Method for removing plasticizers from ethyl esterified fish oil

Abstract

A method for removing plasticizers from ethyl esterified fish oil. The method for removing plasticizers from ethyl esterified fish oil includes providing ethyl esterified fish oil. The plasticizers in the ethyl esterified fish oil are removed by supercritical fluid simulated moving bed chromatography, and at the same time, high-purity eicosapentaenoic acid is obtained.

Description

Technical Field

[0001] The present application relates to a purification method, and more particularly to a method for removing plasticizers from ethyl esterified fish oil. Background Art

[0002] Fish oil is a general term for oily substances extracted from fish. It helps lower cholesterol and triglyceride levels, promotes the metabolism of saturated fatty acids in the body, and thus reduces the risk of cardiovascular disease.

[0003] Among them, eicosapentaenoic acid (EPA) belongs to the ω-3 series of polyunsaturated fatty acids. It is the main nutrient component of fish oil and is also an important nutrient that the human body cannot synthesize on its own but is indispensable.

[0004] The existing methods for separation and purification of fish oil mainly include: molecular distillation, low-temperature crystallization, urea inclusion method, lipase method, silver resin chromatography, silver nitrate complexation method and high performance liquid chromatography.

[0005] The above-mentioned fish oil separation and purification methods are difficult to purify high-purity eicosapentaenoic acid alone and are also unable to effectively remove plasticizers from ethylated fish oil, thereby causing food safety issues.

[0006] Therefore, finding a method to purify high-purity EPA from fish oil and separate plasticizers is an urgent problem to be solved. Summary of the Invention

[0007] The present application provides a method for removing plasticizers from ethyl esterified fish oil, which can effectively separate plasticizers from fish oil and purify high-purity EPA.

[0008] An embodiment of the present application provides a method for removing plasticizers from ethylated fish oil. The method comprises the following steps. First, ethylated fish oil is provided, wherein the ethylated fish oil comprises eicosapentaenoic acid and a mixed component, wherein the mixed component comprises a plasticizer. Then, the eicosapentaenoic acid in the ethylated fish oil is separated from the mixed component by supercritical fluid simulated moving bed chromatography, wherein the supercritical fluid simulated moving bed chromatography comprises: (i) providing a supercritical fluid simulated moving bed, wherein the supercritical fluid simulated moving bed comprises a first section, a second section and a third section in sequence, wherein the supercritical fluid simulated moving bed is composed of a mobile phase and a stationary phase, the stationary phase particles have pores inside, and the mobile phase flows in the same direction from the inlet of the flushing end through the first section, the second section and the third section in the supercritical fluid simulated moving bed. Between the second section and the third section, the stationary phase is simulated to move in the opposite direction relative to the mobile phase, and the mobile phase is a detergent comprising supercritical carbon dioxide and ethanol; (ii) the ethylated fish oil is injected into the supercritical fluid simulated moving bed from the feed inlet between the second section and the third section, so that the eicosapentaenoic acid in the ethylated fish oil moves with the mobile phase to the raffinate end of the third section, and the mixed components move with the stationary phase to the extraction end between the first section and the second section, thereby separating the eicosapentaenoic acid and the plasticizer.

[0009] In one embodiment of the present application, the mixed component further includes docosahexaenoic acid.

[0010] In one embodiment of the present application, the plasticizer includes dibutylphthalate (DBP) and di(2-ethylhexyl)phthalate (DEHP).

[0011] In one embodiment of the present application, the content of the ethanol is 1 wt % to 10 wt % based on the total amount of the cleaning agent.

[0012] In one embodiment of the present application, the content of the ethanol is 8 wt % based on the total amount of the cleaning agent.

[0013] In one embodiment of the present application, the stationary phase is a surface-modified silica filler.

[0014] In one embodiment of the present application, each of the first section, the second section, and the third section comprises at least two columns, and each column is filled with a stationary phase.

[0015] In one embodiment of the present application, the operating pressure of the supercritical fluid simulated moving bed is 130 bar to 150 bar.

[0016] In one embodiment of the present application, the operating temperature of the supercritical fluid simulated moving bed is 40°C to 60°C.

[0017] In one embodiment of the present application, the separation conditions used in the above-mentioned supercritical fluid simulated moving bed are: the carbon dioxide flow rate is 8.0 g / min at the inlet of the wash end, 0.5 g / min at the feed inlet, 2.82 g / min at the extraction end, and 5.68 g / min at the raffinate end, and the ethanol flow rate is 0.882 ml / min at the inlet of the wash end, 0.055 ml / min at the feed inlet, 0.311 ml / min at the extraction end, and 0.626 ml / min at the raffinate end.

[0018] In one embodiment of the present application, the switching time of the supercritical fluid simulated moving bed is 3 minutes and 40 seconds to 4 minutes.

[0019] In one embodiment of the present application, the purity of eicosapentaenoic acid in the sample separated from the raffinate end is greater than 74%.

