Derivatization-electric membrane extraction method for separating polar compounds

Abstract

The invention relates to the technical field of sample pretreatment and analytical chemistry, in particular to a derivatization-electric membrane extraction method for separating polar compounds. According to the specific technical scheme, the derivatization-electromembrane extraction method for separating the polar compound comprises the following steps: adding a derivatization reagent into a sample solution containing a polar target compound, and carrying out a derivatization reaction; taking the reacted sample solution as a donor phase, and migrating the polar target compound after the derivatization reaction into the acceptor phase solution through the supported liquid membrane under the action of an external electric field. According to the invention, the problem of poor extraction efficiency caused by poor hydrophobicity and low partition coefficient of the polar compound in an electric membrane extraction system in the prior art is solved.

Description

Technical Field

[0001] The invention relates to the technical field of sample pretreatment and analytical chemistry, and in particular to a derivatization-electromembrane extraction method for separating polar compounds. Background Art

[0002] Electromembrane extraction is an emerging sample pretreatment technology. Its basic principle is to use an external electric field to drive the migration of charged analytes from the donor phase through the supported liquid membrane to the acceptor phase, thereby achieving selective extraction and enrichment of the target compounds. The system usually consists of three parts: the donor phase, the acceptor phase and the supported liquid membrane. The supported liquid membrane is composed of a hydrophobic porous membrane impregnated with an organic extractant, and has the dual functions of physical isolation between the donor phase and the acceptor phase and selective mass transfer. During the extraction process, the pH values ​​of the donor phase and the acceptor phase are adjusted to promote the ionization of the target analyte, thereby enhancing its migration ability driven by the electric field. Due to its high selectivity, low organic solvent consumption and good method compatibility, this technology has been widely used in the pretreatment of environmental, food and biological samples, and has shown great potential in sample pretreatment.

[0003] In theory, all charged substances can be separated and enriched by electromembrane extraction, but in practical applications, this technology still faces significant difficulties in extracting polar compounds. The main reason is that the distribution coefficient of polar compounds in the organic extractant used in the supported liquid membrane is low, making it difficult to effectively enter the supported liquid membrane, which seriously affects the extraction efficiency. In order to improve the electromembrane extraction efficiency of polar compounds, existing research mainly focuses on optimizing the composition of the supported liquid membrane to improve the adaptability of the system to polar compounds, such as selecting more polar organic solvents (Analytica Chimica Acta, 2020, 1129, 118-125), adding ionic or non-ionic additives (Journal of Chromatography A, 2021, 1639, 461915), constructing a semi-interpenetrating network polymer support membrane (Chinese patent CN112138547A), and even using gel materials to replace traditional supported liquid membranes (Talanta, 2018, 179, 318-325) to improve the distribution performance of polar compounds in the supported liquid membrane. However, these methods often have shortcomings such as poor selectivity, complex operation or limited scope of application, which makes it difficult to meet the needs of efficient and stable pretreatment in complex samples.

[0004] To address the above issues, there is an urgent need to develop a method that can effectively improve the distribution coefficient and migration efficiency of polar compounds in the electromembrane extraction system without significantly increasing the complexity of the operation. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention provides a derivatization-electromembrane extraction method for separating polar compounds. By introducing chemical derivatization into the donor phase, the hydrophobicity of the target compound is enhanced, thereby enhancing its distribution within the supported liquid membrane, achieving efficient extraction of polar compounds. This method addresses the existing problem of poor extraction efficiency of polar compounds in electromembrane extraction systems due to poor hydrophobicity and low partition coefficients.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0007] The present invention discloses a derivatization-electromembrane extraction method for separating polar compounds. The method comprises the following steps: adding a derivatization reagent to a sample solution containing a polar target compound to carry out a derivatization reaction; using the sample solution after the reaction as a donor phase, and under the action of an external electric field, the polar target compound after the derivatization reaction migrates through a supported liquid membrane into an acceptor phase solution.

[0008] Preferably, the polar target compound is a compound containing a carboxyl, amino, hydroxyl or carbonyl functional group.

[0009] Preferably, the derivatization reagent includes but is not limited to at least one of acyl halides, hydrazides, aromatic aldehydes, and isocyanate reagents.

[0010] Preferably, the acyl halide reagents include but are not limited to dansyl chloride, benzoyl bromide, dimethylaminobenzoyl bromide, and acetyl bromide; the hydrazide reagents include but are not limited to dansyl hydrazide and benzoyl hydrazide; the aromatic aldehyde reagents include but are not limited to benzaldehyde and p-nitrobenzaldehyde; the isocyanate reagents include but are not limited to phenyl isocyanate and propyl isocyanate.

