Method for separating EPA and DHA through supercritical fluid chromatography

Abstract

The invention provides a method for separating EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) through supercritical fluid chromatography, and relates to the technical field of separation and purification. The invention provides a method for separating EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) by supercritical fluid chromatography, which comprises the following steps: by taking supercritical carbon dioxide and an organic reagent as mobile phases, and by utilizing the specific mobile phase property of supercritical CO2 after being mixed with the organic reagent (saturated aliphatic hydrocarbon) and matching with a normal-phase chromatographic column, separating the EPA from the DHA by supercritical fluid chromatography. A specific stationary phase (one or more of a silica gel surface bonded propylurea stationary phase, a silica gel surface bonded phenyl stationary phase and a silica gel surface bonded imidazolyl stationary phase) is adopted, effective separation and preparation of the EPA and the DHA in the fish oil can be achieved, the EPA and the DHA in the fish oil can be effectively separated within 50 min, the separation speed is high, the separation effect is good, and the method is suitable for industrial production. The purity of the obtained EPA and DHA is 98% or above, the purity of the EPA and the DHA is high, and the EPA and the DHA have good application prospects in the fields of medicine production and food and health care products.

Description

Technical Field

[0001] This invention relates to the field of separation and purification technology, specifically to a method for separating EPA and DHA by supercritical fluid chromatography. Background Technology

[0002] Fish oil, as an important source of Omega-3 polyunsaturated fatty acids (PUFAs), contains the core active components eicosapentaenoic acid (EPA, structural formula below) and docosahexaenoic acid (DHA, structural formula below), which have irreplaceable physiological functions in areas such as cardiovascular protection, anti-inflammation, and nerve development. EPA and DHA in fish oil usually coexist with other impurities in the form of triglycerides or ethyl esters, requiring efficient separation technology to achieve high-purity extraction.

[0003]

[0004] The current mainstream separation methods include urea inclusion method, molecular distillation method, enzyme catalysis method, supercritical CO2 extraction method, and chromatographic separation method. Among them, supercritical CO2 extraction method can achieve separation at a relatively low temperature, which is particularly suitable for separating low-volatility heat-sensitive substances; supercritical CO2 is non-toxic, inert, and will not cause fish oil to oxidize and deteriorate, and the post-processing is simple. For example, the related technology discloses a method for separating and preparing ethyl eicosapentaenoate and ethyl docosahexaenoate using supercritical fluid chromatography, which includes the following steps: (1) converting gaseous CO2 into supercritical CO2; (2) using supercritical CO2 as the mobile phase and reversed-phase packing as the stationary phase to form a chromatographic column system; (3) injecting crude unsaturated polyenoic acid ethyl ester from the injection valve of the chromatographic column system, which is carried into the chromatographic column by stable supercritical CO2 for separation; (4) performing gas chromatography analysis on the product separated by the supercritical CO2 reversed-phase chromatographic column, and collecting the components that meet the analytical data of ethyl eicosapentaenoate and ethyl docosahexaenoate to obtain the product. However, the purity of EPA obtained by the above-mentioned supercritical fluid chromatography technique in a single separation is 89.85-93.2%, and the purity of DHA is 89.06-91.61%, which are low. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a method for separating EPA and DHA by supercritical fluid chromatography. The method provided by the present invention yields EPA and DHA with high purity.

[0006] This invention provides a method for separating EPA and DHA by supercritical fluid chromatography, comprising the following steps:

[0007] A dissolving reagent is mixed with the sample to be separated to obtain a test solution; the dissolving reagent includes saturated aliphatic hydrocarbons; the sample to be separated contains EPA and DHA;

[0008] The test solution was subjected to supercritical fluid chromatography to separate EPA and DHA. The conditions for supercritical fluid chromatography included: the mobile phase being a mixture of supercritical carbon dioxide and an organic reagent; the organic reagent including saturated aliphatic hydrocarbons; and the chromatographic column being a normal-phase column, wherein the stationary phase of the normal-phase column included one or more of silica gel surface-bonded propylurea stationary phase, silica gel surface-bonded phenyl stationary phase, and silica gel surface-bonded imidazole stationary phase.