[0020] In one embodiment of the present application, the recovery rate of eicosapentaenoic acid in the sample separated from the raffinate end is greater than 71%.

[0021] In one embodiment of the present application, the method further includes performing plasticizer analysis on the sample separated from the raffinate end, wherein the plasticizer analysis includes: extracting the sample separated from the raffinate end using a solid phase extraction column to obtain a first extract; and performing gas chromatography-mass spectrometry analysis on the first extract in a selected ion detection mode.

[0022] Based on the above, the method for removing plasticizers from ethylated fish oil of the present application separates EPA from plasticizers and DHA in fish oil by applying supercritical fluid simulated moving bed chromatography. The method of the present application can be fed continuously, has simple operating steps, and has good stability. Compared with traditional preparation methods, it has the advantages of low solvent consumption and high yield. In addition, the method for removing plasticizers from ethylated fish oil of the present application can effectively remove plasticizers and DHA from fish oil, and is therefore conducive to the industrial production of high-purity EPA. Therefore, the method for removing plasticizers from ethylated fish oil by using supercritical fluid simulated moving bed chromatography technology provided by the present application can solve the problems of severe product dilution, low operational repeatability, and poor stability in traditional technologies.

[0023] In order to make the above features and advantages of the present application more obvious and easy to understand, embodiments are given below and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a pipeline flow chart of a supercritical fluid simulated moving bed device according to an embodiment of the present application;

[0025] Figure 2 is the GC-FID spectrum of ethyl esterified fish oil sample;

[0026] Figure 3 This is the supercritical fluid chromatography analysis spectrum of Example 1 of the present application;

[0027] Figure 4 This is the supercritical fluid chromatography analysis spectrum of Example 2 of the present application;

[0028] Figure 5 This is the supercritical fluid chromatography analysis spectrum of Example 3 of the present application;

[0029] Figure 6 This is the supercritical fluid chromatography analysis spectrum of ethyl esterified fish oil sample under specific conditions;

[0030] Figure 7 This is the result analysis diagram of the supercritical fluid simulation moving bed with a switching time of 3 minutes and 40 seconds;

[0031] Figure 8 This is the result analysis diagram of the supercritical fluid simulation moving bed with a switching time of 3 minutes and 50 seconds;

[0032] Figure 9 This is the result analysis diagram of the supercritical fluid simulation moving bed with a switching time of 3 minutes and 55 seconds;

[0033] Figure 10 This is the analysis result of the supercritical fluid simulation moving bed with a switching time of 4 minutes. DETAILED DESCRIPTION

[0034] The method for removing plasticizers from ethylated fish oil according to the present invention can be used to remove plasticizers from fish oil and simultaneously purify EPA, thereby obtaining high-purity EPA.

[0035] The following examples illustrate the details or conditions of the plasticizer removal method of the present application. The method for removing plasticizers from ethylated fish oil of this embodiment includes the following steps. First, ethylated fish oil is provided, wherein the ethylated fish oil includes eicosapentaenoic acid and a mixed component, wherein the mixed component includes a plasticizer. Next, the eicosapentaenoic acid is separated from the mixed component using supercritical fluid simulated moving bed chromatography. In one embodiment, the mixed component also includes docosahexaenoic acid (DHA). In one embodiment, the plasticizer includes dibutyl phthalate (DBP) and di(2-ethylhexyl)phthalate (DEHP). In one embodiment, the mixed component includes docosahexaenoic acid, dibutyl phthalate, and di(2-ethylhexyl)phthalate. The method for removing plasticizers from ethylated fish oil of this embodiment can effectively remove plasticizers from the fish oil and separate EPA and DHA from the fish oil. Thereby, high-purity EPA can be obtained.

[0036] The following embodiments are not intended to limit the scope of protection of this application. The drawings are schematic diagrams drawn for the convenience of explanation only and are not intended to limit the actual methods, conditions or devices.

[0037] [Configuration design of supercritical fluid simulated moving bed]

[0038] In this embodiment, the Figure 1 The supercritical fluid-simulated moving bed (SF-SMB) apparatus shown is used to perform supercritical fluid simulated moving bed chromatography. Figure 1 This is a pipeline flow chart of a supercritical fluid simulated moving bed device according to an embodiment of the present application. Figure 1 The supercritical fluid simulated moving bed 100 includes a first section, a second section, and a third section. In this embodiment, the first section includes two tubing strings C1 and C2, the second section includes three tubing strings C3 and C4, and the third section includes two tubing strings C5 and C6. These six tubing strings are connected in series, but the present application is not limited thereto. In another embodiment, the first section includes two tubing strings, the second section includes two tubing strings, and the third section includes three tubing strings. These eight tubing strings are connected in series.

[0039] The supercritical fluid simulated moving bed 100 is composed of a mobile phase (not shown) and a stationary phase (not shown). The mobile phase flows from the purge inlet D1 through the first, second, and third sections in the same direction relative to the supercritical fluid simulated moving bed 100, while the stationary phase simulates movement in the opposite direction relative to the mobile phase.

