CN114570065B - Separation method of phenolic acid components in traditional Chinese medicine - Google Patents
Separation method of phenolic acid components in traditional Chinese medicine Download PDFInfo
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- CN114570065B CN114570065B CN202011398600.XA CN202011398600A CN114570065B CN 114570065 B CN114570065 B CN 114570065B CN 202011398600 A CN202011398600 A CN 202011398600A CN 114570065 B CN114570065 B CN 114570065B
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/30—Partition chromatography
- B01D15/305—Hydrophilic interaction chromatography [HILIC]
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
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Abstract
The invention relates to a separation method of phenolic acid components in traditional Chinese medicine, belongs to the field of analytical chemistry, relates to a separation method of phenolic acid components in traditional Chinese medicine samples, and in particular relates to an analysis method of ginkgetin and organic acid active ingredients in honeysuckle based on zwitterionic hydrophilic chromatographic stationary phase of binary anhydride modified amino. The method adopts the materials shown in the following structural schematic diagram, optimizes the condition of a mobile phase and establishes an analysis method of flavonoids and organic acids in the traditional Chinese medicine. The established method has different separation selectivity with common reversed phase C18 chromatographic column and other hydrophilic chromatographic columns, and is suitable for orthogonal separation of traditional Chinese medicine complex system.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a method for separating and analyzing phenolic acid components in traditional Chinese medicines by using a high performance liquid chromatography.
Technical Field
Hydrophilic liquid chromatography mode (HILIC) is a novel chromatographic separation technique developed in recent years for separating strongly polar compounds, and was originally proposed by Alpert in 1990 [ Alpert, A.J.J.chromatogrInd., 1990,499,177-196]. Similar to normal phase chromatography (NPLC), HILIC uses a polar stationary phase and a relatively less polar water/organic solvent as the mobile phase, where water is the strong eluting solvent. HILIC can use a solution system with large water content as a mobile phase, and can solve the problems of poor solubility of mobile phase chromatography relative to water-soluble substances, extremely sensitive retention time to the water content of the mobile phase, compatibility with a mass spectrum detector and the like. Meanwhile, the separation selectivity difference between HILIC and RPLC is larger, and the mobile phase system is similar, so that the system can be used as an effective supplement of RPLC, and can be combined with RPLC to construct a two-dimensional liquid chromatography system for separating complex samples.
Traditional Chinese medicine is a typical complex system, and has various effective components including flavone, alkaloid, organic acid, saponin, terpenoid, etc. The substance basis of traditional Chinese medicines cannot be comprehensively researched by simply relying on one-dimensional reversed phase chromatography, and urgent requirements are put forward on separation materials with orthogonality with reversed phase chromatography. The hydrophilic chromatographic material is compatible with the mobile phase system due to the large difference between the hydrophilic chromatographic material and the stationary phase of the reversed phase chromatography, and is suitable for establishing an orthogonal multidimensional separation system with the reversed phase chromatography. Hydrophilic chromatographic materials are various, chromatographic methods are developed by combining the properties of a stationary phase and a sample, and suitable analytical methods for different classes of compounds in traditional Chinese medicine are established. The phenolic acid components such as organic acid and flavone are important active components in traditional Chinese medicines, and are usually analyzed by adopting reverse phase chromatography, but due to complex components, the problems of insufficient separation selectivity and limited peak capacity are usually caused, and the method is combined with special separation materials to provide different separation selectivities from the reverse phase materials, so that an orthogonal two-dimensional separation system is constructed, and a novel means is hopeful to be provided for the separation of complex phenolic acid components.
The invention establishes a separation method of phenolic acid in traditional Chinese medicine by adopting a novel zwitterionic hydrophilic chromatographic stationary phase, and the target compound has different separation selectivities with reversed phase C18 columns and other hydrophilic chromatographic columns, thereby providing a novel technical means for researching the substance basis of a traditional Chinese medicine complex system.
Disclosure of Invention
The invention aims to provide a chromatographic separation method of phenolic acid in traditional Chinese medicine, which obtains different separation selectivity from reversed phase C18 and other hydrophilic chromatographic materials.
The above object of the present invention is achieved by the following technical solutions:
1) Dissolving the traditional Chinese medicine extract containing phenolic acid components with a solvent to obtain a traditional Chinese medicine extract solution; the solvent is one or more of methanol, acetonitrile and water; the solvent contains one or two of ammonium formate and ammonium acetate with the final concentration of 0-50 mM;
2) Pouring the solution into a high performance liquid chromatograph, eluting with a mobile phase, wherein chromatographic column filler in the high performance liquid chromatograph is a zwitterionic group with a bonding phase end being dibasic acid anhydride modified amino;
the adopted water phase mobile phase is water, wherein additives are added or not, the additives in the water phase mobile phase comprise one or more than two of phosphoric acid, ammonium formate, ammonium acetate, formic acid and acetic acid, the concentration of the additives is 0-250mM (preferably 10-100 mM), and the pH value of the mobile phase is 2.5-7; the adopted organic phase mobile phase is one or more than two of acetonitrile, methanol and acetone;
the elution process is that the volume proportion of the mobile phase organic phase is 95-10%.
