Detection method for chelating degree of crystalline glycine chelate

Abstract

The invention discloses a method for detecting the chelating degree of a crystalline glycine chelate, which comprises the following steps of: firstly, obtaining a crystal structure of glycine chelate salt through a scanning electron microscope, and judging whether the glycine chelate salt is a crystalline product or not; then characterizing through Fourier infrared spectroscopy, and judging whether the glycine chelate is subjected to chelation reaction or not through the change and deviation of a peak value; determining the content of glycine in the glycine chelate salt by adopting a high performance liquid chromatography, and determining the content of metal elements by adopting an inductively coupled plasma spectrometer; and finally, measuring free separated water and crystal water of the glycine chelate salt through thermogravimetry, and adding the glycine content, the metal ion content, the crystal water and the free water measured in each glycine chelate salt, thereby indicating the high purity characteristic of the product. According to the method, accurate detection of the glycine chelate can be realized, the operation is simple, convenient and reliable, and a means is provided for researching the crystalline chelate.

Description

Technical Field

[0001] The present invention relates to the technical field of substance chelation degree analysis, in particular to the detection of the chelation degree of amino acid chelates, and more particularly to a method for detecting the chelation degree of crystalline glycine chelates. Background Art

[0002] Glycine chelates include calcium glycinate, magnesium glycinate, zinc glycinate, and ferrous glycinate. They are a new generation of food nutritional enhancers that have a unique structure of a five-membered ring with a metal coordination bond. Whether glycine and metal elements in the product form a salt or a chelate, the detection of the coordination bond in the five-membered ring is crucial, and the purity of the chelate is an important indicator for its application.

[0003] At present, there is no report on the chelation degree detection method of glycine chelate. In order to strengthen the quality control of the chelation degree of glycine salt, it is necessary to develop a detection method thereof. Summary of the invention

[0004] In view of this, the present invention provides a method for detecting the chelation degree of glycine chelate, which can realize accurate detection of glycine chelate and is simple and reliable to operate, thus providing a means for studying such crystalline chelate.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for detecting the chelation degree of a crystalline glycine chelate comprises the following steps:

[0007] (1) obtaining the crystal structure of the glycine chelate salt by scanning electron microscopy to determine whether it is a crystalline product;

[0008] (2) Characterizing by Fourier transform infrared spectroscopy, judging whether the glycine chelate salt has undergone a chelation reaction by the change and shift of the peak value;

[0009] (3) The glycine content in the glycine chelate salt was determined by high performance liquid chromatography, and the metal element content was determined by inductively coupled plasma spectrometry;

[0010] (4) The free water and crystal water of glycine chelate salts were measured by thermogravimetry, and the glycine content, metal ion content, crystal water and free water measured in each glycine chelate salt were added together to illustrate the high purity characteristics of the product.

[0011] Preferably, the scanning electron microscope in step (1) has a magnification of 50-2000 times, an acceleration voltage of 0.02-30 kV, and a probe current of 3 pA-20 nA.

[0012] Furthermore, the scanning electron microscope in step (1) has a magnification of 100-1000 times, an acceleration voltage of 10-20 kV, and a probe current of 5 pA-15 nA.

[0013] Preferably, the Fourier transform infrared spectrum scanning range in step (2) is: 7800-350cm -1 .

[0014] Furthermore, the Fourier transform infrared spectrum scanning range in step (2) is: 4000-400cm -1 .

[0015] Preferably, the conditions of the high performance liquid chromatography in step (3) are:

[0016] Mobile phase A: sodium acetate solution; Mobile phase B: acetonitrile;

[0017] The volume ratio of mobile phase A to mobile phase B was 84:16;

[0018] Chromatographic column: Agilent, C18 column 4.6*150mm, 4μm; column temperature 35-45℃;

[0019] Flow rate 0.8-1.2 mL / min;

[0020] The injection volume is 15-25 μL;

[0021] The detection wavelength is 350-370nm.

[0022] Furthermore, in step (3), the column temperature is 40° C., the flow rate is 1.0 mL / min, the injection volume is 20 μL, and the detection wavelength is 360 nm.