[0040] Each pipe column is filled with a stationary phase having pores inside the particles. In the present embodiment, the stationary phase is, for example, a surface-modified silica filler, but the application is not limited thereto. In the present embodiment, the mobile phase (or washing agent) is, for example, a washing agent comprising supercritical carbon dioxide and an auxiliary solvent. In the present embodiment, the auxiliary solvent is ethanol (anhydrous ethanol). The washing agent comprising supercritical carbon dioxide and an auxiliary solvent can be formed by generating high-pressure carbon dioxide through a carbon dioxide liquid pump and mixing with the auxiliary solvent.

[0041] Refer again Figure 1 The supercritical fluid simulated moving bed 100 includes two feed ports, namely the sample feed port F1 (i.e., the column C5 inlet) and the purge port D1 (i.e., the column C1 inlet), and two discharge ports, namely, the extraction port E1 (i.e., the column C2 outlet) and the raffinate port R1 (i.e., the column C8 outlet). If all feed ports and discharge ports are simultaneously switched to the next column after a period of time, the stationary phase movement (i.e., the movement of the stationary phase to the next column) can be simulated. Figure 1 For example, the feed inlet is switched from the original position at the inlet of column C5 to the inlet of column C6. The remaining feed and discharge ports are also simultaneously moved to the next column. At the same time, the detergent and feed continue to flow continuously to the raffinate. If the feed and discharge ports are continuously switched, the solids will flow continuously downward and circulate repeatedly, thus achieving a continuous countercurrent contact process between the solids and the supercritical fluid.

[0042] Because the present embodiment uses supercritical carbon dioxide as the scrubbing agent (mobile phase), a high-pressure carbon dioxide supply source 110 is required. The supercritical fluid simulated moving bed 100 utilizes a carbon dioxide liquid pump 115 to generate high-pressure carbon dioxide from the carbon dioxide supply source 110 and temporarily stores it in a high-pressure buffer tank 120. The feed carbon dioxide flow rate is then controlled using a front-end pressure regulating valve 122 or a rear-end pressure regulating valve 123, a mass flow meter, and a control valve (not shown).

[0043] In addition to the control of the carbon dioxide mass flow rate, the input of the auxiliary solvent is controlled from the input port D2 by a high-performance liquid chromatography liquid pump 125a, while the input of the sample is controlled from the input port F2 by a high-performance liquid chromatography liquid pump 125b. In detail, after the sample feed is dissolved in the auxiliary solvent, it is input from the input port F2 by the high-performance liquid chromatography liquid pump 125b and mixed with carbon dioxide before entering the supercritical fluid simulated moving bed 100. Similarly, as a mobile phase, a wash liquid comprising supercritical carbon dioxide and the auxiliary solvent is formed by mixing the high-pressure carbon dioxide generated by the carbon dioxide liquid pump 115 with the auxiliary solvent input from the input port D2. In addition, the step of mixing the high-pressure carbon dioxide and the auxiliary solvent can be achieved by a mixer 130.

[0044] While the supercritical fluid continuously switches the feed port and the discharge port, the supercritical fluid continues to flow upward (i.e. Figure 1 ), but does not directly circulate back to column C1. Conventional simulated moving bed devices using a liquid as the mobile phase often incorporate a fourth section to regenerate the mobile phase for direct recirculation. In this embodiment, a pressure reduction separation method is used to easily achieve supercritical fluid regeneration. Therefore, the supercritical fluid flowing from the raffinate end R1 and the extraction end E1 is simply depressurized in separation tanks 145a and 145b to vaporize the carbon dioxide. The carbon dioxide gas can then be secondary cooled to precipitate the residual auxiliary solvent and solutes, achieving carbon dioxide regeneration. This reduces the use of columns in the fourth section, lowering equipment costs and the cost of packing materials.

[0045] After appropriate pressure regulation and metering, the carbon dioxide in the high-pressure buffer tank 120 is injected into the system via columns C1 and C6, respectively. Prior to injection, it is mixed with a metered amount of auxiliary solvent or feed solution. After separation by the supercritical fluid simulated moving bed, the two outputs flow out of the system via the extraction end E1 and the raffinate end R1. The supercritical fluid exiting the raffinate end R1 first passes through the rear-end pressure regulating valve 123 before being separated into the auxiliary solvent and solute in the separation tank 145b, where the carbon dioxide gas is then recovered. The rear-end pressure regulating valve 123 at the outlet of the raffinate end R1 also controls the operating pressure of the entire SF-SMB. The supercritical fluid exiting the extraction end E1 has its flow rate controlled by a mass flow control valve before entering the separation tank 145a to separate the auxiliary solvent and solute. The carbon dioxide gas exiting the separation tanks at the extraction end E1 and the raffinate end R1 is combined and discharged together.

