Method for measuring chelation degree of crystalline glycine chelate

Abstract

A method for measuring the chelation degree of a crystalline glycine chelate, comprising: first obtaining the crystal structure of a glycine chelate salt by means of a scanning electron microscope, and determining whether the glycine chelate salt is a crystalline product; then performing characterization by means of Fourier-transform infrared spectroscopy, and determining, on the basis of changes and shifts in peak values, whether a chelation reaction has occurred in the glycine chelate salt; then determining glycine content in the glycine chelate salt by means of high performance liquid chromatography, and using an inductively coupled plasma spectrometer to determine metal element content; and finally determining free water and crystal water of the glycine chelate salt by means of thermogravimetric analysis and summing the glycine content, metal ion content, crystal water, and free water measured in each glycine chelate salt to demonstrate the high-purity characteristic of the product.

Description

A method for detecting the chelation degree of crystalline glycine chelates Technical Field

[0001] This invention relates to the field of chelation degree analysis technology, specifically to the detection of chelation degree of amino acid chelates, and more specifically to a method for detecting the chelation degree of crystalline glycine chelates. Background Technology

[0002] Glycine chelates, including calcium glycinate, magnesium glycinate, zinc glycinate, and ferrous glycinate, are a new generation of food fortifiers. They possess the unique structure of a five-membered ring with metal coordination bonds. Whether glycine and the metal element in the product form a salt or chelate, the detection of the coordination bonds in the five-membered ring is crucial. Furthermore, the purity of the chelate is an important indicator for its application.

[0003] There are currently no reports on methods for detecting the chelation degree of glycine chelates. In order to strengthen the quality control of glycine salt chelation degree, it is necessary to develop a detection method. Summary of the Invention

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

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for detecting the chelation degree of crystalline glycine chelates, comprising the following steps:

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

[0008] (2) Characterization was performed by Fourier transform infrared spectroscopy, and the changes and shifts in peak values ​​were used to determine whether the glycine chelate salt had undergone a chelation reaction;

[0009] (3) The glycine content in 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 water of crystallization of glycine chelate salts were measured by thermogravimetric analysis. The glycine content, metal ion content, water of crystallization and free water measured in each glycine chelate salt were summed to illustrate the high purity characteristics of the product.

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

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

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

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

[0015] Preferably, the conditions for high-performance liquid chromatography in step (3) are as follows:

[0016] Mobile phase A: Sodium acetate solution; Mobile phase B: Acetonitrile;

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

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

[0019] The flow rate is 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℃, the flow rate is 1.0mL / min, the injection volume is 20μL, and the detection wavelength is 360nm.

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

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

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

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

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

[0028] Table 1

[0029] Preferably, the thermogravimetric temperature rise range in step (4) is 20-400℃, and the temperature rise rate is 5-20℃ / min.

[0030] Furthermore, the thermogravimetric temperature rise range in step (4) is 30-250℃, and the heating rate is 10℃ / min.

[0031] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for detecting the chelation degree of crystalline glycine chelates, which has the following beneficial effects:

[0032] The chelation degree detection method for crystalline glycine chelates provided by this invention is accurate and reliable, and can effectively indicate the degree of chelation of glycine chelates. Glycine chelate products verified by this method have broad application prospects and can be used in food, health products, pharmaceuticals and other fields to improve product quality and market competitiveness. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0034] Figure 1 shows the SEM image of magnesium glycine;

[0035] Figure 2 shows the FTIR spectra of glycine and glycine magnesium;

[0036] Figure 3 shows the HPLC chromatogram of magnesium glycine;

[0037] Figure 4 shows the TG spectrum of magnesium glycine. Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1

[0040] Methods for determining the glycine magnesium chelation degree:

[0041] (1) The crystal structure of glycine chelate salt was obtained by scanning electron microscopy: Take an appropriate amount of glycine magnesium powder sample, place the sample on the stage with conductive adhesive, gently flatten it with tweezers, spray a thin metal layer, put it into the sample cup, open the sample chamber door, put the sample cup into the chamber, observe and adjust the imaging, adjust the electron gun current and accelerating voltage to achieve the best resolution and clarity, magnification 50x, accelerating voltage: 10kV, probe current: 10nA to obtain a high-quality SEM image, as shown in Figure 1. It can be seen that glycine magnesium has a good crystal form.