The traditional Chinese medicine phenolic acid component comprises ginkgo flavonoid compounds in ginkgo or organic acid compounds in honeysuckle.
The filler is a zwitterionic hydrophilic chromatographic stationary phase bonded on the surface of a silica gel matrix, and the tail end of the bonding phase is a zwitterionic group of a dibasic acid anhydride modified amino group, and the structural schematic formula is as follows:
the preparation method of the zwitterionic hydrophilic chromatographic stationary phase comprises the following steps: adding pyrazine dianhydride into water/methanol, water/ethanol or water/DMF/pyridine mixed solvent with the volume ratio of 1/10-10/1, adding amino silica gel (PAS), and reacting for 10-100 hours at the temperature of 40-70 ℃; filtering, washing with DMF, methanol, water and methanol in sequence, and drying the obtained solid at 60-90 ℃ for 6-48 hours to obtain the stationary phase (ZAC).
The separation method uses an ultraviolet detector with a wavelength of 200-400 nm.
The invention has the following advantages:
(1) The separation selectivity is good. The chromatographic method established by the invention for separating organic acid, flavone and the like in the traditional Chinese medicine has different separation selectivity from reverse phase chromatography and other hydrophilic chromatographic materials, and lays a foundation for separation analysis and purification preparation of traditional Chinese medicine complex compounds.
(2) The analysis method is simple and convenient and is easy to operate.
Drawings
FIG. 1 shows the separation of ginkgo flavone according to example 1 of the present invention on different chromatographic columns.
Peak1 quercetin-3-O-β-D-glucoside；
2-quercetin 3-O-β-D-(6”-p-coumaroyl)glucopyranosyl(1-2)-α-L-rhamnopyranoside；
3-Rutin；
4-quercetin-3-O-2”,6”-rhamnosyl glucoside
FIG. 2 shows the separation of organic acids on different columns according to example 2 of the present invention.
Peak1 isochlorogenic acid A；2-chlorogenic acid；3-neochlorogenic acid；
FIG. 3 shows the separation of the extract of honeysuckle flower in different chromatographic columns according to the embodiment 2 of the present invention.
Peak 1-isochlorogenic acid A；2-chlorogenic acid；3-neochlorogenic acid；4-isochlorogenic acid B or C。
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments.
Example 1
The ginkgetin compound is analyzed by adopting the zwitterionic stationary phase bonded by pyrazine dianhydride, the chromatographic conditions are as follows, the comparison chart of the ginkgetin compound and other chromatographic columns is shown in figure 1, and the zwitterionic stationary phase (ZAC) adopted in the invention has the longest retention time and good peak shape under the same mobile phase condition.
Chromatographic conditions:
chromatographic column: PTZ (4.6X105 mm,3 μm); XBridge Amide (4.6 x 150mm,3.5 μm);
ZIC HILIC(2.1*150mm,3.5μm)；RZ5-ZACE(4.6*150mm,5μm)
mobile phase: a-acetonitrile; b-water (containing 60mM ammonium formate, pH 3.2);
isocratic: A/B (82:18)
Flow rate: 1.0mL/min &0.2mL/min
Detection wavelength: 254nm
Sample preparation: the quercetin-3-O-beta-D-glucoside, quercetin 3-O-beta-D- (6 ' -p-coumaroyl) glucopyranosyl (1-2) -alpha-L-rhamnopyranoside, rutin and quercetin-3-O-2 ', 6 ' -rhamnosyl glucoside were weighed and mixed sample solutions with concentrations of 1mg/mL respectively were prepared with 50% methanol/50% water (v/v).
Example 2
The organic acid compound is analyzed by adopting the pyrazinedicine anhydride bonded zwitterionic stationary phase, the peak shapes and the retention of different chromatographic columns have larger difference, as shown in figure 2, wherein the ZAC chromatographic column has the best effect and mainly shows the phenomena of longest retention time and most symmetrical peak shapes, and peak shape tailing exists when other chromatographic columns are adopted for analysis.
The analysis of the honeysuckle extract by the chromatographic column has obvious difference from the analysis of the C18 chromatographic column, and as shown in figure 3, the peak sequence is different from that of the C18 material, and the ZAC material has better potential in the analysis of organic acid.