[0046] Next, the method of separating plasticizer from fish oil by using supercritical fluid simulated moving bed chromatography will be described below. Figure 1Following the supercritical fluid simulated moving bed 100, ethylated fish oil is injected from feed inlet F1 between the second and third sections of the supercritical fluid simulated moving bed 100. The eicosapentaenoic acid in the ethylated fish oil migrates with the mobile phase to the raffinate end R1 of the third section, while the mixed components, including the plasticizer, migrate with the stationary phase to the extraction end E1 between the first and second sections, thereby separating the eicosapentaenoic acid from the plasticizer. To achieve this separation, the mobile phase comprises a detergent comprising supercritical carbon dioxide and ethanol. In this embodiment, the ethanol content is 1% to 10% by weight, based on the total amount of the detergent. In this embodiment, the ethanol content is 8% to 10% by weight, based on the total amount of the detergent. In one embodiment, the ethanol content is 8% by weight, based on the total amount of the detergent. In another embodiment, the ethanol content is 8% by weight, based on the total amount of the detergent.

[0047] Establishment of analytical method for EPA and DHA

[0048] The composition of ethylated fish oil was analyzed using a gas chromatography-flame ionization detector (GC-FID) (model GC-2014, Shimadzu). A CP-WAX52 CB capillary column (25 mL × 0.5 mm ID, 0.2 μm) was used, and helium at 1.0 mL / min was used as the carrier gas. The GC-FID temperature settings are shown in Table 1.

[0049] [Table 1]

[0050]

[0051] The fish oil sample used in this example has been pre-treated (i.e., ethyl esterification), and the EPA content of the fish oil sample is 71.7%, and the DHA content is 9.7%. The GC-FID spectrum of the fish oil sample is as follows: Figure 2 shown. Figure 2 This is the GC-FID spectrum of the ethyl esterified fish oil sample. Figure 2 The internal standard (IS) used was 2034 mg / L pentadecane. Comparison with the MS database revealed that the peak at a retention time of 11.9 minutes was EPA, while the peak at a retention time of 16.3 minutes was DHA. The positions of the DHA and EPA peaks were clearly identified from the GC-FID spectrum and used as the analytical standard.

[0052] Establishment of plasticizer analysis method

[0053] In this example, ethylated fish oil was extracted and cleaned using a solid phase extraction column (SPE) (Welchrom Silica, 2g / 6mL) and then analyzed using a gas chromatography-mass spectrometer (GC-MS) (Model QP2010, Shimadzu). Selective ion monitoring (SIM) mode was used for qualitative analysis based on retention time and the ion ratio between the qualitative ion fragments, while external standard analysis was used for quantitative analysis. The specific analysis steps are shown below.

[0054] (A) Sample pretreatment

[0055] A1: All containers should be rinsed with methanol to n-hexane in sequence before use and then dried.

[0056] A2: Weigh 0.10 g of fish oil sample and add 2 mL of n-hexane to mix well.

[0057] A3: Take 10 μL of isotope standard working solution and add it to the sample in step A2.

[0058] A4: SPE column cleanup

[0059] A4.1: Add 10 mL of methyl tert-butyl ether (MTBE)-n-hexane solution (5%, v / v) to the solid phase extraction column and discard the effluent.

[0060] A4.2: Add 10 mL of n-hexane to the SPE column and discard the flow-through.

[0061] A4.3: Add the sample prepared in step A2 and discard the flow-through.

[0062] A4.4: Add 15 mL of MTBE-n-hexane solution (3%, v / v) and discard the effluent.

[0063] A4.5: Add 20 mL of MTBE-n-hexane solution (5%, v / v) and collect the effluent in a 20 mL sample bottle.

[0064] A4.6: After drying the effluent from step A4.5 with nitrogen at room temperature, add 1 mL of acetonitrile to the sample vial and use a rotary shaker to allow the acetonitrile to fully contact the vial wall. Then perform gas chromatography-mass spectrometry (GC-MS) analysis.

[0065] A5: Blank test

[0066] A5.1: No sample blank test

[0067] Except that the sample is not weighed in step A2, the rest of the process is carried out according to step A4.

[0068] A5.2: Testing of blank samples with the same matrix

[0069] The remaining process is carried out in accordance with step A4, except that 0.10 g of a commercially available sample that has been verified to be free of plasticizers is weighed in step A2.

[0070] A6: Add tests

[0071] High concentration addition test

[0072] The remaining procedures were carried out in accordance with step A4, except that in step A2, 0.10 g of a commercially available sample that had been verified to be free of plasticizers was weighed and 0.1 mL of the standard working solution was added.

[0073] (B) Instrumental analysis

[0074] B1: Gas chromatography conditions

[0075] -Capillary: 5% phenyl-methylpolysiloxane quartz capillary column, 30 m in length, 0.25 mm in inner diameter, 0.25 μm in film thickness, or equivalent.

[0076] -Inlet temperature: 280°C.

[0077] - The heating conditions are shown in Table 2 below.