[0009] Preferably, the saturated aliphatic hydrocarbons in the dissolving reagent and the mobile phase independently include n-hexane and / or n-heptane.

[0010] Preferably, the concentration of the sample to be separated in the test solution is ≤500 mg / mL;

[0011] The sample to be separated includes crude fish oil.

[0012] Preferably, the injection volume for supercritical fluid chromatography is 5–50 μL.

[0013] Preferably, the normal phase chromatographic column has a size of 10 mm × 250 mm and a column temperature of 30–35 °C.

[0014] Preferably, the volume ratio of supercritical carbon dioxide to organic reagent in the mobile phase is 85-98:2-15.

[0015] Preferably, the flow rate of the moving phase in the supercritical fluid chromatography separation is 5–15 mL / min.

[0016] Preferably, the elution method for the supercritical fluid chromatography separation is isocratic elution.

[0017] Preferably, the back pressure for the supercritical fluid chromatography separation is 8–15 MPa.

[0018] Preferably, the detection wavelength for the supercritical fluid chromatography separation is 205 nm.

[0019] This invention provides a method for separating EPA and DHA using supercritical fluid chromatography (SFC). The method uses supercritical carbon dioxide and organic reagents as the mobile phase, leveraging the unique properties of the mobile phase resulting from the mixture of supercritical CO2 and organic reagents (saturated aliphatic hydrocarbons). It also utilizes a normal-phase chromatographic column and a specific stationary phase (one or more of silica gel surface-bonded propylurea, silica gel surface-bonded phenyl, and silica gel surface-bonded imidazole). This method enables the effective separation and preparation of EPA and DHA from fish oil. It can effectively separate EPA and DHA from fish oil within 50 minutes, exhibiting fast separation speed, good separation effect, and EPA and DHA purity exceeding 98%. The high purity of EPA and DHA has excellent application prospects in the pharmaceutical and food / health product fields.

[0020] The method provided by this invention can purify raw materials of different purities into products suitable for medical pharmaceuticals and health food products by adjusting the process parameters of SFC separation. Furthermore, the method uses less organic reagent during separation, effectively reducing reagent costs and making it more environmentally friendly. In addition, it effectively simplifies post-processing of samples, further improving environmental friendliness and achieving cost reduction and efficiency improvement. It has broad application prospects in the separation and purification of EPA and DHA in fish oil. Attached Figure Description

[0021] Figure 1 The separation spectrum of the fish oil sample in Example 1 in the supercritical fluid chromatography system;

[0022] Figure 2 The separation spectrum of the fish oil sample in Example 2 in the supercritical fluid chromatography system;

[0023] Figure 3 The separation spectrum of the fish oil sample in Example 3 in the supercritical fluid chromatography system;

[0024] Figure 4 This is the preparation chromatogram of the fish oil sample in Example 4 using a supercritical fluid chromatography system;

[0025] Figure 5 This is the preparation chromatogram of the fish oil sample in Example 5 using a supercritical fluid chromatography system;

[0026] Figure 6 The chromatogram of the EPA eluent collected in Example 5 is obtained by detection and analysis in a high-performance liquid chromatography system.

[0027] Figure 7 The chromatogram of the DNA eluent collected in Example 5 is obtained by detection and analysis in a high-performance liquid chromatography system.

[0028] Figure 8This is the preparation chromatogram of the fish oil sample in Example 6 using a supercritical fluid chromatography system;

[0029] Figure 9 This is the preparation chromatogram of the fish oil sample in Example 7 using a supercritical fluid chromatography system;

[0030] Figure 10 The image shows the detection and analysis spectrum of the combined eluent of EPA and DNA collected in Example 7 in a gas chromatography system.