Analysis conditions of organic acid control:
chromatographic column: PTZ (4.6X105 mm,3 μm); XBridge Amide (4.6 x 150mm,3.5 μm);
ZIC HILIC(2.1*150mm,3.5μm)；RZ5-ZACE(4.6*150mm,5μm)
mobile phase: a-acetonitrile; b-water (containing 150mM ammonium formate, pH 2.7);
flow rate: 1.0mL/min &0.2mL/min
Elution conditions (isocratic): A/B/C (87:13)
Detection wavelength: 325nm
Sample preparation: an appropriate amount of isochlorogenic acid A, chlorogenic acid and neochlorogenic acid were weighed and dissolved in water to prepare mixed sample solutions each having a compound concentration of 1 mg/mL.
Analysis conditions of honeysuckle extract:
ZAC chromatographic column:
mobile phase (ZAC): a-acetonitrile (containing 10mM ammonium formate); b-water (containing 10mM ammonium formate, pH 3.3);
gradient (ZAC): 0-40min,85-75% A;40-50min,75-50% A.
Collecting corresponding phenolic acid components according to peaks;
c18 chromatographic column
Mobile phase (C18): c-16mM FA water; d-16mM FA acetonitrile.
Gradient (C18): 0-50min,5-30% D;
flow rate: 1.0mL/min &0.2mL/min
Honeysuckle sample: weighing appropriate amount of flos Lonicerae, adding 50% methanol water to concentration of 5mg/mL, ultrasonic treating for 5 hr, and filtering to obtain flos Lonicerae extract solution.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the above detailed method, i.e. it does not mean that the present invention must rely on the above detailed method to be practiced. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (3)
1. A separation method of phenolic acid components in traditional Chinese medicine comprises the following steps:
1) Dissolving the traditional Chinese medicine extract containing phenolic acid components with a solvent to obtain a traditional Chinese medicine extract solution; the solvent is one or more of methanol, acetonitrile and water; the solvent contains one or two of ammonium formate and ammonium acetate with the final concentration of 0-50 mM;
2) Pouring the solution into a high performance liquid chromatograph, eluting with a mobile phase, wherein chromatographic column filler in the high performance liquid chromatograph is a zwitterionic group with a bonding phase end being dibasic acid anhydride modified amino;
the adopted water phase mobile phase is water, wherein additives are added or not, the additives in the water phase mobile phase comprise one or more than two of phosphoric acid, ammonium formate, ammonium acetate, formic acid and acetic acid, the concentration of the additives is 0-250mM, and the pH value of the mobile phase is 2.5-7; the adopted organic phase mobile phase is one or more than two of acetonitrile, methanol and acetone;
the elution process is that the volume proportion of the mobile phase organic phase is 95-10%;
the chromatographic column filler is a zwitterionic hydrophilic chromatographic stationary phase bonded on the surface of a silica gel matrix, and the tail end of the bonding phase is a zwitterionic group of a dibasic acid anhydride modified amino group, and the structural schematic formula is as follows:
the preparation method of the zwitterionic hydrophilic chromatographic stationary phase comprises the steps of adding pyrazine dianhydride into a water/methanol, water/ethanol or water/DMF/pyridine mixed solvent with the volume ratio of 1/10-10/1, adding amino silica gel (PAS), and reacting for 10-100 hours at the temperature of 40-70 ℃; filtering, washing with DMF, methanol, water and methanol in sequence, and drying the obtained solid at 60-90 ℃ for 6-48 hours to obtain the stationary phase.
2. The separation method according to claim 1, wherein the phenolic acid component of the traditional Chinese medicine comprises ginkgo flavonoids in ginkgo or organic acids in honeysuckle.
3. The separation method according to claim 2, wherein: an ultraviolet detector with a wavelength of 200-400nm is used.
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| CN106861662A (en) * | 2017-04-01 | 2017-06-20 | 大连理工大学 | Amphion hydrophilic Interaction Chromatography fixing phase, preparation method and applications that zwitterion is bonded respectively |
| CN107029686A (en) * | 2017-04-01 | 2017-08-11 | 大连理工大学 | Hydrophilic Interaction Chromatography stationary phase, preparation method and applications based on amine functionalized imidazole ion liquid |
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| CN103170311A (en) * | 2013-03-20 | 2013-06-26 | 华东理工大学 | Novel amino serial chromatographic support and preparation method |
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| CN106861662A (en) * | 2017-04-01 | 2017-06-20 | 大连理工大学 | Amphion hydrophilic Interaction Chromatography fixing phase, preparation method and applications that zwitterion is bonded respectively |
| CN107029686A (en) * | 2017-04-01 | 2017-08-11 | 大连理工大学 | Hydrophilic Interaction Chromatography stationary phase, preparation method and applications based on amine functionalized imidazole ion liquid |
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