[0078] [Table 2]

[0079]

[0080] -Carrier gas: high purity helium (purity> 99.999%)

[0081] -Flow rate: 1 ml / min

[0082] -Injection method: non-split

[0083] -Injection volume: 0.5 μL B2: Mass spectrometer setting conditions -Ion source: Electron Impact (EI)

[0084] -Ion source temperature: 230℃

[0085] -Detector temperature: 280℃

[0086] -Detector voltage: 0.7Kv

[0087] -Solvent delay: 5 minutes

[0088] - The ion table is shown in Table 3 below.

[0089] [Table 3]

[0090]

[0091] B3: Preparation of calibration curve

[0092] In this embodiment, the calibration curve is prepared as shown in Table 4 below.

[0093] [Table 4]

[0094]

[0095] B4: Qualitative confirmation

[0096] B4.1: Detention time

[0097] After sample analysis, the retention times of DBP and DEHP shall not exceed ±0.5% of the retention times of their corresponding standards.

[0098] B4.2: Ion Ratio

[0099] After sample analysis, the mass-to-charge ratio of the sample and the mass-to-charge ratio of the standard must meet the specifications in Table 5 below.

[0100] [Table 5]

[0101] Mass-to-charge ratio >50％ 50％～20％ 20％～>10％ ≦10％ Allowable error ±10％ ±15％ ±20％ ±50％

[0102] (C) Calculation of analysis results

[0103] C1: Concentration Calculation - Quantification Using Calibration Curve

[0104] In this embodiment, the concentration of the plasticizer is calculated and quantified using the calibration curve. The calculation method of the plasticizer concentration is shown in formula (1):

[0105]

[0106] CPAEs = plasticizer concentration (μg / g)

[0107] c = Plasticizer concentration calculated using the calibration curve (μg / mL)

[0108] V = final volume (mL)

[0109] m = sample weight (g)

[0110] C2: Concentration Calculation - Quantification Using Isotope Standards

[0111] In this embodiment, the concentration of the plasticizer is calculated and quantified using an isotope standard. The calculation method of the plasticizer concentration is shown in formula (2):

[0112]

[0113] C PAEs = Plasticizer concentration (μg / g)

[0114] Area PAEs =Plasticizer analysis area

[0115] Area PAEs-d4 =Plasticizer isotope analysis area

[0116]

[0117] RF PAEs =Plasticizer Sensing Factor

[0118] V = final volume (mL)

[0119] m = sample weight (g)

[0120] [Retention behavior of EPA, DHA, and plasticizers in 2-EP filler tested using supercritical fluid chromatography]

[0121] In order to set the operating conditions for supercritical fluid simulated moving bed chromatography, before the actual separation of EPA and plasticizers, a suitable supercritical fluid chromatography system was screened and the retention behavior of EPA, DHA, and plasticizers was tested.

[0122] In this embodiment, a preparative-grade surface-modified silica filler was used as the stationary phase and filled in a 4.6 mm ID × 250 mm L stainless steel column. The column was installed in a supercritical fluid chromatograph (SFC) instrument, and the operating conditions were screened. In this embodiment, ethanol was used as an auxiliary solvent to separate fish oil and plasticizer. The retention behavior of fish oil and plasticizer in silica was discussed with respect to operating conditions such as pressure, temperature and auxiliary solvent concentration. Since DEHP has a higher retention than DBP, under the operating conditions of low pressure, high temperature and low auxiliary solvent concentration, DEHP and DBP will be clearly separated into two peaks. Therefore, the selectivity of the separation of fish oil and plasticizer can be calculated by dividing the retention time of DBP by the retention time of fish oil.

[0123] Example 1: Pressure Effect

[0124] In this example, under the operating conditions of 40°C, a carbon dioxide flow rate of 2.0 g / min, and an auxiliary solvent (i.e., ethanol) concentration of 10 wt%, the retention behavior of fish oil and plasticizer on 2-EP filler at operating pressures of 130 bar, 140 bar, and 150 bar was investigated. The results are shown in FIG. Figure 3 As shown in Table 6.

[0125] [Table 6]

[0126]

[0127] Figure 3 This is the supercritical fluid chromatography analysis spectrum of Example 1 of this application. Please refer to Figure 3 As shown in Table 6, when the pressure is increased from 130 bar to 140 bar and 150 bar, the retention time of fish oil is shortened from 2.46 minutes to 2.42 minutes and 2.34 minutes, respectively. The retention time of DBP is extended from 2.55 minutes to 2.86 minutes, and then shortened to 2.64 minutes. The selectivity of fish oil and DBP separation is 1.036, 1.182 and 1.149 at operating pressures of 130 bar, 140 bar and 150 bar, respectively. From the above results, it can be seen that the separation effect of fish oil and plasticizer is best when the operating pressure is 140 bar. However, considering the system pressure during the subsequent operation of SF-SMB, the subsequent examples use 130 bar as the operating pressure.