[0031] Figure 11 The image shows the preparation chromatogram of the fish oil sample in Comparative Example 1 in a supercritical fluid chromatography system. Detailed Implementation

[0032] This invention provides a method for separating EPA and DHA by supercritical fluid chromatography, characterized by comprising the following steps:

[0033] A dissolving reagent is mixed with the sample to be separated to obtain a test solution; the dissolving reagent includes saturated aliphatic hydrocarbons; the sample to be separated contains EPA and DHA;

[0034] The test solution was subjected to supercritical fluid chromatography to separate EPA and DHA. The conditions for supercritical fluid chromatography included: the mobile phase being a mixture of supercritical carbon dioxide and an organic reagent; the organic reagent including saturated aliphatic hydrocarbons; and the chromatographic column being a normal-phase column, wherein the stationary phase of the normal-phase column included one or more of silica gel surface-bonded propylurea stationary phase, silica gel surface-bonded phenyl stationary phase, and silica gel surface-bonded imidazole stationary phase.

[0035] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0036] The present invention mixes a dissolving reagent with the sample to be separated to obtain a test solution.

[0037] In this invention, the sample to be separated contains EPA and DHA; the concentration of EPA in the sample to be separated is preferably 50-75%, specifically 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 72%, or 75%; the concentration of DHA in the sample to be separated is preferably 10-20%, specifically 10%, 12%, 15%, 18%, or 20%. In this invention, the sample to be separated preferably includes crude fish oil.

[0038] In this invention, the dissolving agent comprises saturated aliphatic hydrocarbons, which preferably include n-hexane and / or n-heptane.

[0039] In this invention, the concentration of the sample to be separated in the test solution is preferably ≤500 mg / mL, more preferably 100-500 mg / mL, and may specifically be 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 350 mg / mL, 400 mg / mL, 450 mg / mL or 500 mg / mL.

[0040] After obtaining the test solution, the present invention performs supercritical fluid chromatography to separate the test solution to obtain EPA and DHA.

[0041] In this invention, the mobile phase used in the supercritical fluid chromatography separation is a mixture of supercritical carbon dioxide and organic reagents; the volume ratio of supercritical carbon dioxide to organic reagents in the mobile phase is preferably 85-98:2-15, specifically 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, or 98:2. In this invention, the organic reagents include saturated aliphatic hydrocarbons, preferably including n-hexane and / or n-heptane. In this invention, the elution method for the supercritical fluid chromatography separation is preferably isocratic elution. In this invention, the flow rate of the mobile phase during the supercritical fluid chromatography separation process is preferably 5 to 15 mL / min, more preferably 8 to 15 mL / min, and can specifically be 5 mL / min, 6 mL / min, 7 mL / min, 8 mL / min, 9 mL / min, 10 mL / min, 11 mL / min, 12 mL / min, 13 mL / min, 14 mL / min or 15 mL / min.

[0042] In this invention, the supercritical fluid chromatography separation uses a normal-phase column. The stationary phase of the normal-phase column includes one or more of the following: silica-bonded propylurea stationary phase (MSU), silica-bonded phenyl stationary phase (Phenyl), and silica-bonded imidazole stationary phase (IMU). The column size is preferably 10 × 250 mm. In this invention, the column preferably includes Nucifera Phenyl, Nucifera MSU, Nucifera IMU, or Nucifera MSU and Nucifera IMU in series (abbreviated as Nucifera MSU-IMU). In this invention, the column temperature for supercritical fluid chromatography separation is preferably 30–35°C, specifically 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C.

[0043] In this invention, the back pressure of the supercritical fluid chromatography separation is preferably 8 to 15 MPa, more preferably 10 to 15 MPa, and may specifically be 8 MPa, 9 MPa, 10 MPa, 11 MPa, 12 MPa, 13 MPa, 14 MPa or 15 MPa.

[0044] In this invention, the injection volume for supercritical fluid chromatography is preferably 5 to 50 μL, specifically 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL or 50 μL.