[0023] Preferably, the inductively coupled plasma spectrometer conditions in step (3) are:

[0024] RF power: 1000-1500W, plasma gas flow: 10-20L / min, nebulizer pressure: 150-250kPa, auxiliary gas flow: 0.2-0.4L / min, nebulizer gas flow: 0.5-1.0L / min, pump injection volume: 1-2mL / min, instrument stabilization delay: 10-20s, injection delay: 20-40s.

[0025] Furthermore, the inductively coupled plasma spectrometer conditions in step (3) are:

[0026] RF power: 1300 W, plasma gas flow: 15 L / min, nebulizer pressure: 200 kPa, auxiliary gas flow: 0.3 L / min, nebulizer gas flow: 0.8 L / min, pump injection volume: 1.50 mL / min, instrument stabilization delay: 15 s, injection delay: 30 s.

[0027] Furthermore, the crystalline glycine chelate can be a crystalline glycine cap chelate, a crystalline glycine magnesium chelate, a crystalline glycine iron chelate, and a crystalline glycine zinc chelate. The analysis spectra of the four elements are shown in Table 1:

[0028] Table 1

[0029] element Analytical line wavelength nm Calcium 317.933 Magnesium 279.077 Iron 239.562 Zinc 206.200

[0030] Preferably, the thermogravimetric temperature rise range in step (4) is 20-400°C, and the heating rate is 5-20°C / min.

[0031] Furthermore, in step (4), the thermogravimetric temperature rise range is 30-250°C, and the heating rate is 10°C / min.

[0032] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for detecting the chelation degree of a crystalline glycine chelate, which has the following beneficial effects:

[0033] The chelation degree detection method of the crystalline glycine chelate provided by the present invention is accurate and reliable, and can effectively illustrate the chelation degree of the glycine chelate. The glycine chelate product verified by the method has a wide range of application prospects and can be used in the fields of food, health care products, medicine, etc., to improve the quality and market competitiveness of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0035] Figure 1 is the SEM image of magnesium glycinate;

[0036] Figure 2 FTIR graphs of glycine and magnesium glycinate;

[0037] Figure 3 is the HPLC chart of magnesium glycinate;

[0038] Figure 4 This is the TG diagram of magnesium glycinate. DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Example 1

[0041] Detection method of magnesium glycinate chelation degree:

[0042] (1) The crystal structure of glycine chelate salt was obtained by scanning electron microscopy: an appropriate amount of magnesium glycine powder sample was taken, the sample was placed on a stage with conductive glue attached, the sample was gently flattened with tweezers, a thin metal layer was sprayed, the sample cup was placed, the sample hatch was opened, the sample cup was placed in the hatch, and the imaging was observed and adjusted. The electron gun current and acceleration voltage were adjusted to achieve the best resolution and clarity. The magnification was 50 times, the acceleration voltage was 10 kV, and the probe current was 10 nA to obtain a high-quality SEM image, such as Figure 1 As shown, it can be seen that magnesium glycinate has a good crystal form;

[0043] (2) Characterize by Fourier transform infrared spectroscopy, and judge whether the glycine chelate salt has undergone chelation reaction by the change and offset of the peak value: take an appropriate amount of sample and scan it, and compare the infrared spectra of glycine and magnesium glycinate, such as Figure 2 As shown, glycine is at 2500 cm -1 and 3100cm -1 There is a strong broad absorption peak between them, which is NH3 + Characteristic absorption peak at 1521.87cm -1 and 1332.93cm -1 The absorption peaks appearing at are the asymmetric and symmetric stretching vibration peaks of carboxylate groups;

[0044] In the Fourier transform infrared spectrum of magnesium glycinate, we found that 2500 cm -1 and 3100cm -1 The strong broad absorption peak between + The characteristic absorption peak of 1577.63cm -1 and 1340.33 -1 The asymmetric and symmetric stretching vibration peaks of carboxylate appear at 1521.87 cm in the infrared spectrum of glycine. -1 To 1332.93cm -1 Compared with the peaks of NH2 at 3211 cm-1 and 3342cm -1 The peak at indicates that the Mg-NH2 group and COO-Mg group of magnesium glycinate are generated. The changes and shifts of the peaks on the FT-IR spectrum indicate that our glycinate has undergone a chelation reaction.