[0128] Example 2: Temperature Effect

[0129] In this example, under the operating conditions of 130 bar, a carbon dioxide flow rate of 2.0 g / min, and an auxiliary solvent (i.e., ethanol) concentration of 10 wt%, the retention behavior of fish oil and plasticizer on 2-EP filler was studied at operating temperatures of 40°C, 45°C, and 50°C. The results are shown in FIG. Figure 4 As shown in Table 7.

[0130] [Table 7]

[0131]

[0132] Figure 4 This is the supercritical fluid chromatography analysis spectrum of Example 2 of this application. Please refer to Figure 4 As shown in Table 7, increasing the temperature from 40°C to 45°C and 50°C increases the retention time of fish oil from 2.46 minutes to 2.75 minutes and 3.23 minutes, respectively. The retention time of DBP increases from 2.55 minutes to 3.06 minutes and 3.52 minutes, respectively. The selectivity for separation of fish oil and DBP is 1.036, 1.113, and 1.090 at operating temperatures of 40°C, 45°C, and 50°C, respectively. These results indicate that the separation of fish oil and plasticizer is optimal at 45°C. Therefore, the subsequent examples will use 130 bar and 45°C as the operating conditions.

[0133] Example 3: Auxiliary solvent effect

[0134] In this example, under the operating conditions of 130 bar, 45 ° C, and a carbon dioxide flow rate of 2.0 g / min, the retention behavior of fish oil and plasticizer on the filler was studied for the auxiliary solvent (i.e., ethanol) concentrations of 8 wt% and 10 wt%. The results are as follows: Figure 5 As shown in Table 8.

[0135] [Table 8]

[0136]

[0137] Figure 5 This is the supercritical fluid chromatography analysis spectrum of Example 3 of this application. Please refer to Figure 5 As shown in Table 8, under the operating conditions of 130 bar, 45°C, and 2.0 g / min, increasing the auxiliary solvent concentration from 8 wt% to 10 wt% reduced the retention time of fish oil from 3.06 minutes to 2.46 minutes, while the retention time of DBP decreased from 3.52 minutes to 2.55 minutes. The selectivity for the separation of fish oil and DBP was 1.150 and 1.036 at auxiliary solvent concentrations of 8 wt% and 10 wt%, respectively.

[0138] In summary, under the operating conditions of 130 bar and 45°C, the present application achieves the best effect of separating fish oil and plasticizer using supercritical carbon dioxide and 8wt% ethanol as an auxiliary solvent, and the residence time is within a reasonable operating range. These operating conditions can be used as the setting for subsequent SF-SMB operating conditions.

[0139] Figure 6 This is the supercritical fluid chromatography analysis spectrum of ethyl esterified fish oil sample under specific conditions. Specifically, Figure 6 The supercritical fluid chromatography analysis spectrum is obtained under the operating conditions of 130 bar, 45 ° C and 8 wt% auxiliary solvent. Figure 6 It can be seen that under the operating conditions of 130 bar, 45°C and 8 wt% auxiliary solvent, the peaks of EPA and DHA partially overlap, and the retention times of DBP and DHA are similar, while the retention time of DEHP is longer. Figure 6 The dotted line in the figure not only separates EPA and DHA, but also removes plasticizers. The raffinate end will produce high-purity EPA and a trace amount of plasticizer, while the extraction end will produce EPA, DHA and a large amount of plasticizer.

[0140] In subsequent SF-SMB separation experiments, GC-FID will be used to analyze samples collected from the SF-SMB to confirm the separation of EPA and DHA. This separation of EPA and DHA will then serve as a basis for the separation of fish oil and plasticizers. In other words, a decrease in DHA content in the raffinate should indicate a decrease in plasticizer content. Subsequently, a larger number of samples will be collected from both the raffinate and extract ends, and further analysis of DBP and DEHP using the aforementioned plasticizer analysis method will be performed.

[0141] [SF-SMB separation test]

[0142] [Calculation of EPA purity and recovery rate]

[0143] First, the ethylated fish oil sample was directly dissolved in pure ethanol as the feed solution. It was then pumped into the SF-SMB system for separation into two samples of varying composition. The EPA and DHA contents of the sample collected from the outlet were calculated and compared with the EPA and DHA contents at the feed inlet to assess the effectiveness of the separation. In this example, the average fish oil contents in the ethanol solutions collected from the extraction and raffinate outlets were 1.8 g / L and 7.6 g / L, respectively.

[0144] A certain volume of sample was taken from the outlet and subjected to GC-FID and plasticizer analysis. The purity and recovery of EPA and DHA were calculated using the wave front area. The EPA and DHA content was calculated using the formula shown in Equation 3 below. In this embodiment, the EPA and DHA content can be defined as purity.

[0145]

[0146] In formula 1, A EPA With A DHA are the peak areas of EPA and DHA in the GC spectrum, and Ai represents the sum of the peak areas of all components in the spectrum.

[0147] After EPA and DHA are separated from ethyl esterified fish oil, their recovery rates are estimated according to the following formula 4.