[0011] Preferably, the derivatization reaction is carried out at pH 8-10, at a temperature of 40-70° C., and for 20-60 min.

[0012] Preferably, the supported liquid membrane is made of a membrane carrier material impregnated with an organic extractant.

[0013] Preferably, the membrane carrier material is polypropylene, polyvinylidene fluoride or polytetrafluoroethylene membrane with a pore size of 0.1-0.2 μm and a thickness of 100-200 μm.

[0014] Preferably, the organic extractant is one or both of alcohol and ketone solvents, or the organic extractant is one or both of ether and phosphate solvents.

[0015] Preferably, the alcohol solvent includes but is not limited to n-octanol and n-nonanol, the ketone solvent includes but is not limited to 2-heptanone and 2-nonanone, the ether solvent includes but is not limited to 2-nitrophenyl octyl ether and 2-nitrophenyl pentyl ether, and the phosphate solvent includes but is not limited to triethyl phosphate and tributyl phosphate.

[0016] Preferably, the pH of the donor phase and acceptor phase solutions are adjusted according to the properties of the target compound. When the target compound is an alkaline compound, the pH of the donor phase and the acceptor phase is adjusted to 2-6; when the target compound is an acidic compound, the pH of the donor phase and the acceptor phase is adjusted to 8-12. During extraction, oscillation is performed at a speed of 300-1000 rpm, a voltage of 10-100 V, and an extraction time of 5-30 min.

[0017] The present invention has the following beneficial effects:

[0018] 1. This method modifies the structure of polar target compounds through chemical derivatization, introducing benzene rings or alkyl hydrophobic groups. This significantly improves their distribution between the donor phase and the supported liquid membrane, enabling efficient extraction under electric field drive. This method is suitable for the extraction of polar small molecules from complex biological matrices such as serum and urine, and has promising application prospects and promotional value.

[0019] 2. The present invention introduces a derivatization reaction in the donor phase to convert polar target compounds into derivatives with hydrophobic functional groups, thereby enhancing their distribution performance in the organic membrane phase, thereby significantly improving the overall extraction efficiency and being suitable for the efficient extraction of polar compounds in complex biological matrices.

[0020] 3. The present invention uses chemical derivatization to allow the polar target compound to react specifically with the derivatization reagent to generate derivatives with specific structures, effectively avoiding the interference of other non-target substances in complex biological samples and improving the selectivity of the extraction process.

[0021] 4. The derivatives after the introduction of hydrophobic groups in the present invention have significantly improved distribution coefficients in the supporting liquid membrane. Driven by the electric field, they can quickly and efficiently migrate from the donor phase to the acceptor phase, thereby improving the extraction efficiency of polar compounds.

[0022] 5. The entire extraction process is carried out in an electro-membrane extraction device with a simple structure and easy-to-control and optimize operation steps. The derivatization reaction conditions are mild, eliminating the need for complex equipment and tedious operations, making it easy to apply in actual sample analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the derivatization-electromembrane extraction process. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

[0025] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0026] refer to Figure 1 As shown, the present invention discloses a derivatization-electromembrane extraction method for separating polar compounds. A derivatization reagent is added to a sample solution containing a polar target compound to perform a derivatization reaction. The derivatization reaction is carried out under conditions of pH = 8-10, a reaction temperature of 40-70°C, and a reaction time of 20-60 minutes, so that the polar target compound generates a highly hydrophobic derivative before migration. The sample solution after the derivatization reaction is used as a donor phase, thereby significantly improving its distribution behavior in the supported liquid membrane. Then, under the action of an external electric field, the polar target compound after the derivatization reaction migrates through the supported liquid membrane into the acceptor phase solution, thereby achieving efficient extraction of the polar target compound in a complex biological matrix.

[0027] Furthermore, the polar target compound is a compound containing a carboxyl, amino, hydroxyl, or carbonyl functional group. The derivatization reagent promotes a selective chemical reaction of the polar functional groups (such as carboxyl, hydroxyl, amino, or carbonyl) on the polar target compound molecule, introducing a hydrophobic substituent group, thereby generating a relatively hydrophobic derivative.

[0028] The derivatization reagent includes, but is not limited to, at least one of an acyl halide, hydrazide, aromatic aldehyde, and isocyanate reagent. The specific selection can be determined based on the functional group characteristics and reaction selectivity of the target compound. As one embodiment, the acyl halide reagent includes, but is not limited to, dansyl chloride, benzoyl bromide, dimethylaminobenzoyl bromide, and acetyl bromide; the hydrazide reagent includes, but is not limited to, dansyl hydrazide and benzoyl hydrazide; the aromatic aldehyde reagent includes, but is not limited to, benzaldehyde and p-nitrobenzaldehyde; and the isocyanate reagent includes, but is not limited to, phenylisocyanate and propyl isocyanate.