[0042] (2) Characterization was performed using Fourier transform infrared spectroscopy. The change and shift of peak values ​​were used to determine whether the glycine chelate salt had undergone a chelation reaction: A suitable amount of sample was scanned, and the infrared spectra of glycine and glycine magnesium were compared. As shown in Figure 2, glycine showed a peak value at 2500 cm⁻¹. -1 and 3100cm -1 There is a very strong broad absorption peak between them, which is NH3. + The characteristic absorption peak is at 1521.87 cm⁻¹. -1 and 1332.93cm -1 The absorption peaks appearing at this point are the peaks of the asymmetric and symmetric stretching vibrations of the carboxylate group;

[0043] In the Fourier transform infrared spectrum of magnesium glycine, we found 2500 cm⁻¹ -1 and 3100cm -1 The strong and broad absorption peak between them, i.e., NH3 + The characteristic absorption peak disappeared at 1577.63 cm⁻¹. -1 and 1340.33 -1 Asymmetric and symmetric stretching vibration peaks of the carboxylate group appear at cm⁻¹. This corresponds to the peak at 1521.87 cm⁻¹ in the glycine infrared spectrum. -1 Up to 1332.93cm -1 Compared to the peaks at 3211 cm⁻¹, these peaks shift to higher wavelengths, indicating that carboxyl groups are involved in coordination. NH₂ at 3211 cm⁻¹... -1 and 3342cm -1The peak values ​​indicate the formation of Mg-NH2 and COO-Mg groups in magnesium glycinate. The variations and shifts in the peaks on the FT-IR spectrum suggest that our glycinate underwent a chelation reaction.

[0044] (3) The glycine content in glycine chelate salt was determined by high performance liquid chromatography, and the metal element content was determined by inductively coupled plasma atomic emission spectrometry.

[0045] Among them, the instruments and detection conditions for high performance liquid chromatography are as follows:

[0046] Chromatographic column: Agilent C18 column, 4.6*150mm, 4μm;

[0047] Mobile phase: 0.1 mol / L sodium acetate solution: acetonitrile = 84:16;

[0048] Column temperature: 40℃;

[0049] Flow rate: 1.0 mL / min;

[0050] Detection wavelength: 360nm;

[0051] Injection volume: 20 μL;

[0052] Preparation of standard solutions:

[0053] Glycine standard stock solution (0.600 mg / mL): Weigh approximately 60 mg of glycine standard and dissolve it in purified water to a final volume of 100 mL.

[0054] Glycine standard working solution: Accurately pipette 0.25 mL, 0.50 mL, 1.00 mL, 2.00 mL, and 4.00 mL of glycine standard stock solution, and dissolve and dilute to 25 mL with purified water. The glycine concentrations in the standard working solutions are 0.006 mg / mL, 0.012 mg / mL, 0.024 mg / mL, 0.048 mg / mL, and 0.096 mg / mL, respectively.

[0055] Derivatization reaction of glycine standard working solution: Transfer 2.0 mL to 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 water bath at 60 °C for 50 min, cool, and then dilute to the mark with pure water. Filter through a 0.45 μm filter membrane and collect the filtrate as the standard curve solution.

[0056] Preparation of sample solution:

[0057] Preparation of solid sample solution: Weigh 60 mg of solid sample and dissolve it in purified water to a final volume of 100 mL.

[0058] Sample dilution: Transfer 1.0 mL to a 25 mL volumetric flask, dissolve and dilute to the mark with purified water.

[0059] Sample derivatization: Transfer 2.0 mL to 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 at 60 °C for 50 min, cool, and then dilute to the mark. Filter through a 0.45 μm filter membrane and collect the filtrate as the sample solution.

[0060] Note: Blank synchronous derivative operation.