[0148]

[0149] In formula 2, A EPA With A DHA are the peak areas of EPA and DHA in the GC spectrum, Q is the volume flow rate of ethanol, and the superscripts E and R represent the extraction end and the raffinate end, respectively.

[0150] Experimental Example 4

[0151] [SF-SMB operating conditions]

[0152] In Experimental Example 1, the ethyl esterified fish oil raw material was first prepared into a 100 g / L ethanol solution. Figure 1 The supercritical fluid simulated moving bed apparatus shown is used to remove plasticizers from ethyl esterified fish oil. The packed column is 10 mm ID ×250mm L The stainless steel columns were designed with surface-modified silica filler as the stationary phase and a detergent containing supercritical carbon dioxide and 8 wt% pure ethanol as the mobile phase. The six packed columns were designed as three-section supercritical fluid simulated moving beds, and each section had two columns. The separation conditions were: the temperature was fixed at 45°C, the outlet pressure at the raffinate end was 130 bar, and the inlet pressure of the detergent was 170 bar. The carbon dioxide flow rates at each inlet and outlet were set as follows: 8.00 g / min at the scrubbing end inlet; 0.50 g / min at the feed inlet; 2.82 g / min at the extraction end; and 5.68 g / min at the raffinate end (calculated by mass conservation). The pure ethanol flow rates at the feed inlet were set as follows: 0.882 ml / min at the scrubbing end inlet; and 0.055 ml / min at the feed inlet. According to the law of conservation of mass, the ethanol flow rates at the extraction end and the raffinate end were 0.311 ml / min and 0.626 ml / min, respectively. In addition, in Experimental Example 4, under the condition of fixed flow rates at each inlet and outlet, the switching time interval of the valves on the SF-SMB device was changed (3 minutes and 40 seconds, 3 minutes and 50 seconds, 3 minutes and 55 seconds, and 4 minutes), and then the composition of the samples collected from the two outlets was observed as the switching time changed. The results obtained by supercritical fluid simulated moving bed chromatography under the above conditions are shown in the figure. Figures 7 to 10 The results of EPA and DHA contents (defined as purity), recovery rates, and plasticizer contents in the samples calculated by Formula 3 and Formula 4 are shown in Table 9.

[0153] [Table 9]

[0154]

[0155] Figure 7 This is the analysis result of the supercritical fluid simulation moving bed with a switching time of 3 minutes and 40 seconds. Figure 8 This is the analysis result of the supercritical fluid simulation moving bed with a switching time of 3 minutes and 50 seconds. Figure 9 This is the analysis result of the supercritical fluid simulation moving bed with a switching time of 3 minutes and 55 seconds. Figure 10 This is the analysis result of the supercritical fluid simulation moving bed with a switching time of 4 minutes.

[0156] Please refer to Figures 7 to 10As shown in Table 9, when the switching time was 3 minutes and 40 seconds, a sample with high-purity EPA (74.2%) was collected at the raffinate end with a recovery rate of 71%, while the DBP and DEHP contents were 0.8 mg / kg and 2.2 mg / kg, respectively. However, the sample collected from the extraction end still contained a large amount of EPA and DHA, with purities of 59.1% and 32.1%, respectively, and DBP and DEHP contents of 3.6 mg / kg and 17.0 mg / kg, respectively.

[0157] When the switching time was extended to 3 minutes and 50 seconds, a sample with high-purity EPA (purity increased to 80.0%) was still collected at the raffinate end with a recovery rate of 94%, while the DBP and DEHP contents were 0.5 mg / kg and 2.4 mg / kg, respectively. Furthermore, the EPA content in the sample collected from the extraction end was significantly reduced, with EPA and DHA purities of 16.7% and 68.7%, respectively, and DBP and DEHP contents of 8.7 mg / kg and 29.5 mg / kg, respectively.

[0158] When the switching time was extended to 3 minutes and 55 seconds, a sample with high-purity EPA (76.9%) and DHA (3.6%) could be collected from the raffinate end, with a recovery rate of 81.8%. The DBP and DEHP contents were 0.3 mg / kg and 1.9 mg / kg, respectively. Furthermore, the EPA content in the sample collected from the extraction end was significantly reduced, with EPA and DHA purities of 36.4% and 54.3%, respectively, and DBP and DEHP contents of 10.0 mg / kg and 35.9 mg / kg, respectively.

[0159] When the switching time was extended to 4 minutes, a sample with high-purity EPA (75.3% purity) was still collected at the raffinate end with a recovery rate of 94.9%. The purity of DHA was 6.5%, and the levels of DBP and DEHP were 0.1 mg / kg and 1.1 mg / kg, respectively. Furthermore, the EPA content in the sample collected from the extraction end was also reduced, with the purity of EPA and DHA reaching 27.6% and 64.8%, respectively, and the levels of DBP and DEHP reaching 14.6 mg / kg and 46.2 mg / kg, respectively.