[0045] In this invention, the detection wavelength for supercritical fluid chromatography separation is preferably 205 nm.

[0046] To further illustrate the present invention, the method for separating EPA and DHA by supercritical fluid chromatography provided by the present invention will be described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0047] In the following examples and comparative examples, the target eluent obtained by supercritical fluid chromatography was subjected to high performance liquid chromatography or gas chromatography to verify the purity of the fish oil.

[0048] The high-performance liquid chromatography (HPLC) detection conditions included: a Nucifera C18M column (5 μm, 10 × 250 mm), a mobile phase of acetonitrile:water (volume ratio) of 88:12, isocratic elution, and an elution time of 20 min; a mobile phase flow rate of 1 mL / min; a detection wavelength of 205 nm; and a column temperature of 22 °C.

[0049] The gas chromatography detection conditions included: FID detector; CP-WAX 52CB quartz capillary column; N2 carrier gas; 250℃ injection port temperature; 270℃ detector temperature; 1 mL / min flow rate; temperature program of 170℃ for 2 min, then increasing to 240℃ at 3℃ / min and holding for 8 min; split ratio of 200:1; and injection volume of 1 μL.

[0050] The supercritical fluid chromatograph is a supercritical fluid chromatograph from Jiangsu Hanbang Technology Co., Ltd., and the temperature of carbon dioxide entering the pump is 8℃. The chromatographic column Nucifera MSU and the chromatographic column Nucifera IMU are connected in series, abbreviated as Nucifera MSU-IMU.

[0051] Fish oil sample 1: UV detection showed that the EPA purity was 70% and the DHA purity was 10%.

[0052] Fish oil sample 2: UV detection showed that the EPA purity was 50% and the DHA purity was 20%.

[0053] Example 1

[0054] Fish oil sample 1 was ultrasonically dissolved in n-heptane to obtain a test solution with a fish oil concentration of 100 mg / mL. The test solution was then subjected to SFC separation according to Table 1 to obtain EPA and DHA, respectively.

[0055] Table 1. SFC separation conditions in Example 1

[0056]

[0057] The separation effect diagram of fish oil sample 1 is shown below. Figure 1 As shown. By Figure 1 It can be seen that under these conditions, EPA and DHA in this fish oil have been effectively separated.

[0058] Example 2

[0059] Fish oil sample 1 was ultrasonically dissolved in n-hexane to obtain a test solution with a fish oil concentration of 100 mg / mL. The test solution was then subjected to SFC separation according to Table 2 to obtain EPA and DHA, respectively.

[0060] Table 2. SFC separation conditions in Example 2

[0061]

[0062] The separation effect diagram of fish oil sample 1 is shown below. Figure 2 As shown. By Figure 2 It can be seen that when the type of stationary phase is changed, the distribution of impurities is slightly different. The MSU has a better separation effect on intermediate impurities and retains them more strongly on the MSU.

[0063] Example 3

[0064] Fish oil sample 1 was ultrasonically dissolved in n-hexane to obtain a test solution with a fish oil concentration of 100 mg / mL. The test solution was then subjected to SFC separation according to Table 3 to obtain EPA and DHA, respectively.

[0065] Table 3. SFC separation conditions in Example 3

[0066]

[0067] The separation effect diagram of fish oil sample 1 is shown below. Figure 3 As shown. By Figure 3 It can be seen that, under this combination of stationary and mobile phases, although EPA and DHA in fish oil did not achieve complete baseline separation, the number of components that could be separated was relatively large, and the separation of impurities was relatively good.

[0068] Example 4

[0069] Fish oil sample 1 was ultrasonically dissolved in n-hexane to obtain a test solution with a fish oil concentration of 100 mg / mL. The test solution was then subjected to SFC separation according to Table 4 to obtain EPA and DHA, respectively.