[0045] (3) The glycine content in the glycine chelate was determined by high performance liquid chromatography, and the metal element content was determined by inductively coupled plasma spectrometry:

[0046] Among them, the HPLC instrument and detection conditions are:

[0047] Chromatographic column: Agilent, C18 column 4.6*150mm, 4μm;

[0048] Mobile phase: 0.1 mol / L sodium acetate solution: acetonitrile = 84:16;

[0049] Column temperature: 40°C;

[0050] Flow rate: 1.0 mL / min;

[0051] Detection wavelength: 360nm;

[0052] Injection volume: 20 μL;

[0053] Preparation of standard solution:

[0054] Glycine standard stock solution (0.600 mg / mL): Weigh about 60 mg of glycine standard and dissolve it in purified water to make up to 100 mL.

[0055] Glycine standard working solution: Accurately pipette 0.25mL, 0.50mL, 1.00mL, 2.00mL, and 4.00mL of glycine standard stock solution and dissolve it in purified water to make up to 25mL. The concentration of glycine in the standard working solution is 0.006mg / mL, 0.012mg / mL, 0.024mg / mL, 0.048mg / mL, and 0.096mg / mL.

[0056] Derivatization reaction of glycine standard working solution: Pipette 2.0 mL into a 25 mL volumetric flask, add 2.0 mL of sodium acetate solution (0.1 mol / L), then add 0.5 mL of derivatization reagent solution, incubate in a 60°C water bath for 50 min, cool, and add pure water to the mark. Filter with a 0.45 μm filter membrane, collect the filtrate, and use it as the standard curve solution.

[0057] Preparation of sample solution:

[0058] Preparation of solid sample solution: Weigh 60 mg of solid sample and dissolve it in purified water to make up to 100 mL.

[0059] Sample dilution: Pipette 1.0 mL into a 25 mL volumetric flask and dissolve to volume with purified water.

[0060] Sample derivatization: Pipette 2.0 mL into a 25 mL volumetric flask, add 2.0 mL of sodium acetate solution (0.1 mol / L), then add 0.5 mL of derivatization reagent solution, place in a 60°C water bath for 50 min, cool, and add constant volume solution to the mark. Filter with a 0.45 μm filter membrane, and collect the filtrate as the sample solution.

[0061] Note: Blank synchronous derivative operation.

[0062] Test results such as Figure 3 and Table 2 below:

[0063] Table 2

[0064]

[0065] Inductively coupled plasma spectrometer instrument and detection conditions:

[0066] RF power: 1300W, plasma gas flow: 15L / min, nebulizer pressure: 200kPa, auxiliary gas flow: 0.3L / min, nebulizer gas flow: 0.8L / min, pump injection volume: 1.50mL / min, instrument stabilization delay: 15s, injection delay: 30s, analysis line 279.077nm;

[0067] Accurately weigh 0.5g of magnesium glycinate chelate in a polytetrafluoroethylene digestion tank. For samples containing ethanol or carbon dioxide, first heat them on a hot plate at low temperature to remove the ethanol or carbon dioxide, and add 10mL of nitric acid-perchloric acid (10+1) mixed solution. Digest on a CNC hot plate and add nitric acid in time (conditions: keep at 120℃ for 1h~2h, heat to 150℃ for 2h~4h, heat to 180℃~200℃ for 2h). If it turns brown-black during the digestion process, add 5mL of nitric acid-perchloric acid (10+1) mixed solution until white smoke is emitted and the digestion liquid becomes colorless, transparent and slightly yellow. Cool, transfer to a 25mL volumetric flask and dilute to the scale with nitric acid solution (the dilution multiple depends on the content of the element to be measured in the sample), mix well for use, and perform a blank test at the same time.

[0068] The test results are shown in Table 3:

[0069] Table 3

[0070]

[0071] (4) The free water and crystal water of glycine chelate salts were measured by thermogravimetry, and the glycine content, metal ion content, crystal water and free water measured in each glycine chelate salt were added together to illustrate the high purity characteristics of the product:

[0072] Thermogravimetric heating rate is 10℃ / min, and the temperature is 30-250℃.