[0061] The test results are shown in Figure 3 and Table 2 below:

[0062] Table 2

[0063] Inductively Coupled Plasma Spectrometer Instrumentation and Detection Conditions:

[0064] RF power: 1300W, plasma gas flow rate: 15L / min, nebulizer pressure: 200kPa, auxiliary gas flow rate: 0.3L / min, nebulizer gas flow rate: 0.8L / min, pump injection rate: 1.50mL / min, instrument stabilization delay: 15s, injection delay: 30s, analytical spectral line: 279.077nm;

[0065] Accurately weigh 0.5 g of glycine magnesium chelate into a polytetrafluoroethylene digestion vessel. For samples containing ethanol or carbon dioxide, first heat on a hot plate at low temperature to remove the ethanol or carbon dioxide, then add 10 mL of a nitric acid-perchloric acid (10+1) mixed solution. Digest on a CNC hot plate, adding nitric acid as needed (conditions: maintain at 120℃ for 1-2 hours, raise to 150℃ and maintain for 2-4 hours, raise to 180℃-200℃ and maintain for 2 hours). If the solution turns brownish-black during digestion, add 5 mL of the nitric acid-perchloric acid (10+1) mixed solution until white fumes are emitted and the digest is colorless, transparent, and slightly yellow. Cool, transfer to a 25 mL volumetric flask, and dilute to the mark with nitric acid solution (the dilution factor depends on the content of the analyte in the sample). Mix well and set aside. Perform a blank test simultaneously.

[0066] The test results are shown in Table 3:

[0067] Table 3

[0068] (4) The free water and water of crystallization of glycine chelate salts were determined by thermogravimetric analysis. The glycine content, metal ion content, water of crystallization, and free water content of each glycine chelate salt were summed to demonstrate the high purity characteristics of the product:

[0069] Thermogravimetric analysis (TGA) heating rate is 10℃ / min, and temperature ranges from 30 to 250℃.

[0070] All components are summed, as shown in Figure 4 and Table 4:

[0071] Table 4

[0072] SEM images show that glycine salt exists in a regular crystal structure. In the Fourier transform infrared spectrum of the glycine magnesium chelate, NH3... + The complete disappearance of the specific absorption peak indicates that all free glycine has been consumed through the chelation reaction. Peaks appearing in the Mg-NH2 and COO-Mg groups confirm that glycinate salts were indeed formed through the chelation reaction. The magnesium content was tested by ICP-OES, and the glycine content was tested by HPLC. The free water and water of crystallization of the glycine magnesium chelate were determined using TG. The combined results of glycine content, mineral content, water of crystallization, and free water were greater than 99.5%, conforming to the standard molecular formula of glycine acid chelates, indicating the high purity of the glycine magnesium chelate.

[0073] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0074] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting the chelation degree of crystalline glycine chelates, characterized in that, Includes the following steps: (1) Obtain the crystal structure of glycine chelate salt by scanning electron microscopy and determine whether it is a crystalline product; (2) Characterization was performed by Fourier transform infrared spectroscopy, and the changes and shifts in peak values ​​were used to determine whether the glycine chelate salt had undergone a chelation reaction; (3) The glycine content in 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 water of crystallization of glycine chelate salts were measured by thermogravimetric analysis. The glycine content, metal ion content, water of crystallization and free water measured in each glycine chelate salt were summed to illustrate the high purity characteristics of the product.

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

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

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

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

6. The method for detecting the chelation degree of crystalline glycine chelates according to claim 1, characterized in that, The conditions for high-performance liquid chromatography described in step (3) are as follows: Mobile phase A: Sodium acetate solution; Mobile phase B: Acetonitrile; The volume ratio of mobile phase A to mobile phase B is 84:16; Chromatographic column: Agilent C18 column, 4.6*150mm, 4μm; column temperature 35-45℃; The flow rate is 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 crystalline glycine chelates according to claim 6, characterized in that, The column temperature in step (3) is 40℃, the flow rate is 1.0mL / min, the injection volume is 20μL, and the detection wavelength is 360nm.

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

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

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

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

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