[0070] Table 4. SFC separation conditions in Example 4

[0071]

[0072]

[0073] The separation effect diagram of fish oil sample 1 is shown below. Figure 4 As shown. By Figure 4 It can be seen that when chromatographic columns are connected in series, more impurity components can be separated. The separation effect is better than that of a single chromatographic column.

[0074] Example 5

[0075] Fish oil sample 1 was ultrasonically dissolved in n-heptane to obtain a test solution with a fish oil concentration of 100 mg / mL. The test solution was then subjected to SFC separation according to Table 5 to obtain EPA and DHA, respectively.

[0076] Table 5. SFC separation conditions in Example 5

[0077]

[0078] The separation effect diagram of fish oil sample 1 is shown below. Figure 5 As shown. By Figure 5 It can be seen that when using MSU and IMU columns in series, the sample loading volume is increased from 10ul to 30ul, and EPA and DHA still have good separation.

[0079] The EPA eluent collected from SFC separation was analyzed by high-performance liquid chromatography (HPLC). The results are as follows: Figure 6 As shown in Table 6, the purity of EPA in the EPA eluent is 99.17%.

[0080] Table 6. High-performance liquid chromatography peak information for EPA eluent.

[0081] Serial Number Retention time / min area area / % high 1 19.019 6900 0.03 364 2 22.629 27366 0.10 1163 3 23.454 26618184 99.17 989900 4 24.677 176498 0.66 5129 5 33.577 12642 0.05 355

[0082] The DNA eluent collected by SFC was analyzed by high-performance liquid chromatography (HPLC). The results are as follows: Figure 7 As shown in Table 7, the purity of DHA in the DNA elution buffer is 98.08%.

[0083] Table 7. High-performance liquid chromatography peak information for DNA elution buffer.

[0084] Serial Number Retention time / min area area / % high 1 22.032 9280 0.11 372 2 23.363 53533 0.62 2104 3 28.662 8401634 98.08 255736 4 29.900 32304 0.38 995 5 33.350 6476 0.08 232 6 38.755 62974 0.74 1506

[0085] Example 6

[0086] Fish oil sample 2 was ultrasonically dissolved in n-heptane to obtain a test solution with a fish oil concentration of 450 mg / mL. The test solution was then subjected to SFC separation according to Table 8 to obtain EPA and DHA, respectively.

[0087] Table 8. SFC separation conditions in Example 6

[0088]

[0089] The separation effect of fish oil sample 2 is shown in the figure below. Figure 8 As shown. By Figure 8 It can be seen that when using EPA and DHA at a ratio of approximately 50:20, there are more impurities under these conditions, but the separation effect can still be guaranteed. EPA and DHA still have a good degree of separation.

[0090] Due to variations in the purity of the raw materials, different types of products can be produced. Fish oil samples with an EPA to DHA mass content ratio of 70:10 can be used for the production of pharmaceutical-grade high-purity EPA, while fish oil samples with an EPA to DHA mass content ratio of 50:20 can be used for the production of food and health supplements. There are no specific purity requirements for EPA and DHA in food and health supplements; a combined purity of 90% or higher is sufficient.

[0091] Example 7

[0092] Fish oil sample 2 was ultrasonically dissolved in n-heptane to obtain a test solution with a fish oil concentration of 450 mg / mL. The test solution was then subjected to SFC separation according to Table 9 to obtain EPA and DHA, respectively.

[0093] Table 9. SFC separation conditions in Example 7

[0094]

[0095]

[0096] The separation effect of fish oil sample 2 is shown in the figure below. Figure 9 As shown. By Figure 9 It can be seen that when preparing EPA and DHA using fish oil sample 2, the separation effect was still maintained even after the sample loading volume was increased from 10 μL to 50 μL. EPA and DHA still maintained good separation.

[0097] The EPA and DNA eluents collected from SFC were combined and analyzed by gas chromatography. The results are as follows: Figure 10 As shown in Table 10, the combined purity of the collected EPA and DHA was 92.9%, and the yield was 80%, which meets the requirements of health supplements.

