C was once studied the use of options of dye and Triton X-114. As shown in Fig. 9, the absorption elevated first with increasing temperature and reached a most temperature of 70. After that is, the absorption decreased due to “higher temperature partial dissociation.” Therefore, the temperature of 70 °C was chosen as the highest quality temperature for the adsorption of the natural layer of the dye for it at the wavelength of the maximum absorption =603 nm. 

Effect of amount of nanomaterials
TEOS-modified Fe3O4-NPs were added to the mixture content sample solution in amounts ranging from (0.01 to 0.06) g. According to Fig. 10, the extraction effectiveness was improved up to (0.05)g of adsorbent,. It is possible to execute an accurate extraction with a very tiny amount of the adsorbent because to the high surface to volume ratio of NPs Consequently, (0.05) g was used for further experiments.

Analytical performance of the optimized method
Below the finest experimental conditions, the calibration curve was linear over the concentration range of 10-90 µg Kg-1 with R2 of 0.9956. Solutions for the construction of calibration curve were prepared by spiking appropriate amounts of (CAR) working solutions and subjected to the recommended CPE-MSPE procedure following the enhanced measurements. The limit of detection (LOD=3.3Sb/m, where Sb is the standard deviation of nine replicate measurements of blank solution and m is the slope of the calibration curve) was found to be 10.6851 µg g-1.

CONCLUSION
The combination of (CPE) and - TEOS modified Fe3O4 - MSPE was effectively used as an capable sample pretreatment procedure for extraction and determination CAR dye. The analytical process has numerous benefits, including easiness of operation, slight organic solvent consumption, and high pre-concentration component, additionally, the approach has ability for the detection of CA dye traces in various samples.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.