[0029] The present invention uses the sample solution after the derivatization reaction as the donor phase. The electromembrane extraction device used includes a donor phase container, an acceptor phase container and a supported liquid membrane sandwiched between the two. The supported liquid membrane is made of a membrane carrier material (i.e., a hydrophobic porous membrane) infiltrated with an organic extractant. The membrane carrier material is polypropylene, polyvinylidene fluoride or polytetrafluoroethylene membrane with a pore size of 0.1-0.2μm and a thickness of 100-200μm. Such materials have good physical and chemical stability and uniform pore size, which can effectively avoid the introduction of non-target macromolecular substances and impurities such as particulate matter during the extraction process.

[0030] Furthermore, the organic extractant is one or both of an alcohol or ketone solvent, or the organic extractant is one or both of an ether or phosphate solvent. Specifically, the alcohol solvent includes but is not limited to n-octanol and n-nonanol, the ketone solvent includes but is not limited to 2-heptanone and 2-nonanone, the ether solvent includes but is not limited to 2-nitrophenyl octyl ether and 2-nitrophenyl pentyl ether, and the phosphate solvent includes but is not limited to triethyl phosphate and tributyl phosphate. The organic extractant is adjusted according to the charge properties of the polar target compound; if the polar target compound is an anionic substance after derivatization, an alcohol or ketone solvent is preferred; if it is a cationic substance, an ether or phosphate solvent is preferred.

[0031] In some embodiments, the donor phase and acceptor phase solutions are selected based on the principle of the extraction method and the acidity and alkalinity of the target. In electromembrane extraction, when the polar target compound is an alkaline substance, the acceptor phase solution is selected from acidic solutions such as hydrochloric acid, formic acid, and trifluoroacetic acid, and the pH value of the donor phase and the acceptor phase is adjusted to 2-6; when the polar target compound is an acidic substance, the acceptor phase solution is selected from alkaline solutions such as sodium hydroxide and ammonia water, and the pH value of the donor phase and the acceptor phase is adjusted to 8-12. The specific value is set according to the charge state of the target. The extraction voltage can be set to 10-100V, the extraction time can be set to 5-30min, and the oscillation condition is provided by a constant temperature mixer with a rotation speed of 300-1000rpm.

[0032] In some embodiments, the sample solution includes complex biological matrices such as serum and urine.

[0033] Example 1

[0034] Under the optimized experimental conditions of the derivatization-electromembrane extraction method described in the present invention, L-carnitine and its acetylcarnitine, propionylcarnitine, butyrylcarnitine, hexanoylcarnitine, octanoylcarnitine and decanoylcarnitine derivatives were efficiently extracted from human serum samples.

[0035] The specific process of electromembrane extraction is:

[0036] (1) Reagent preparation: The derivatization reagent dimethylaminobenzoyl bromide was prepared as a 40 mg / mL acetonitrile solution; triethanolamine was prepared as a 750 mM acetonitrile solution to adjust the pH of the derivatization reaction; formic acid was prepared as a 30 mg / mL acetonitrile solution to consume the residual derivatization reagent in the reaction.

[0037] (2) Take 25 μL of serum sample and place it in a 2 mL polypropylene centrifuge tube (donor phase container). After adding 10 ng of isotope internal standard, add 25 μL of triethanolamine solution and 25 μL of dimethylaminobenzoyl bromide solution in sequence. Adjust the pH of the reaction system to 10, mix well, and react at 65°C for 60 min. After the reaction is completed, add 25 μL of formic acid to consume the remaining derivatization reagent. Then, add an appropriate amount of hydrochloric acid solution to adjust the pH of the reaction mixture to 5.

[0038] (3) A polypropylene membrane with a pore size of 0.2 μm and a thickness of 100 μm was selected and fixed to the bottom of the connecting end of a 1 mL pipette tip by thermal bonding to achieve a seal. The excess membrane on the outer circle was then cut off. For ease of operation, the tip of the pipette tip was cut off by about 2 cm. The resulting sealed pipette tip was used as the acceptor phase container. 5 μL of binary organic extractant (1-ethyl-2-nitrobenzene and 2-nonanone, volume ratio 1:1) was evenly coated on the surface of the polypropylene membrane at the bottom of the pipette tip to form a supported liquid membrane.

[0039] (4) Considering that all seven carnitine derivatives are alkaline compounds, 200 μL of hydrochloric acid solution (pH = 5) was added to the acceptor phase. The tip of the gun containing the acceptor phase solution (with the membrane end facing downward) was inserted into the donor phase container, ensuring that a gap of about 1 mm was maintained between the support liquid membrane and the donor phase solution. Two platinum wire electrodes were inserted into the donor phase and the acceptor phase respectively, and connected to a DC regulated power supply, thereby forming a complete flat-plate membrane electro-membrane extraction device. The device was placed on a constant temperature mixer, the speed was set to 500 rpm and an operating voltage of 80 V was applied for extraction. After 20 minutes of extraction, the acceptor phase solution was directly analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS).