[0073] Add all the components together, such as Figure 4 and Table 4:

[0074] Table 4

[0075]

[0076] SEM images show that glycinate exists in a regular crystal structure. In the Fourier transform infrared spectrum of magnesium glycinate chelate, NH3 + The special absorption peak of completely disappeared, indicating that all free glycine was consumed by chelation reaction. Peaks appeared in Mg-NH2 and COO-Mg groups, indicating that glycinate was indeed generated by chelation reaction. The magnesium content was tested by ICP-OES, and the glycine content was tested by HPLC. The free water and crystal water of magnesium glycinate chelate were measured by TG. The glycine content, mineral content, crystal water and free water together, the result is greater than 99.5%, which conforms to the standard molecular formula of glycine acid chelate, indicating the high purity chelation degree of magnesium glycinate.

[0077] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0078] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting the chelation degree of a crystalline glycine chelate, characterized in that: The following steps are involved: (1) obtaining the crystal structure of the glycine chelate salt by scanning electron microscopy to determine whether it is a crystalline product; (2) Characterizing by Fourier transform infrared spectroscopy, judging whether the glycine chelate salt has undergone a chelation reaction by the change and shift of the peak value; (3) The glycine content in the glycine chelate salt was determined by high performance liquid chromatography, and the metal element content was determined by inductively coupled plasma spectrometry; (4) The free water and crystal water of glycine chelate salts were measured by thermogravimetry, and the glycine content, metal ion content, crystal water and free water measured in each glycine chelate salt were added together to illustrate the high purity characteristics of the product.

2. The method for detecting the chelation degree of a crystalline glycine chelate according to claim 1, characterized in that: The scanning electron microscope described in step (1) has a magnification of 50-2000 times, an acceleration voltage of 0.02-30 kV, and a probe current of 3 pA-20 nA.

3. The method for detecting the chelation degree of a crystalline glycine chelate according to claim 1, characterized in that: The scanning electron microscope described in step (1) has a magnification of 100-1000 times, an acceleration voltage of 10-20 kV, and a probe current of 5 pA-15 nA.

4. The method for detecting the chelation degree of a crystalline glycine chelate according to claim 1, characterized in that: The Fourier transform infrared spectrum scanning range in step (2) is: 7800-350cm -1 .

5. The method for detecting the chelation degree of a crystalline glycine chelate according to claim 1, characterized in that: The Fourier transform infrared spectrum scanning range in step (2) is: 4000-400cm -1 .

6. The method for detecting the chelation degree of a crystalline glycine chelate according to claim 1, characterized in that: The conditions of the high performance liquid chromatography in step (3) are: Mobile phase A: sodium acetate solution; Mobile phase B: acetonitrile; The volume ratio of mobile phase A to mobile phase B was 84:16; Chromatographic column: Agilent, C18 column 4.6*150mm, 4μm; column temperature 35-45℃; Flow rate 0.8-1.2 mL / min; The injection volume is 15-25 μL; The detection wavelength is 350-370nm.

7. The method for detecting the chelation degree of a crystalline glycine chelate according to claim 6, characterized in that: In step (3), the column temperature is 40° C., the flow rate is 1.0 mL / min, the injection volume is 20 μL, and the detection wavelength is 360 nm.

8. The method for detecting the chelation degree of a crystalline glycine chelate according to claim 1, characterized in that: The inductively coupled plasma spectrometer conditions in step (3) are: RF power: 1000-1500W, plasma gas flow: 10-20L / min, nebulizer pressure: 150-250kPa, auxiliary gas flow: 0.2-0.4L / min, nebulizer gas flow: 0.5-1.0L / min, pump injection volume: 1-2mL / min, instrument stabilization delay: 10-20s, injection delay: 20-40s.

9. The method for detecting the chelation degree of a crystalline glycine chelate according to claim 1, characterized in that: The inductively coupled plasma spectrometer conditions in step (3) are: RF power: 1300 W, plasma gas flow: 15 L / min, nebulizer pressure: 200 kPa, auxiliary gas flow: 0.3 L / min, nebulizer gas flow: 0.8 L / min, pump injection volume: 1.50 mL / min, instrument stabilization delay: 15 s, injection delay: 30 s.

10. The method for detecting the chelation degree of a crystalline glycine chelate according to claim 1, characterized in that: The thermogravimetric temperature rise range in step (4) is 20-400°C, and the heating rate is 5-20°C / min.

11. The method for detecting the chelation degree of a crystalline glycine chelate according to claim 1, characterized in that: The thermogravimetric temperature rise range in step (4) is 30-250°C, and the heating rate is 10°C / min.

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