[0160] It is worth mentioning that the raffinate samples collected at switching times of 3 minutes 40 seconds and 3 minutes 50 seconds shown in Table 9 contain a small amount of DHA, which is a residual phenomenon caused by the dead volume in the system and can be ignored.

[0161] The above results demonstrate that the present method for removing plasticizers from ethylated fish oil, which utilizes supercritical fluid simulated moving bed chromatography to remove plasticizers from fish oil, not only effectively removes plasticizers but also effectively improves the separation efficiency of EPA and DHA, yielding high-purity EPA. In this example, the purity of the eicosapentaenoic acid isolated from the raffinate end was greater than 74%. The recovery rate of the eicosapentaenoic acid isolated from the raffinate end was greater than 71%.

[0162] In summary, the supercritical fluid simulated moving bed technology provided in this application allows for continuous feeding, simple operation steps, and excellent stability. Compared with traditional preparation methods, it has advantages such as low solvent consumption and high yield. In addition, the method for removing plasticizers from ethylated fish oil in this application can effectively remove plasticizers, thereby facilitating the industrial production of high-purity EPA. Therefore, the method for removing plasticizers from ethylated fish oil using supercritical fluid simulated moving bed chromatography technology provided in this application can solve the problems of severe product dilution, low operational repeatability, and poor stability in traditional technologies.

[0163] Although the present application has been disclosed as above with reference to the embodiments, it is not intended to limit the present application. Any person skilled in the art should be able to make slight changes and modifications without departing from the spirit and scope of the present application. Therefore, the scope of protection of the present application should be based on the definition of the appended claims.

Claims

1. A method for removing plasticizers from ethylated fish oil, comprising: Provided is an ethylated fish oil comprising eicosapentaenoic acid and a mixed component, wherein the mixed component comprises a plasticizer; and separating the eicosapentaenoic acid in the ethylated fish oil from the mixed component by supercritical fluid simulated moving bed chromatography. The supercritical fluid simulated moving bed chromatography method comprises: (i) providing a supercritical fluid simulated moving bed, the supercritical fluid simulated moving bed sequentially comprising a first section, a second section, and a third section, wherein the supercritical fluid simulated moving bed is composed of a mobile phase and a stationary phase, wherein the stationary phase particles have pores, the mobile phase flows from the wash end inlet in the same direction as the supercritical fluid simulated moving bed through the first section, the second section, and the third section, and the stationary phase simulates movement in the opposite direction relative to the mobile phase, wherein the mobile phase is a wash agent comprising supercritical carbon dioxide and ethanol; and (ii) injecting the ethylated fish oil from the feed inlet into the space between the second section and the third section of the supercritical fluid simulated moving bed, so that the eicosapentaenoic acid in the ethylated fish oil moves with the mobile phase to the raffinate end of the third section, and the mixed components move with the stationary phase to the extraction end between the first section and the second section, thereby separating the eicosapentaenoic acid and the plasticizer. The operating temperature of the supercritical fluid simulated moving bed is 40°C to 45°C. The separation conditions used in the supercritical fluid simulated moving bed are as follows: the carbon dioxide flow rate is 8.0 g / min at the inlet of the wash end, 0.5 g / min at the inlet of the feed, 2.82 g / min at the extraction end, and 5.68 g / min at the extraction end, and the ethanol flow rate is 0.882 ml / min at the inlet of the wash end, 0.055 ml / min at the feed inlet, 0.311 ml / min at the extraction end, and 0.626 ml / min at the extraction end.

2. The method for removing plasticizers from ethylated fish oil according to claim 1, wherein the plasticizers comprise di-n-butyl phthalate and di-(2-ethylhexyl) phthalate.

3. The method for removing plasticizers from ethylated fish oil according to claim 1, wherein the content of the ethanol is 1 wt% to 10 wt% based on the total amount of the detergent.

4. The method for removing plasticizers from ethylated fish oil according to claim 1, wherein the stationary phase is a surface-modified silica filler, and the first section, the second section, and the third section each comprise at least two columns, and each column is filled with the stationary phase.

5. The method for removing plasticizers from ethyl esterified fish oil according to claim 1, wherein the operating pressure of the supercritical fluid simulated moving bed is 130 bar to 150 bar.

6. The method for removing plasticizers from ethylated fish oil according to claim 1, wherein the switching time of the supercritical fluid simulated moving bed is from 3 minutes 40 seconds to 4 minutes. 7 . The method for removing plasticizers from ethyl esterified fish oil according to claim 1 , wherein the purity of eicosapentaenoic acid in the sample separated from the raffinate end is greater than 74%.

8. The method for removing plasticizers from ethyl esterified fish oil according to claim 1, further comprising subjecting the sample separated from the raffinate end to a plasticizer analysis, wherein the plasticizer analysis comprises: extracting the sample separated from the raffinate end using a solid phase extraction column to obtain a first extract; as well as The first extract was subjected to gas chromatography-mass spectrometry analysis in a selected ion detection mode.

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