[0040] Chromatographic conditions: chromatographic column: C18 column; column temperature: 40°C; mobile phase A: 0.1% formic acid, mobile phase B: methanol; gradient elution program: 0-2 min, 90% A; 2-10 min, 90% A-0% A; 10-15 min 0% A, 15.1-20 min 90% A; flow rate: 0.6 mL / min, injection volume: 5 μL.

[0041] Mass spectrometry conditions: electrospray ionization-positive ionization mode (ESI+); detection method: selected reaction monitoring (SRM); spray voltage: 3200 V; evaporation temperature: 350°C; ion transfer tube temperature: 320°C; sheath gas: 30 Arb; auxiliary gas: 10 Arb.

[0042] As shown in Table 1, the calculated recoveries of the seven carnitines were: L-carnitine 65.3%, acetylcarnitine 66.0%, propionylcarnitine 66.1%, butyrylcarnitine 74.1%, hexanoylcarnitine 73.4%, octanoylcarnitine 82.4%, and decanoylcarnitine 94.2%.

[0043] Table 1 Comparison of water-n-octanol partition coefficient and recovery rate before and after carnitine derivatization

[0044]

[0045] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A derivatization-electro-membrane extraction method for separating polar compounds, characterized in that: A derivatization reaction is performed by adding a derivatization reagent to a sample solution containing a polar target compound; The sample solution after the reaction is used as the donor phase. Under the action of an external electric field, the polar target compound after the derivatization reaction migrates into the acceptor phase solution through the supported liquid membrane.

2. The derivatization-electromembrane extraction method for separating polar compounds according to claim 1, characterized in that: The polar target compound is a compound containing a carboxyl group, an amino group, a hydroxyl group or a carbonyl functional group.

3. The derivatization-electromembrane extraction method for separating polar compounds according to claim 1, characterized in that: The derivatization reagent includes but is not limited to at least one of acyl halides, hydrazides, aromatic aldehydes, and isocyanate reagents.

4. The derivatization-electromembrane extraction method for separating polar compounds according to claim 3, characterized in that: The acyl halide reagents include but are not limited to dansyl chloride, benzoyl bromide, dimethylaminobenzoyl bromide, and acetyl bromide; the hydrazide reagents include but are not limited to dansyl hydrazide and benzoyl hydrazide; the aromatic aldehyde reagents include but are not limited to benzaldehyde and p-nitrobenzaldehyde; the isocyanate reagents include but are not limited to phenyl isocyanate and propyl isocyanate.

5. The derivatization-electromembrane extraction method for separating polar compounds according to claim 1, characterized in that: The derivatization reaction is carried out at pH 8-10, at a temperature of 40-70° C., and for 20-60 minutes.

6. The derivatization-electromembrane extraction method for separating polar compounds according to claim 1, characterized in that: The supported liquid membrane is made of a membrane carrier material impregnated with an organic extractant.

7. The derivatization-electromembrane extraction method for separating polar compounds according to claim 6, characterized in that: The membrane carrier material is polypropylene, polyvinylidene fluoride or polytetrafluoroethylene membrane with a pore size of 0.1-0.2 μm and a thickness of 100-200 μm.

8. The derivatization-electromembrane extraction method for separating polar compounds according to claim 6 or 7, characterized in that: The organic extractant is one or both of alcohol and ketone solvents, or the organic extractant is one or both of ether and phosphate solvents.

9. The derivatization-electromembrane extraction method for separating polar compounds according to claim 8, characterized in that: The alcohol solvents include but are not limited to n-octanol and n-nonanol, the ketone solvents include but are not limited to 2-heptanone and 2-nonanone, the ether solvents include but are not limited to 2-nitrophenyl octyl ether and 2-nitrophenyl pentyl ether, and the phosphate solvents include but are not limited to triethyl phosphate and tributyl phosphate.

10. The derivatization-electromembrane extraction method for separating polar compounds according to claim 1, characterized in that: The pH of the donor phase and the acceptor phase solutions are adjusted according to the properties of the target compound. When the target compound is an alkaline compound, the pH of the donor phase and the acceptor phase is adjusted to 2-6; When the target compound is an acidic compound, the pH of the donor phase and the acceptor phase is adjusted to 8-12. During extraction, the rotation speed is 300-1000 rpm, the voltage is 10-100 V, and the extraction time is 5-30 minutes.

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