[0098] Table 10 Gas chromatographic peak information of the combined EPA eluent and DNA eluent

[0099] Serial Number Time / min Peak area (pA.s) Peak area % Peak height (PA) 1 18.577 28.651 0.291 4.58 2 18.743 5.615 0.057 0.96 3 19.438 1.900 0.019 0.50 4 19.639 114.116 1.160 14.16 5 20.239 6013.831 61.138 593.36 6 20.502 3.874 0.039 1.29 7 20.513 5.764 0.059 1.33 8 20.729 1.036 0.011 0.56 9 20.752 1.222 0.012 0.60 10 20.904 5.519 0.056 0.88 11 22.473 9.523 0.097 1.32 12 22.905 1.397 0.014 0.56 13 22.922 1.439 0.015 0.56 14 23.463 0.844 0.009 0.29 15 23.482 0.613 0.006 0.28 16 23.745 4.798 0.049 0.81 17 23.984 0.791 0.008 0.19 18 24.490 0.543 0.006 0.28 19 24.705 454.504 4.621 66.46 20 25.346 3126.315 31.783 371.36 21 25.519 0.868 0.009 0.88

[0100] Comparative Example 1

[0101] Fish oil sample 1 was ultrasonically dissolved in ethanol to obtain a test solution with a fish oil concentration of 100 mg / mL. The test solution was then subjected to SFC separation according to Table 11 to obtain EPA and DHA, respectively.

[0102] Table 11 SFC separation conditions in Comparative Example 2

[0103]

[0104] The separation effect diagram of fish oil is shown below. Figure 11 As shown. By Figure 11 It can be seen that when ethanol is used as an entrainer, EPA and DHA in the sample cannot be effectively separated.

[0105] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for separating EPA and DHA by supercritical fluid chromatography, characterized in that, Includes the following steps: The dissolving reagent is mixed with the sample to be separated to obtain a test solution; the dissolving reagent includes saturated aliphatic hydrocarbons; The sample to be separated contains EPA and DHA; The test solution was subjected to supercritical fluid chromatography to separate EPA and DHA; the conditions for supercritical fluid chromatography included: the mobile phase being a mixture of supercritical carbon dioxide and organic reagents; the organic reagents included saturated aliphatic hydrocarbons; The chromatographic column is a normal-phase chromatographic column, and the stationary phase of the normal-phase chromatographic column includes one or more of the following: silica gel surface-bonded propylurea stationary phase, silica gel surface-bonded phenyl stationary phase, and silica gel surface-bonded imidazole stationary phase.

2. The method according to claim 1, characterized in that, The dissolving reagent and the saturated aliphatic hydrocarbons in the mobile phase independently include n-hexane and / or n-heptane.

3. The method according to claim 1 or 2, characterized in that, The concentration of the sample to be separated in the test solution is ≤500 mg / mL; The sample to be separated includes crude fish oil.

4. The method according to claim 1 or 2, characterized in that, The injection volume for supercritical fluid chromatography separation is 5–50 μL.

5. The method according to claim 4, characterized in that, The normal phase chromatographic column has a size of 10 mm × 250 mm and a column temperature of 30–35 °C.

6. The method according to claim 1 or 2, characterized in that, The volume ratio of supercritical carbon dioxide to organic reagent in the mobile phase is 85–98:2–15.

7. The method according to claim 6, characterized in that, The flow rate of the moving phase in the supercritical fluid chromatography separation is 5–15 mL / min.

8. The method according to claim 1, 5, or 7, characterized in that, The elution method for supercritical fluid chromatography separation is isocratic elution.

9. The method according to claim 8, characterized in that, The back pressure for the supercritical fluid chromatography separation is 8–15 MPa.

10. The method according to claim 1, 5, 7 or 9, characterized in that, The detection wavelength for the supercritical fluid chromatography separation is 205 nm.

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